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CONTENTS

Dedications

Acknowledgements

About the Authors

SECTION I: THE BASIC MOLECULES OF LIFE

CHAPTER 1 Amino Acids and Proteins

Overview

Amino Acids—Structure and Functional Groups

Essentia l and Non-Essentia l

Bas ic Structure

Characteris tics of R-Groups

Bas ic Protein Structure

Levels of Protein Structure

Categories of Proteins

Amino Acid and Peptide-Derived Hormones and Neurotransmitters

Enzymes

Structura l Proteins

Motor Proteins

Transport/Channel Proteins

Review Questions

CHAPTER 2 Carbohydrates

Overview

Bas ic Carbohydrate Structure and Function

Monosaccharides and Disaccharides

Glycogen and Starches

Glycoproteins

Glycosaminoglycans

Review Questions

CHAPTER 3 Lipids

Overview

Bas ic Lipid Functions

Bas ic Membrane Lipid Structure

Complex Lipids

Glycol ipids/Sphingol ipids

Eicosanoids

Cholesterol

Lipoproteins

Bi le Sa l ts

Lipid-Derived Hormones/Vitamin D

Corticosteroids (Adrenal Gland)

Androgens (Testes ) and Estrogens (Ovaries )

Vi tamin D

Review Questions

CHAPTER 4 Nucleos ides , Nucleotides , DNA, and RNA

Overview

Nucleos ides and Nucleotides

Components of Nucleos ides and Nucleotides

Synthes is of Purine Nucleos ides and Nucleotides

Synthes is of Pyrimidine Nucleos ides and Nucleotides

Formation of Deoxy Nucleos ides and Nucleotides

Breakdown of Purines and Pyrimidines

RNA and DNA—Bas ic Structure and Function

RNA

DNA

Review Questions

SECTION I: Integrated USMLE-Style Questions and Answers

Questions

Answers

SECTION II: FUNCTIONAL BIOCHEMISTRY

CHAPTER 5 Enzymes and Amino Acid/Protein Metabol i sm

Overview

Enzymes

Enzyme Reactions

Cofactors

Regulation

Amino Acid Metabol i sm

Amino Acid Synthes is

Amino Acid Degradation

The Urea Cycle

Review Questions

CHAPTER 6 Carbohydrate Metabol i sm

Overview

Glycolys is

Ci tric Acid Cycle

Oxidative Phosphorylation

Gluconeogenes is

The Pentose Phosphate Pathway

Glycogen Synthes is

Glycogen Breakdown

Modified Carbohydrates (Glycoproteins , GAGs)

Review Questions

CHAPTER 7 Lipid Metabol i sm

Overview

Fatty Acid Metabol i sm

Fatty Acid Synthes is

Fatty Acid Degradation

Metabol i sm of Complex Lipids

Triacylglycerol Synthes is

Phosphoglyceride Synthes is

Ketone Body Synthes is

Ceramide/Sphingol ipids Synthes is

Cholesterol Synthes is

Review Questions

CHAPTER 8 Membranes

Overview

Membrane Structure

Lipids

Proteins

Membrane Functions

Membrane Channels

Membrane Signal ing

Review Questions

CHAPTER 9 DNA/RNA Function and Protein Synthes is

Overview

Structure of the Nucleus

His tones

Nuclear Matrix/Scaffold

Nucleolus and Ribosome Synthes is

DNA Repl ication and Transcription

DNA Repl ication

Transcription

Protein Synthes is

Posttrans lational Trafficking/Modification

Control of Gene Express ion

Mutations and Repair Mechanisms

Regulation of Cel l Growth and Di fferentiation

Review Questions

SECTION II: Integrated USMLE-Style Questions and Answers

Questions

Answers

SECTION III: APPLIED BIOCHEMISTRY

CHAPTER 10 Metabol i sm and Vi tamins/Minera ls

Co-authors/Editors: Maria L. Valencik and Cynthia C. Mastick

Overview

Metabol ic Roles of Major Biochemica l Molecules

Integration and Regulation of Metabol i sm

Glucose-6-phosphate

Pyruvate

Acetyl -CoA

Hormonal Control of Metabol i sm

Insul in

Glucagon

Catecholamines

Glucocorticoids

Diabetes Mel l i tus (DM)

Vitamins and Minera ls

Vi tamins

Minera ls

Review Questions

CHAPTER 11 The Digestive System

Editor: Kshama Jaiswal

Overview

Summary of the Digestive System

Mouth

Stomach

Liver

Lipid Metabol i sm in the Liver

Gal l Bladder

Pancreas

Smal l Intestine (Duodenum, Jejunum, and I leum)

Large Intestine/Anus

Review Questions

CHAPTER 12 Muscles and Moti l i ty

Co-author/Editor: Darren Campbell

Overview

The Bas ic Components of Muscle

Actin

Tropomyos in–Troponins

Myos in

Myos in Light Chains

Actin-Binding Proteins

Exci tation–Contraction Coupl ing

Skeleta l Muscle

Structure and Genera l Overview

Skeleta l Muscle Types

Cardiac Muscle

Smooth Muscle

Energy Production and Use in Muscles

Microtubule-Based Moti l i ty

Intermediate Fi laments

Nonmuscle Cel l s

Review Questions

CHAPTER 13 Connective Tissue and Bone

Editor: Jacques Kerr, BSc, MB, BS, FRCS, FCEM

Overview

Connective Tissue

Components of Bone

Bone Growth and Remodel ing

Regulation of Ca lcium Levels

Markers of Bone Formation and Resorption

Review Questions

CHAPTER 14 Blood

Co-authors/Editors: Matthew Porteus, MD, PhD and Tina Mantanona

Overview

Bas ic Components of Blood

Red Blood Cel l (RBC) Functions

Diseases Associated with Inadequate Synthes is of Hemoglobin Components

Oxygen Binding

Tense and Relaxed Hgb

Al losteric Binding of O2 by Hgb

Regulation of O2 Binding

Phys iologic Response to Inadequate O2 Del ivery

Sickle Cel l Disease (SCD)

Iron

Iron Metabol i sm

Transferrin

Ferri tin

Regulation of Iron Avai labi l i ty by Hepcidin

Clotting

Platelet Plug Formation Cons is ts of Adhes ion, Aggregation, and Activation of Platelets

The Clotting Cascade

The Fibrin Meshwork

Di fference between Platelet Plug Formation and Clot Formation

Regulation of Clot Formation

Plasmin and Clot Dissolution

Review Questions

CHAPTER 15 The Immune System

Editor: Eric L. Greidinger, MD

Overview

Overview of the Immune System

Antigen

Antibody

Cel l s Associated with the Immune System

T Lymphocytes

B Lymphocytes/Plasma Cel l s

Natura l Ki l ler (NK) Cel l s

Monocytes and Macrophages

Neutrophi l s

Eos inophi l s

Basophi l s

Dendri tic Cel l s (DCs)

Cytokines

Innate Immunity

Complement system

Hypersens i tivi ty Reactions

Review Questions

CHAPTER 16 The Cardiovascular System

Editor: Ralph V. Shohet, MD

Overview

Cardiac Muscle Structure and Function

Sinoatria l and Atrioventricular Nodes

The Cardiac Cycle

Blood Vessels

Endogenous Cholesterol/Lipoprotein Metabol i sm and Transport

VLDL

Intermediate-Dens i ty Lipoprotein (IDL) and LDL

HDL

Atheroscleros is

Biochemica l Mechanisms Associated with Heart Attack

Review Questions

CHAPTER 17 The Respiratory System

Editor: Howard J. Huang, MD

Overview

Bas ic Anatomy and Development

Pulmonary Surfactant and the Developing Lung

O2–CO2 Exchange in the Lung and Acid–Base Ba lance

Noninfective Diseases of the Respiratory System

Obstructive Diseases—Emphysema

Obstructive Diseases—Bronchiti s

Obstructive Diseases—Asthma

Biochemica l Bas is of Asthma Medications

Restrictive Diseases—Acute Respiratory Disease Syndrome

Restrictive Diseases—Occupational Exposures

Restrictive Diseases—Intersti tia l Lung Diseases

Infective Diseases of the Respiratory System

Review Questions

CHAPTER 18 The Urinary System

Editor: Armando J. Lorenzo, MD, MSc, FRCSC, FAAP Overview

Bas ic Anatomy and Phys iology

Renal Fi l tration

The Renal Corpuscle

Nephron

Inul in/Creatinine Clearance

Renin–Angiotens in–Aldosterone System (RAAS)

Renin and Blood Pressure

Macula Densa and Blood Flow/Osmolari ty

Angiotens inogen/Angiotens in I and II

Aldosterone

Vasopress in

Atria l Natriuretic Peptide (ANP)

Acid–Base Ba lance

NH3 and Acid–Base Ba lance

Synthetic Functions

Synthes is of Erythropoietin

Role in Vi tamin D Synthes is

Review Questions

CHAPTER 19 The Nervous System

Editor: Kathryn Beck-Yoo, MD

Overview

Components of the Nervous System

Nerve Impulse Conduction

Neuron at Rest

Nerve Impulse

Repolarization

Autonomic Nervous System

Sympathetic Nervous System

Parasympathetic Nervous System

Neurotransmitters

Dopamine

NE/Epinephrine

Serotonin

Acetylchol ine (Ach)

Regulation of Catecholamines

Glycine, Glutamate, and GABA

Neuropeptides

Biochemistry of Vis ion

Anesthes ia

Review Questions

CHAPTER 20 The Reproductive System

Editor: Catrina Bubier, MD

Overview

Bas ic Anatomy and Development

Female Reproductive System

GnRH

FSH

LH

Estrogens

Progesterone

hCG

The Menstrua l Cycle

Menstruation (Days 1–4)

Fol l i cular/Prol i ferative Phase (Days 5–13)

The Lutea l/Secretory Phase (Days 15–28)

Ferti l i zation

Breast Development and Lactation

Oxytocin

Prolactin

Male Reproductive System

Testosterone

FSH and LH

Review Questions

SECTION III: Integrated USMLE-Style Questions and Answers

Questions

Answers

SECTION IV: APPENDICES

APPENDIX I: Biochemica l Bas is of Diseases

Contributing Editor: Harold Cross, MD, PhD

Amino Acid Synthes is/Degradation

Amino Acid Transport

Urea Cycle Disorders

Structura l Proteins

Carbohydrates

Glycogen Storage

Mitochondria l Enzymes (Excluding Urea Cycle and Fatty Acid Oxidation)

Lipids and Fatty Acid Oxidation Errors

Nucleotide Metabol i sm

Defective DNA

Bi l i rubin Metabol i sm

Blood Clotting Factor Defects

Steroid Hormone Synthes is

Vi tamins/Minera ls and Electrolytes

APPENDIX II: Biochemica l Methods

Polyacrylamide Gel Electrophores is (PAGE) [Sodium Dodecyl Sul fate (SDS)/Non-SDS]

Immunoassays

RIA

ELISA or EIA

Chromatography

Thin Layer (Paper) Chromatography (TLC)

Column Chromatography

Gel Fi l tration Chromatography

Ion-Exchange Chromatography

Affini ty Chromatography

High-Performance/Pressure Liquid Chromatography (HPLC)

Protein and Deoxyribonucleic Acid (DNA)/Ribonucleic Acid (RNA) Precipi tation

DNA and RNA Sequencing

Southern, Northern, and Western Blots

Southern Blotting

Northern Blotting

Western Blotting

PCR

Cloning

Flow Cytometry

Laboratories

APPENDIX III: Organic Chemistry Primer

Overview

Introduction

The Six Organic Elements (C, H, N, O, P, and S)

Carbon (C)

Hydrogen (H)

Nitrogen (N)

Oxygen (O)

Phosphorus (P)

Sul fur (S)

Biochemica l Functional Groups (H, OH, COOH, NH3, PO3, S—S, COH, and )

Hydrogen (Partia l ly Charged and Ionic Forms, H+)

Hydroxyl Group (—OH−)

Carboxyl Group (—COOH)

Amine Group (—NH2)

Phosphate Group (PO3 and PO4)

Sul fur–Sul fur Bonds (—S—S—)

Aldehyde Group (—COH)

Ketone (— )

Summary

Index

DEDICATIONS

To my fami ly: my dear wi fe, Meryl , who has unfa i l ingly supported my profess ional endeavors and endured my countless evening hours at thecomputer on this project; my daughters , Rebecca, Laura , and Miriam, who bring me incredible “naches”; my mother and father (may they rest inpeace) for thei r support in my formative years ; my brothers Howard and Matthew; my mother-in-law Martha for her ever-present accolades ; my

father-in-law Ed (may he rest in peace); and my father-in-law Lou for the profess ional respect a lways accorded me.

Fina l ly to the more than 3000 medica l s tudents I have taught who inspi red my success as a teacher and educator and who preceded thosestudents who I trust wi l l benefi t from this textbook

— Marc E. Tischler

To my parents , fami ly, and friends who persevered throughout the wri ting, proofing, and publ ication of this book as wel l as the manyinstructors , from high school to univers i ty to graduate school to medica l school and beyond, who insti l led in me not only a love for learning

but a lso for teaching.

This book i s personal ly dedicated to one such person, Cass ie Murphy-Cul len, PhD (may she rest in peace), who served as a teacher, counselorand, most-of-a l l , constant and dedicated friend to a l l of her s tudents , including mysel f.

— Lee W. Janson

ACKNOWLEDGEMENTS

Sincere thanks to the current authors of Harper’s I l lus trated Biochemistry for thei r reviews and comments with specia l thanks to Dr. RobertMurray, edi tor of Harper’s , for his patience, kindness , and exceptional efforts in reviewing this book. The authors a lso wish to thank AndreaAguirre, Chineyne Anako, Martin Benjamin, Natasha Bhuyan, Joseph Carrol l , Katharine Flannery, Si lvi ja Gottesman, Michael Ori , CharlesRappaport, Chris topher Ri ley, Alan Schumacher, and Karen Stern, medica l s tudents at the Univers i ty of Arizona, who served on a focus group toprovide va luable ins ight for this text from a medica l s tudent perspective.

ABOUT THE AUTHORS

Marc E. Tischler received an undergraduate degree in biology from Boston Univers i ty, a master degree in chemistry from the Univers i ty of SouthCarol ina , and his doctorate degree in biochemistry from the Univers i ty of Pennsylvania . After serving in a postdoctora l pos i tion in phys iologyat Harvard Medica l School , he joined the facul ty at the Univers i ty of Arizona in 1979 where he i s currently a professor of biochemistry andmolecular biophys ics holding joint appointments in phys iology and in internal medicine. Having served as coordinator of the medica lbiochemistry course for a decade, he was recrui ted to play a major role in the development of the revised medica l curriculum at the Univers i tyof Arizona, which debuted in 2006 and offers an integrated, organ-based approach akin to the second ha l f of this textbook. In that newcurriculum, he des igned and serves as di rector of the medica l block enti tled Digestion, Metabol i sm, and Hormones . He has taught more than3000 medica l s tudents during his tenure in Arizona.

Lee W. Janson received a BS in Biochemistry/minor in Latin from the Univers i ty of Rochester fol lowed by a PhD in biologica lsciences/biochemistry from Carnegie Mel lon Univers i ty. After a postdoctora l fel lowship at NASA-Johnson Space Center doing research onimmunologica l activation in microgravi ty, he entered medica l school at the Univers i ty of Texas Southwestern Medica l Center at Dal las ,continuing with a fami ly practice res idency in both Dal las and San Antonio. He entered the active duty Ai r Force and served as a fami lyphys ician and a fl ight surgeon, including tours of Korea, Iraq, and Afghanis tan. After mi l i tary service, he permanently moved to Edinburgh,Scotland in 2007, where he practices in the National Heal th Service as an emergency room doctor and a genera l phys ician in the UnitedKingdom with occas ional work in Austra l ia and other parts of the world. In his free time, he wri tes and does research. Past book publ icationsinclude Brew Chem 101: The Basics of Home-brewing Chemistry (Storey Publ i shing).

SECTION ITHE BASIC MOLECULES OF LIFE

CHAPTER 1AMINO ACIDS AND PROTEINS

Amino Acids—Structure and Functional GroupsBas ic Protein StructureCategories of ProteinsReview Questions

OVERVIEWAmino acids are the bas ic bui lding blocks of proteins and serve as biologica l molecules in thei r own right with a variety of functions . Aminoacids are often categorized as essentia l or non-essentia l , depending on the abi l i ty of the body to manufacture each amino acid versusrequirement for ingestion in the diet. Al though severa l hundred amino acids exis t, 20 play the predominant role in the human body. Eachamino acid has a characteris tic R-group that determines i ts chemica l nature and, therefore, how i t wi l l interact with other amino acids , othermolecules , and with i ts envi ronment.

Amino acids l ink together via peptide bonds to form peptides and proteins . These peptides and proteins fold into their fina l three-dimens ional shape as the resul t of hydrophobic, hydrophi l i c, hydrogen bonding, and ionic bonding forces (among others ) that resul t from theamino acids in the peptide cha in, including the characteris tics of thei r R-groups . Proteins may be categorized as enzymes , s tructura l proteins ,motor proteins , and transport/channel proteins . The speci fic roles of amino acids and proteins in the synthes is of other molecules and in thefunctions of organ systems and the human bodies wi l l be explored in deta i l in subsequent chapters .

AMINO ACIDS—STRUCTURE AND FUNCTIONAL GROUPS

ESSENTIAL AND NON-ESSENTIAL

Amino acids are the bas ic bui lding blocks of proteins . Twenty amino acids make up proteins in l iving organisms; severa l hundred more aminoacids perform specia l i zed functions in human and non-human biology. Amino acids are often described as Essential (must be obta ined di rectly from food) Non-essential (the human body i s able to produce them on i ts own).

There i s some debate about the exact defini tions of these terms, but 8–10 amino acids are usual ly deemed essentia l and 10–12 are non-essentia l (see Table 1-1).

TABLE 1-1. Amino Acids—R-Group Class i fi cations

Succotash, a combination of corn and l ima beans , was used by Native American hunters and warriors . Light in weight, easy to carry, ands imple to prepare, succotash conta ins a l l the essentia l required amino acids needed by humans and kept these travelers wel l nourishedand heal thy during long trips away from their settlements .

BASIC STRUCTURE

Every amino acid has four components l inked together with a centra l carbon atom (Figure 1-1):

1. Amino group

2. Carboxyl ic acid group

3. Hydrogen atom

4. R-group, which varies with each amino acid

Figure 1-1. Basic Structure of an Amino Acid. A central alpha (α) carbon is bonded to an amino group (NH2), a carboxyl group (COOH), and an R group.[Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

CHARACTERISTICS OF R-GROUPS

As amino acids are identica l except for thei r R-group, four R-group characteris tics class i fy the amino acids (see a lso Table 1-1).

1. Hydrophobic (Water Hating) R-groups: These amino acids prefer to be ins ide a folded protein or covered by another part of a protein or a l ipidmembrane (Chapter 8) where they are hidden from the external water envi ronment.

2. Hydrophilic (Water Loving) R-groups: With partia l charges from the hydroxyl (OH−) or amino (NH3+) parts of these R-groups , these amino acids

prefer to be at or near the surface of a protein where they interact with surrounding water molecules . An exception would be the surfaceportion of a membrane protein that interacts with the hydrophobic region of the phosphol ipid bi layer (Chapter 8). Hydrophi l i c R-groups areoften important at the active s i te of an enzyme (Chapter 5).

3. Charged R-groups: Ei ther pos i tively or negatively charged, these amino acids prefer to be at the surface of a folded protein or in contact withother charged atoms/molecules .

4. Special R-groups: There are four amino acids with specia l qual i ty R-groups .

• Prol ine has a glutamate R-group that has bonded onto i tsel f (see Figure 1-2A) forming an “imino” acid. Prol ine i s often found at sharpturns of folded proteins (Figure 1-2B).

Figure 1-2. A–B. Proline. A. Prol ine forms from the amino acid glutamate when the amino group ni trogen (blue) creates a bond with carbon 2 onthe R-group (red), making an amino acid that i s “folded” onto i tsel f. The carbon atoms are individual ly numbered to a l low the reader to fol lowthe process that takes three s teps (indicated by arrows). B. Prol ine’s specia l s tructure a l lows the formation of a “ha i rpin” β-turn via formationof an imino bond. The amino group from prol ine i s shaded in blue. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, JaypeeBrothers Medica l Publ i shers (P) Ltd., 2009.]

• Cysteine has a sul fhydryl group that can bond with another cysteine sul fhydryl group to form a cystine “disul fide” bond (Figure 1-3). Thisbond can be ei ther within one protein or between two di fferent proteins and i s important in making s trong s tructures such as the proteinkeratin found in fingernai l s .

Figure 1-3. Formation of Cystine Disulfide Bond. The sul fhydryl groups (SH) from two cysteine amino acid res idues from di fferent parts of a s ingleor two separate amino acid sequences may lose one hydrogen atom each to become a cystine res idue by the formation of a disul fide bond (S—S). [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

• Methionine has a sul fur atom conta ined within i ts R-group. Al though i t does not make disul fide bonds , this sul fur i s seen at the s i te ofsome enzyme reactions or at specia l areas of protein s tructure.

• His tidine has a unique imidazole ring conta ining two ni trogen atoms, which can be uncharged or pos i tively charged. This uniquecharacteris tic makes the his tidine R-group important in enzyme reactions that make or break bonds .

Hair and Cystine Double Bonding: Ha i r, which i s composed of l inear protein sequences , i s curly or s tra ight depending on the number of cystinedisul fide bonds . The number and location of cysteine res idues and accessory proteins speci fic for each person affect the number ofdisul fide bonds resul ting in this very individual i zed characteris tic. Chemica ls that promote these disul fide bonds are used for “perms” and,a l ternatively, chemica ls that break these bonds are used in ha i r s tra ightening treatments .

BASIC PROTEIN STRUCTURESevera l factors affect bas ic protein s tructure, including the fol lowing: Amino Acid Composition: Whether the R-group of each amino acid wants to be away from, near, or in contact with water at the surface of the

folded protein. Special Amino Acids: Prol ine, cysteine, and/or methionine exert effects on protein folding due to resul ting bends and disul fide bonds . Functional Sites: Structura l proteins or proteins that cata lyze a reaction (i .e., enzymes; Chapter 5) usual ly conta in speci fic amino acids that are

important for that protein’s particular function. The R-groups at these s i tes wi l l a ffect the protein’s fina l folded shape. Final Modifications: Most proteins are ini tia l ly made with extra amino acids at thei r beginning and/or end. These extra amino acids may be

modified or removed during the maturation of the protein, resul ting in changes to the fina l s tructure. Final Destination: Whether the protein wi l l end up in an aqueous solution or in a membrane wi l l a l so change the folding, as proteins that go

to membranes wi l l ul timately want thei r hydro-phobic, “water hating” R-groups on the outs ide in contact with the hydrophobic membrane(see below, Chapter 8).

LEVELS OF PROTEIN STRUCTURE

When describing protein s tructure, we use the fol lowing terms: primary (1°), secondary (2°), tertiary (3°), and/or quaternary (4°) s tructure. Primary (1°) Structure: The particular sequence of amino acids in a protein (a lso ca l led a polypeptide) i s termed as primary structure. The amino

acids within a polypeptide are termed residues and are l inked via peptide bonds (Figure 1-4A), formed when the carboxyl ic acid group of oneamino acid interacts with the amino group of a second amino acid. The combination produces the peptide bond and one molecule of water.Repeated peptide bonds form a cha in of peptide bond l inkages with the ni trogen from the amino group, the centra l carbon from the aminoacid, and the carbon from the carboxyl ic acid forming the protein “backbone.” The primary s tructure i s often shown as “beads on a s tring”with each bead representing an amino acid res idue (Figure 1-4B).

Figure 1-4. A. Formation of a Peptide Bond. A peptide bond i s formed between the carboxyl ic acid (COOH) group and the amino ni trogen (HHN)group to form a new C—N bond. The process releases one molecule of water (OH and H as shown in pink ci rcle). By convention a protein i sa lways drawn s tarting with the N-terminus on the left. [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica lPhys iology, 23rd edi tion, McGraw-Hi l l , 2010.] B. Primary (1°) Structure. The primary s tructure of a protein i s the cha in of amino acids from the N-terminal (NH2 group of amino acid 1) to i ts C-terminal end (COOH group of the fina l amino acid). Individual amino acids are denoted as green“beads” and bonds are red “s trings” in this s tyl i zed representation. Proteins vary greatly in length from a few amino acids to severa l hundred.[Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Secondary (2°) Structure: From the l inear cha in of amino acids , the C—N and C—C bonds rotate around the centra l carbon atom (see Figure 1-1).This rotation forms secondary structures (Figure 1-5A–C) ca l led an α-helix, β-strand, or β-turn (see a lso Figure 1-2B above) depending on the

hydrophi l i c and hydrophobic charge and s ize influences of the R-groups as wel l as hydrogen and ionic bonding. Parts of the peptide that donot form conventional secondary s tructures are referred to as “random coi l .”

Figure 1-5. A–C. Secondary (2°) Structure. A. The α-hel ix s tructure i s s tabi l i zed by hydrogen bonds (blue dotted l ine) between the N—H of one

amino acid and the of another amino acid. The hydrophobic and hydrophi l i c nature of the R-groups from each amino acid in the hel ixa lso plays a part in formation of the α-hel ix. B. The β-s trands form when the one sequence of success ive amino acids form hydrogen bondsbetween the N—H and the C═O of another group of success ive amino acids . R-groups of these amino acids a lso influence the formation of β-s trands by both charge and s teric forces . β-Strands can be ei ther para l lel (left) or antipara l lel (right) as noted by the arrows. C. A β-turn formsat the juncture between two antipara l lel β-s trands and usual ly involves amino acids with smal l R-groups , including glycine, a lanine, andval ine. β-turns can a lso form via prol ine’s unique s tructure (Figure 1-2B). [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion,Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Tertiary (3°) Structure: Secondary s tructure and the remaining amino acid sequences continue to fold and interact with other parts of the aminoacid sequence to form tertiary (3°) s tructure. These processes aga in are driven by hydrophobic and hydrophi l i c forces of the individual partsof the sequence, as wel l as hydrogen bonding and ionic bonding between charged amino acid R-groups . In particular, R-groups and thepartia l pos i tive charge of the ni trogen and negative charge of oxygen (OH− and C═O−) molecules often form hydrophi l i c and hydrophobic“s ides” of the α-hel ix or β-s trand. As a resul t, these secondary s tructures wi l l pos i tion themselves with each other and with other s imi larareas of the folded protein to keep their hydrophobic areas away from and their hydrophi l i c areas exposed to the external waterenvironment. Examples are shown in Figure 1-6. Additional ly, tertiary s tructure s tarts to develop at active s i tes of proteins where cri tica lactions and interactions wi l l take place. These active s i tes wi l l be discussed in subsequent chapters .

Figure 1-6. Examples of Tertiary (3°) Structure. Secondary s tructures , including α-hel ices , β-sheets , β-turns/bends , and loop regions combine toform tertiary s tructure domains . Severa l common forms of tertiary s tructure have been characterized and are i l lus trated in the figure.[Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Globular vs. Fibrous Proteins: Proteins are often grouped into the broad categories of “globular”—having an approximately spherica l three-dimens ional shape—or “fibrous”—being long and fa i rly s tra ight overa l l . A vast majori ty of proteins are globular, with fibrous proteins oftenplaying s tructura l or very specia l i zed functional roles . Examples of fibrous proteins include actin, col lagen, and keratin, which play

s tructura l roles in muscle, connective ti ssue, and skin/nai l s .

Quaternary (4°) Structure: Al though many proteins are made of only one peptide cha in (ca l led “monomers” or “subunits”), two or more foldedchains may combine together to make a fina l active “ol igomer” protein. The subunits in a multimeric protein may have identica l (homo-oligomer) or di fferent (hetero-oligomer) amino acid sequences and interact to optimize hydrophobic and hydrophi l i c areas and hydrogen/ ionicbonding. The combination of the multiple protein subunits makes quaternary (4°) s tructure (Figure 1-7).

Figure 1-7. Quaternary (4°) Structure. Protein monomers may join, held together by hydrogen bonding (shown) and/or hydrophobic–hydrophi l i cinteractions , to make ol igomers . Identica l protein subunit monomers may join together to make a homo-ol igomer. Nonidentica l proteinsubunit monomers may join together to make a hetero-ol igomer. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, JaypeeBrothers Medica l Publ i shers (P) Ltd., 2009.]

The process of protein folding i s not a truly l inear one; a protein never exis ts as a long amino acid s tring. Instead, protein folding i s acomplex interaction of these processes and secondary and tertiary s tructures actua l ly form somewhat in para l lel . Many more complex factorsnot discussed here a lso occur to help in determining the fina l protein conformation. The process of making and trafficking a protein wi l l beexplored more ful ly in Chapters 5 and 9.

Dystrophin and Muscular Dystrophy: Muscular dystrophies (including Duchenne and Becker muscular dystrophies ) are diseases in whichskeleta l muscle rapidly breaks down, resul ting in muscle weakness and wasting, decreased motor ski l l s , and, eventual ly, the inabi l i ty towalk (usual ly by the age of 12 years ). Of approximately, 30 di fferent types of muscular dystrophies , the Duchenne and Becker forms show Xchromosome-l inked inheri tance and, therefore, a lmost a lways affect males at a rate of approximately one in 3500 boys worldwide. Both arecaused by the absence or mutation of the protein dystrophin, which provides s trength for skeleta l muscle cel l s by anchoring the internal cel lcomponents to the extracel lular matrix.

Reproduced with permiss ion from Kandel l E, et a l .: Principles of Neuroscience, 4th edi tion, McGraw-Hi l l , 2000.

The interaction of dystrophin with severa l other proteins involved in this s tructura l support network i s a prime example of protein–protein interactions involving the forces of thei r hydrophobic and hydrophi l i c regions . More importantly, the mutations in the dystrophinprotein are bel ieved to s igni ficantly a l ter one or more of these regions , resul ting in breakdown of the anchoring complex and thus disease.

CATEGORIES OF PROTEINS

AMINO ACID AND PEPTIDE-DERIVED HORMONES AND NEUROTRANSMITTERS

Amino acids and the peptides/proteins that they form serve severa l cri ti ca l roles in human biochemistry and l i fe. The major role of aminoacids i s to provide the bui lding blocks for proteins and a vast majori ty of the body’s amino acids are involved in this function. However, aminoacids are a lso the major precursors of severa l biologica l ly important molecules , as noted in Table 1-2.

TABLE 1-2. Amino Acids—Components of Various Biologica l Molecules

Quaternary (4°) Structure: Quaternary (4°) s tructure i s exempl i fied in many other ways than those given above, where two secondary s tructura lelements are s imply in close proximity to each other. Many fibrous proteins expand on this concept, with two or more α-hel ices woundaround each other for most of thei r amino acid sequence. Examples include F-actin and the ta i l section of myos in, keratin in ha i r, andintermediate fi laments , which play an important s tructura l role ins ide a l l cel l s .

Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010

Collagen, another fibrous s tructura l protein of skin and connective ti ssue, i s composed of three intertwined hel ica l protein sequences(see the figure on right), which di ffer from the α-hel ica l s tructure. Unl ike an α-hel ix where hydrogen bonding within the same proteinsequence predominates , col lagen uti l i zes hydrogen bonding between the three hel ica l protein cha ins . Col lagen optimizes this very uniquestructure by having the smal l amino acid glycine as every thi rd amino acid to a l low the three hel ices to fi t very close together (see the figurebelow). In addition, col lagen has an abundance of prol ine res idues to promote the hel ica l protein s tructure and a specia l form of prol ine(hydroxyprol ine) with an extra hydroxyl (OH–) to form the interprotein hydrogen bonds with the glycine NH groups . Hydroxylys ine res idueshelp to s tabi l i ze the s tructure as wel l .

Adapted (left) and reproduced (right) with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd.,2009.

Osteogenesis imperfecta (OI) i s a genetic disease that affects col lagen-conta ining ti ssues such as bone, skin, joints , eyes , ears , and teethbecause of point mutations that destabi l i ze or a l ter col lagen’s important triple hel ix s tructure. Patients with OI often display frequentfractures and easy bruis ing (sometimes mistaken for chi ld abuse); weak joints ; a bluish color to the normal ly white part of thei r eyes ;hearing loss due partly to abnormal i ties of the inner ear bones ; and poorly shaped, smal l , blue-yel low teeth.

As a resul t, ei ther the excess or deficiency of amino acids can lead di rectly to disease that may resul t in centra l nervous system defects ,dietary and metabol i sm problems, l iver and kidney fa i lure, skin and eye les ions , and even death.

ENZYMES

Enzymes are specia l i zed proteins that accelerate a chemica l reaction by serving as a biologica l cata lys t. By cata lyzing these reactions ,enzymes cause them to take place one mi l l ion or more times faster than in thei r absence. Enzymes are usual ly identi fied by the ending of“ase” to thei r name (e.g., hexokinase, the fi rs t enzyme in the breakdown of glucose). There are exceptions for enzymes that were discoveredbefore this naming scheme was adopted (e.g., tryps in, peps in, and thrombin). Enzyme reactions wi l l be discussed in greater deta i l in Chapter5.

STRUCTURAL PROTEINS

Proteins a lso serve an important role as s tructura l elements of cel l s and ti ssues . The best examples of these proteins are actin and tubul in,which form actin fi laments and microtubules , respectively (Figure 1-8A-B). In skeleta l muscle, actin fi laments provide the “scaffolding” aga instwhich the motor protein myos in can generate force to produce muscle contraction. In smooth muscle and non-muscle (e.g., skin and immunesystem), actin fi laments create the mechanica l s tructure of the cel l and are di rectly associated with l inkages to surrounding cel l s a l lowingintercel lular s igna l ing. The actin fi laments a lso provide tracks on which specia l i zed myos in molecules move ves icles and organel les (seeChapter 12). Fina l ly, actin fi laments are intimately involved in cel l moti l i ty, a wide array of cel lular movements such as wound heal ing (themovement of skin cel l s into cuts ), the immune response (the process of white blood cel l s contacting and recognizing each other in the highlyselective process of immune reactions), and cytokines is (the divis ion of one cel l into two during mitos is ).

Figure 1-8. A–B. A. Actin and B. Tubulin in Monomer and Filamentous Forms. [Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review ofMedica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

The s tructura l protein tubul in creates microtubule tracks for the movement by two molecular motor proteins , dynein and kines in, ofves icles , granules , organel les , and chromosomes. Microtubules are a lso the s tructura l component in flagel la and ci l ia involved in functionssuch as sperm moti l i ty, the movement of the egg down the Fa l lopian tubes , and the expuls ion of di rt and mucus out of the lungs and trachea.Nonmoti le ci l ia are a lso important in rod cel l s in the eye and neurons involved in ol faction (smel l ). Microtubules a lso serve a mechanica l–structura l role in the cel l s imi lar to actin microfi laments and are respons ible for the movement and separation of chromosomes duringmitos is (see Chapter 12).

MOTOR PROTEINS

“Motor” proteins transport molecules ins ide a cel l , provide movement of certa in parts of individual cel l s involved in specia l i zed functions(e.g., immune responses and wound heal ing), generate larger sca le movements of fluids and semisol ids such as the ci rculation of blood andmovement of food through the digestive tract, and fina l ly provide movement of the human body through their roles in skeleta l muscles .

Myos in i s a protein with a hydrophobic ta i l ; a head group, which can attach and detach from actin fi laments ; and a “hinge” section, whichmoves the head group back and forth resul ting in movement. Two major types of myos in exis t. Myos in I i s composed of one molecule with anadditional area on i ts short ta i l that can bind to other proteins and membranes . Myos in II (severa l subtypes exis t) i s composed of a long ta i lthat binds via hydrophobic interactions to other myos in II molecules , resul ting in a compos i te molecule that can shorten skeleta l muscles byi ts interaction with actin fi laments in those muscles . Kines in and dynein are very much l ike myos in I in form and function (see Chapter 12).

Microtubules, Cilia, and Flagella—Roles in Disease Processes: Al though usual ly rare, defects in ci l ia/flagel la , known as ci l iopathies , lead tosevera l diseases/syndromes including the fol lowing:Kartagener Syndrome/Primary Ciliary Dyskinesia—defective ci l ia in the respiratory tract, Eustachian tube, and Fa l lopian tubes leading to chronic

lung infections , ear infections and hearing loss , and inferti l i ty. Poss ible association with “s i tus inversus ,” a condition in which majorinternal organs are “fl ipped” left to right.

Senior–Loken Syndrome/Nephronophthisis—eye disease and formation of cysts in the kidneys leading to renal fa i lure.Bardet–Biedl Syndrome—dysfunction of ci l ia throughout the body leading to obes i ty due to inabi l i ty to sense satiation, loss of eye

pigment/visua l loss and/or bl indness , extra digi ts and/or webbing of fingers and toes , menta l and growth retardation and behaviora l/socia l problems, smal l and/or misshaped genita l ia (male and female), enlarged and damaged heart muscle, and kidney fa i lure.

Alstrom Syndrome—chi ldhood obes i ty, breakdown of the retina leading to bl indness , hearing loss , and type 2 diabetes .Meckel–Gruber Syndrome—formation of cysts in kidneys and bra in leading to renal fa i lure and neurologica l defici ts , extra digi ts and

bowing/shortening of the l imbs .Increased Ectopic (Tubal) Pregnancies/Male Infertility—deficient ci l ia in Fa l lopian tubes or deficient flagel la/ sperm ta i l moti l i ty.Autosomal Recessive Polycystic Kidney Disease—much rarer than the autosomal dominant form, dys function of basa l bodies and ci l ia in renal

cel l s leads to a l terations of the lungs and kidneys leading to a variety of secondary medica l conditions and often death.Parkinson’s and Alzheimer’s diseases—Although work i s s ti l l ongoing, researchers now feel that some forms of Parkinson’s and Alzheimer’s

diseases may resul t, in part, from damage to microtubules and associated proteins . Treatments a imed at s tabi l i zing microtubules mayhelp many sufferers of these maladies .

TRANSPORT/CHANNEL PROTEINS

Another group of proteins fold into a tertiary or quaternary s tructure that creates channels for the movement of molecules into and out of thenucleus , various cel l organel les , and from cel l s to thei r outs ide envi ronment such as the blood s tream. The hydrophobic and hydrophi l i cnature of the amino acids that make up these channel proteins a l lows an exterior of the protein that can exis t ins ide the extremelyhydrophobic envi ronment of a membrane bi layer and a hydrophi l i c interior that can a l low charged molecules to move through the membrane(Chapter 8). Channels are essentia l for the transportation of nutrients into and out of cel l s as wel l as for nerve s ignals and the selectivefi l tration of molecules in the kidneys . These specia l i zed functions of channels wi l l be discussed in deta i l in Chapter 8 and Section II I .

REVIEW QUESTIONS1. What i s the meaning and s igni ficance of essentia l and non-essentia l amino acids?2. What i s the s igni ficance of each amino acid R-group (hydrophobic, hydrophi l i c, and charged)?3. What are the four major types of s tructura l elements of proteins and how are they defined?4. What i s an enzyme and how do the terms cata lys t and active s i te relate to enzymes?5. What i s the bas ic s tructure of amino acids?6. How do the elements peptide bond, peptides , α-hel ix, β-s trand, β-turn, ha i rpin turn, and disul fide bond relate to the s tructure of proteins?7. How does an amino acid sequence fold?8. What are the roles of R-groups and primary to quaternary s tructure in the fina l conformation of proteins?9. What are the di fferent categories of proteins and how are they defined?

CHAPTER 2CARBOHYDRATES

Bas ic Carbohydrate Structure and FunctionMonosaccharides and DisaccharidesGlycogen and StarchesGlycoproteinsGlycosaminoglycansReview Questions

OVERVIEWCarbohydrates are vastly important in human biology, including roles as a major energy source, s tructura l molecules when combined withother carbohydrates , proteins , and other molecules , and binding and s ignal ing between molecules and cel l s . As a resul t of a l l theseimportant functions , carbohydrate biochemistry i s involved in a large number of disease s tates . Al though multiple carbohydrates exis t, only afew sugar molecules and polysaccharides are important to human phys iology (e.g. only eight di fferent carbohydrates are found asconsti tuents of glycoproteins and glycol ipids ). However, a number of addi tional molecules created by l inkages of carbohydrates to proteinsplay various roles in cel l–cel l interactions and biologica l s tructures .

BASIC CARBOHYDRATE STRUCTURE AND FUNCTIONCarbohydrates, whose names end in “-ose,” have a formula of (CH2O)x where x i s a number from three to seven (giving the names of triose,tetrose, pentose, hexose, and heptose). Al l carbohydrates conta in a ketone or an a ldehyde group, as wel l as one or more hydroxyl groups (Figure2-1A–B; Appendix I I I). The oxygen atoms of the ketone and a ldehyde groups have s imi lar reactive qual i ties to that of the carboxyl ic acid groupseen in amino acids and are the s i tes of chemica l reactions within the carbohydrate molecule, as wel l as with other carbohydrate, protein, orl ipid molecules . Often, the ketone or a ldehyde reacts with a hydroxyl group from the same sugar molecule to form a carbohydrate ringstructure as shown.

Figure 2-1. A–B. Basic Carbohydrate Structures. A. The reactive ketone group of carbon 2 (green carbon group) from the hexose fructose reacts withthe hydroxyl group of carbon 5 to form a new bond and a five-s ided (pentose) ring s tructure. Al l carbon atoms are numbered for clari ty. Thisreaction i s ful ly revers ible as indicated by the bidi rectional arrows. As a resul t, the l inear and ring s tructures are constantly changing insolution. B. The reactive a ldehyde group of carbon 1 (green carbon group) from the hexose glucose reacts with the hydroxyl group of carbon 5 toform a new bond and a s ix-s ided (hexose) ring s tructure. Al l carbon atoms are numbered for clari ty. This reaction i s ful ly revers ible asindicated by the bidi rectional arrows. As a resul t, the l inear and ring s tructures are constantly changing in solution. [Adapted with permiss ionfrom Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Carbohydrates play a major role in humans as energy sources and s torage, and their role in diet and nutri tion, a l though sometimescontrovers ia l , i s a lways one of supreme importance. However, carbohydrates play other roles as noted in Table 2-1.

TABLE 2-1. Biochemica l Roles of Carbohydrates

MONOSACCHARIDES AND DISACCHARIDESAlthough there are multiple trioses , pentoses , hexoses and heptoses depending on the various arrangements of hydroxyl groups andhydrogens around the centra l carbon backbone, only a few of these s ingle res idue sugars are commonly seen in human biology. Commons ingle sugar groups , ca l led monosaccharides, include the triose glyceraldehyde; the pentose ribose; and the hexoses fructose, glucose, andgalactose (shown in Figure 2-2).

Figure 2-2. Common Monosaccharides in Human Biology. The common monosaccharide carbohydrates found in humans are shown above, includingthe three-carbon triose glycera ldehyde; the five-carbon pentose ribose; and the s ix-carbon hexoses fructose, glucose, and ga lactose. Note theonly s tructura l di fference between glucose and ga lactose i s the placement of the hydrogen atom and hydroxyl group at carbon 4. Carbon atomsare numbered for clari ty. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Hydroxyl groups (OH) are often locations of enzyme reactions , especia l ly the formation of a new bond between two carbohydrate moleculeswith the resul ting release of a water molecule. When monosaccharides form such bonds , the resul ting molecules are a disaccharide,trisaccharide, and so on. The various combinations of a l l the di fferent monosaccha-rides would produce a vast mixture of these new moleculesbut, in fact, only a few are common in humans (Figure 2-3), namely lactose (the primary sugar found in mi lk), trehalose [found in plants (e.g.,sunflower seeds), animals (e.g., shrimp), Baker’s yeast, and severa l types of mushrooms)], maltose [(found in many foods made from gra ins(e.g., barley)], and sucrose (found natura l ly in plants ; usual ly arti fi cia l ly made for human consumption as common table sugar). The ringstructure of monosaccharides a lso has reactive hydroxyl groups at each of thei r carbon atoms, especia l ly at the fi rs t and s ixth carbons , whichcan bond to other sugar molecules and amino groups and proteins (discussed below and in the fol lowing chapters ).

Figure 2-3. Common Disaccharides in Human Biology. The common disaccharide carbohydrates found in humans are shown above, includinglactose, trehalose, maltose, and sucrose. Component monosaccharides and speci fic α- and β-bond configurations are indicated immediatelyunder each disaccharide. Note that the second glucose molecule in trehalose and the fructose molecule in sucrose are fl ipped horizonta l ly inthe fina l disaccharide molecule (see carbon numbers ). [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

Carbohydrate Intolerance: The digestion of carbohydrates from food involves severa l processes relying on proteins , both enzymes andtransport/channel types . Defects in any of these proteins lead to disease s tates in which a particular carbohydrate cannot be tolerated inthe patient’s diet. One of the best known examples i s lactose intolerance, which develops in adolescence or adulthood and i s caused by theinabi l i ty to digest the sugar lactose, the primary carbohydrate in cow’s mi lk. Decreased digestion increases bacteria l fermentation of theexcess sugar molecules producing intestina l gas , bloating, nausea, and pa inful cramping. The osmotic effect of the excess monosaccharideand disaccharide molecules leads to increased water retention and absorption in the large intestine, caus ing watery diarrhea.

In fact, his torica l evidence indicates that humans only recently evolved the abi l i ty to digest lactose when cow’s mi lk became a s taple oftheir diet. In addition, some races of humans are less able to digest lactose, leading to increased incidence of lactose intolerance in thatracia l group.

Other types of carbohydrate intolerance include enzyme deficiencies in the digestion of sucrose, maltose, and trehalose—the latter seenpredominately in Inui t and Greenland populations—and the absence or decreased action of transporter/channel proteins that can causeprofound effects on digestion of glucose, fructose, and ga lactose. Some carbohydrate intolerance can lead to serious problems of fa i lure tothrive and kidney and/or l iver disease. Treatment of carbohydrate intolerances i s normal by avoidance of the offending sugar or bysupplementation of the affected enzyme.

GLYCOGEN AND STARCHESLinkages of monosaccharides and disaccharides form long carbohydrate cha ins ca l led polysaccharides . The common polysaccharides found innature are glycogen and starch. In fact, over ha l f of a l l the carbohydrates in the human diet are s tarch molecules . Al though glycogen i s a lways abranched polysaccharide molecule, s tarch can be ei ther branched (ca l led amylopectin) or unbranched (ca l led amylose). The glycogen moleculeand s tarch molecules are shown in Figure 2-4.

Figure 2-4. A–B. Glycogen and the Plant Starch Forms Amylopectin and Amylose. A. Glucose molecules , bonding both l inearly between the carbons 1and 4 (green bonds) and as branches between carbons 1 and 6 (red bond) of success ive glucose molecules , create glycogen (branchesapproximately every 10 glucose res idues) or the form of plant s tarch ca l led amylopectin (branches approximately every 30 glucose molecules ).Glycogen and s tarch are usual ly thousands of glucose molecules long and are extremely important forms of carbohydrate s torage in thehuman body and plants , respectively. B. Glucose molecules , bonding only l inearly between the carbons 1 and 4 (green bonds) of success iveglucose molecules , create the form of plant s tarch ca l led amylose, which has no branching. Amylose i s an important form of carbohydratestorage in plants . [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Glycogen Storage Diseases: Glycogen s torage diseases include 11 classes of inborn errors in the production and breakdown of these longcarbohydrate cha ins . The genetic errors , a l though rare, resul t in an inabi l i ty of the body to respond to the increased need for glucosemolecules . As a resul t, patients suffering from glycogen s torage diseases are l imited in thei r abi l i ty to exercise or develop low blood sugarafter a relatively short period of food deprivation. These glycogen s torage diseases wi l l be explored in ful ler deta i l in later chapters .

Cellulose, the another major plant polysaccharide bes ides s tarch, di ffers from amylose only in the bonds between carbons 1 and 4. Changesin the way the a ldehyde and hydroxyl groups of the two glucose molecules form the bond resul ts in ei ther an amylose α-bond [indicated by adownward bond (Figure 2-4A)] or a cel lulose β-bond [indicated by an upward bond (Figure 2-5)]. Al though the di fference may seemins igni ficant, this β-bond resul ts in a markedly di fferent overa l l s tructure of the polysaccharide. Al though α-l inkages create an overa l l hel ica ls tructure of the cha in, β-l inkages create a s tra ight cha in. The α-l inked hel ica l s tructure i s important for access ing the carbohydrate moleculesfor metabol i sm, whereas the β-l inked l inear cha in i s s tronger and, therefore, wel l sui ted for cel lulose-conta ining s tructures such as plantwal l s (e.g., wood). In addition, the β-l inkages cannot be digested by humans but can be digested by animals such as cows and termites , and i ti s the reason humans cannot l ive on grass or wood, both of which are composed of β-bonded glucose molecules .

Figure 2-5. Cellulose. Note the β-l inkages (upward-going green bond) between carbons 1 and 4 of the two glucose molecules that di ffer from theα-l inkages seen in glycogen and s tarches . Cel lulose i s composed of multiple repeats of this bas ic uni t as indicated by the subscript “n.”[Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

GLYCOPROTEINSCarbohydrates may a lso form bonds with proteins via any of the carbohydrate hydroxyl groups combining with the amino acid hydroxyl groupsof serine or threonine or the amine ni trogen of asparagine. The resul ting carbohydrate–protein molecules are referred to as glycoproteins, andapproximately ha l f of the proteins in the human body are estimated to be glycoproteins . The divers i ty of glycoproteins seen in nature andhumans i s immense with complex mixtures of proteins , amino acids , and sugars (monosaccharides , disaccharides , and tri saccha-rides ) l inked

together in a vast number of l inear and branched formations generica l ly ca l led oligosaccharides. These ol igosaccharides play a diverse numberof functional roles in binding, s igna l ing, and regulation that wi l l be more ful ly explored in Section II I .

GLYCOSAMINOGLYCANSEven more complex than s imple ol igosaccharides are the glycosaminoglycans (often referred to as “GAGs”), which conta in repeatingdisaccharide chains of a modi fied glucose and/or ga lactose. Prior to thei r l inkage to the GAG molecule, these carbohydrate molecules have anamine group added with an additional acetyl (e.g., NHCOCH3) or negatively charged sul fate (e.g., NHSO3

−) group, producing glucosamine and

galactosamine molecules . In addition, negatively charged sul fate (SO3−), via the reactive hydroxyl groups , and/or carboxylate (COO−) groups are

l inked to at least one of the sugars of the repeating cha in (Figure 2-6). A very wel l known GAG is heparin, a potent inhibi tor of blood clotformation, used in patients with heart attacks , s trokes , and clotting diseases . Other GAGs include chondroitin and hyaluronic acid (the longest ofthe GAGs), which bond with a centra l l inear core protein to create proteoglycans. These molecules are important as s trong s tructura l elementsin connective ti ssue and carti lage (Figure 2-6C and Table 2-2).

Figure 2-6. A–C. Examples of Glycosaminoglycans (GAGs) and of Proteoglycan Associated with Collagen. A. Heparin, a GAG and an important moleculein blood clot regulation, i s composed of two carbohydrate molecules l inked via a β-l inkage between carbons 1 and 4 and the addition of aCOO− group, as wel l as sul fate and ni trogen and sul fate groups at various other carbon atoms. Al though multiple forms of heparin exis t,depending on the particular carbon that i s modi fied, one of the most common is shown above. B. Chondroi tin, a GAG important in carti lage,tendon, and bone s tructure, i s composed of a l ternating carbohydrate molecules (glucuronic acid N-acetyl -ga lactosamine; note COO− andnitrogen groups) l inked via an α-l inkage between carbons 1 and 3. Each carbohydrate molecule may be sul fated (once or twice) or left unsul -fated. The more common sul fated forms are referred to as chondroi tin sul fate and are fel t to be mainly respons ible for the molecule’sbiologica l activi ty. Chondroi tin has recently become popular to ingest in pi l l form by some patients with knee and other joint pa in in hopes of“replacing” carti lage that has been depleted by time and wear and tear. C. Col lagen, the main protein in connective ti ssue, i s s tronglyassociated with proteoglycans composed of severa l GAGs. Common components include hya luronic acid, and long, l inear cha ins of repeti tiveunits of uronic acids (left) and chondroi tin sul fate or keratan sul fate bound together by l ink and core proteins . The resul ting protein andcarbohydrate molecules as wel l as interactions between the charged GAG groups and surrounding water molecules create an overa l l s tructure(right) that i s both s trong and rigid but a lso flexible and conformable. These qual i ties help to make col lagen the perfect substance to providestructure but a lso a l low movement in joints . [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

TABLE 2-2. Biochemica l Roles of Glycosaminoglycans

Glycosaminoglycans are respons ible for a vast number of functions in human biology, mostly involving s tructure outs ide the cel l and/orattachment of cel l s to external s tructures . An example of this s tructura l moti f i s shown in Figure 2-7.

Figure 2-7. Example of Extracellular Matrix Structure. The role of col lagen, chondroi tin, proteoglycans , and l ink proteins in the formation of theextracel lular matrix i s i l lus trated. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion,McGraw-Hi l l , 2010.]

Carbohydrates and Fertilization: The ferti l i zation of a human egg rel ies on carbohydrate binding and s ignal ing. Binding of a receptor on thesperm’s surface to a galactose molecule within an ol igosaccharide on the egg’s surface s ignals the sperm to release molecules that a l lowsperm entry into the egg, resul ting in ferti l i zation.

Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.

The l i s t below i l lus trates just a few of these molecules and their functions , which wi l l be explored further in later chapters .

REVIEW QUESTIONS1. What are triose, pentose, and hexose carbohydrates and their key features?2. What are ketone and a ldehyde groups and their key features?3. What are monosaccharides and disaccharides and their key features?4. What are the bas ic s tructures of glycera ldehyde, ribose, glucose, ga lactose, fructose, maltose, lactose, sucrose, glycogen, s tarch,

amylopectin, amylase, glycoproteins , and glycosaminoglycans?5. What are the bas ic roles and functions of carbohydrate molecules , including the various other types of molecules to which they may bond

and the resul ting s tructura l characteris tics?

CHAPTER 3LIPIDS

Bas ic Lipid FunctionsBas ic Membrane Lipid StructureComplex LipidsLipid-derived Hormones/Vitamin DReview Questions

OVERVIEWLipids are the thi rd major type of biochemica l molecule found in humans . Al though one of thei r major functions relates to the formation ofbiologica l membranes (phosphol ipids and cholesterol ), l ipid molecules are a lso essentia l for energy s torage and transport (triacylglycerols ),cel lular binding and recognition and other biologica l processes (glycol ipids ), s igna l ing (s teroid hormones), digestion (bi le sa l ts ), andmetabol i sm (fatty acids , ketone bodies , and vi tamin D). Lipid molecules are mainly hydrophobic and are, therefore, found in areas away fromwater molecules or are involved in mechanisms such as l ipoprotein complexes that a l low their movement in and through water envi ronments .The smal ler hydrophi l i c parts of l ipids are, themselves , important in formation of biologica l membranes and in the severa l speci fic functionsof l ipids and l ipid-derived molecules .

BASIC LIPID FUNCTIONSA major role of l ipid molecules i s to provide the bui lding blocks for biologica l membranes , including phospholipids, glycolipids, and cholesterol.However, other l ipids known as triacylglycerols (a l so referred to as triglycerides or fats ) function in the s torage of biologica l energy and bilesalts, derived in the l iver from cholesterol and serve in the digestion of dietary fat. Fina l ly, severa l l ipid-derived molecules serve as importanthormones and intracel lular messengers .

It i s important to note that the major part of every l ipid molecule i s hydrophobic in nature and, l ike the hydrophobic parts of proteinsdiscussed earl ier (Chapter 1), prefers to be away from and protected aga inst interaction with water molecules . This hydrophobic character i sfundamenta l in membrane formation, l ipid transport, and in many of the functions that the various types of l ipid molecules perform.

BASIC MEMBRANE LIPID STRUCTUREA membrane l ipid i s composed of three bas ic components that are as fol lows:

1. Fatty acids are composed of long chains of carbon molecules with a carboxyl ic acid (COOH) at carbon 1 and a CH3 (methyl ) group at the end ofthe chain (Figure 3-1A). The carboxyl ic acid group i s involved in bonding of the fatty acid to the other components of a l ipid molecule. Inhumans , fatty acids are usual ly 12–24 carbons long and most often the number of carbons in the fatty acid backbone i s even. Fatty acids can

conta in s ingle (C—C), double (C═C), or triple carbon–carbon bonds .

Figure 3-1. A. Common Saturated and Unsaturated Fatty Acids. Pa lmitate (16-carbon), s tearate (18-carbon), and arachidate (20-carbon), fatty acids ,shown by the carbon backbone and in “s tick diagram” form for arachidate often used for s impl ici ty. The carbon atoms of fatty acids arenumbered from the carboxyl ic acid (COOH) to the terminal methyl (CH3) group. Hydrogen atoms are not shown for clari ty. B. Fatty Acid ChainDouble Bonding. Deta i l of unsaturated fatty acid carbon chain, i l lus trating trans double bond (left: hydrogen atoms on oppos i te s ides of thebond and resul ting l inear carbon chain) and unsaturated cis double bond (right: hydrogen atoms on the same s ide of the bond and resul ting“kinked” carbon chain). C. Common Unsaturated Fatty Acids. (Top and middle) Two 18-carbon unsaturated acids showing a cis double bond (oleicacid) and a trans double bond (ela idic acid) both at carbon 9. Arrows i l lus trate di fferent conformations of fatty acid cha in that resul t from thetwo types of saturated bonds . (Bottom) An 18-carbon unsaturated fatty acids with two cis double bonds (l inoleic acid) at carbons 9 and 12 (seearrows). Note how the addition of a second cis double bond creates an even more nonl inear fatty acid cha in, which causes increased disorderof packing and, therefore, increased fluidi ty of biologica l membranes . [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus tratedBiochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

• Saturated fatty acids conta in only s ingle carbon–carbon bonds , and a l l of the carbon molecules are bonded to the maximum number ofhydrogen molecules .

• Unsaturated fatty acids have at least one double carbon–carbon bond with the potentia l for addi tional hydrogen atom bonding s ti l lexis ting for some of the carbon atoms in the backbone chain. If more than one double bond i s present, the term polyunsaturated i s used.These double bonds can exis t in ei ther a “kinked” cis double bond or a more l inear trans double bond (Figure 3-1B-C).

2. Glycerol i s a s imple three-carbon molecule with hydroxyl groups at each carbon (Figure 3-2A). These hydroxyl groups are the reactive locationwhere fatty acids and other components of a l ipid molecule bond to form diacylglycerol (Figure 3-2A) and triacylglycerol (Figure 3-2B)molecules .

Figure 3-2. A–B. A. Glycerol, Diacylglycerol, and Triacylglycerol. Glycerol i s a s imple, three-carbon chain molecule (green) with a hydroxyl group (OH)bonded to each of the carbon atoms. The hydroxyl groups at carbons 1 and 2 of glycerol bond react with the carboxyl ic acid groups (COO–) of thefatty acid cha ins , resul ting in two new bonds and two water (H2O) molecules . In genera l , unsaturated fatty acids bond to carbon 1, whereassaturated fatty acids bond to carbon 2. The resul ting molecule i s ca l led “diacylglycerol” and i s involved in important s igna l ing pathways(Chapter 8). B. Triacylglycerol i s formed when a thi rd fatty acid bonds to the thi rd glycerol hydroxyl group. This resul tant molecule i s animportant s torage form of energy (Chapter 8). [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

Double Bonds and Melting Temperatures: The number and type of double bonds in a particular l ipid molecule’s fatty acid affects how that fattyacid “packs” with other fatty acids and, therefore, the temperature at which the particular l ipid molecule melts . For example, the saturatedfatty acids l i s ted in Table 3-1 have melting points between 44°C and 77°C. The unsaturated fatty acids have far lower melting points , rangingfrom 13°C to –50°C, decreas ing as the number of double bonds (polyunsaturation) increases . “Kinked” cis double bonds make the packing offatty acids even more disorganized and lower the melting temperature even further. The effects of saturated/unsaturated/polyun-saturatedand cis/trans fatty acids are readi ly seen in the di fferent melting temperatures of butter, composed of a highly saturated l ipids , andmargarine, composed of unsaturated l ipids . The l inear nature of trans fatty acids makes them s imi lar to the s tructure of saturated fattyacids . This s tructura l feature, relative to the cis configuration, seems to make metabol i sm of trans fats di ffi cul t. Consequently, trans fatsremain longer in the ci rculation, thereby contributing to arteria l depos i tion and subsequent development of coronary heart disease. Ingenera l , unsaturated fats are heal thier for the human body and, therefore, dietary fats composed mostly of cis double bonded,polyunsaturated fats are recommended by dieticians and cl inicians to help avoid heart disease and other medica l problems.

TABLE 3-1. Common Fatty Acids Found in Humans

Essential Fatty Acids: Much l ike there are essential amino acids that the body can only get from dietary sources , certa in fatty acids are a lsodeemed essentia l . Two particular essentia l fatty acids , l inoleate and l inolenate, have double bonds at the s ixth and thi rd carbon atomscounting from the methyl end of thei r cha ins , respectively, and are needed to produce certa in 20-carbon long fatty acids conta ining doublebonds . These two fatty acids are known as omega-6 (ω-6) and omega-3 (ω-3) fatty acids . Humans do not have the abi l i ty to produce doublebonds at these locations and, therefore, must obta in these two required fatty acid bui lding blocks from vegetable oi l s . Arachidonate with a20-carbon chain and four cis double bonds i s a lso an essentia l fatty acid involved in severa l important biologica l functions . Recently, longercarbon chain ω-3 fatty acids have been proposed to decrease heart attacks and s trokes ; supplements and some food products are nowavai lable, which conta in these fatty acids . Interestingly, excess ive dietary intake of ω-6 fatty acids has been impl icated in an increased ri skof heart attacks , s trokes , some cancers , and even depress ion.

3. Head Group The fina l component of a l ipid molecule varies with each type of l ipid and, a long with the two speci fic fatty acids , defines eachparticular l ipid. This thi rd part of the l ipid molecule i s often ca l led the “head group,” aptly named i f one envis ions the end methyl group ofthe fatty acid cha ins to be the ta i l of the l ipid molecule (Figure 3-3A). Most l ipids in a biologica l membrane have a phosphate group (PO4

–3)attached to the thi rd glycerol carbon and are, therefore, ca l led phospholipids. Usual ly, an additional molecule (severa l common examplesfound in humans and the resul ting phosphol ipid molecules are shown in Figure 3-3B) i s attached to the phosphate molecule, resul ting inthe fina l head group of the l ipid molecule. This head group i s usual ly charged, creating a part of the l ipid that i s hydrophi l i c, and wants tobe near water, a qual i ty that i s essentia l for the formation of biologica l membranes (Chapter 8) and many l ipid functions .

Figure 3-3. A. Phospholipid Components and Formation. Bonding between hydroxyl (OH) of a phosphate group with the hydroxyl (OH) of glycerolcarbon 3 (green) resul ts in a phosphol ipid molecule and one water molecule. The bas ic phosphol ipid i l lus trated above i s termedphosphatidic acid and i s the bui lding block of common phosphol ipids found in the cel l membrane (see below). Fatty acid cha ins are depictedin black. B. Common Phospholipids Found in Humans. Common head groups , which form phosphol ipids (top and middle) and are shown in blue,bonded to the hydroxyl (OH) of a phosphate group (purple), which i tsel f i s bonded to the hydroxyl (OH) of glycerol carbon 3 (green). Thiss tructure resul ts in a phosphol ipid molecule that i s genera l ly found in membranes . Lipid molecules are often represented in “s tick diagram”form (bottom) with the charged phosphate group and hydrophi l i c head group shown as a ci rcle and ova l and with the fatty acid “ta i l s ,”depicted as ei ther s tra ight or jagged l ines , forming the hydrophobic region. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion,Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

The common fatty acids found in humans are l i s ted in Table 3-1.

COMPLEX LIPIDS

GLYCOLIPIDS/SPHINGOLIPIDS

Just as carbohydrate and protein molecules can bind together (discussed in Chapter 2), carbohydrates can a lso bind to l ipids to form aglycolipid. However, in a human glycol ipid, the glycerol backbone i s genera l ly replaced by a backbone of sphingosine [made from the amino acidserine and the 16-carbon fatty acid pa lmitate (Figure 3-4A)] and i s , therefore, referred to as a sphingolipid. Sphingos ine can bind two othermolecules with the remaining hydroxyl (OH) and amino (NH3) groups from the serine amino acid. In human sphingol ipids , the amino group i sa lways bound to another fatty acid to make the molecule ceramide. From ceramide, the particular molecule(s ) attached to the remaininghydroxyl group defines both the name and the characteris tics of the resul ting sphingol ipid. For example, the molecule sphingomyelin, whichcan make up to 20% of the tota l phosphol ipid in many biologica l membranes , i s made of ceramide and a phosphoryl chol ine head group(Figure 3-4B).

Figure 3-4. A-B. Ceramide and Sphingolipids. A. Ceramide i s produced from the combination of the amino acid serine and the 16-carbon, fatty acid

palmitate (green) with the subsequent addition of a second fatty acid (black) to the amino (NH3) group (referred to as N-acetylation). Othermolecules can then bind to the ceramide hydroxyl (OH) to produce a wide range of sphingol ipids important to humans , an example of which i ssphingomyel in (B) shown in the right panel and discussed in the text. C. Common Cerebrosides. Cerebros ides are composed of sphingos ine anda s ingle glucose or ga lactose molecule attached via the hydroxyl group at carbon 4. D. A Sulfatide. Sul fatides are s imply cerebros ides with theaddition of a carbohydrate-l inked sul fate molecule. Compare with ga lactocerebros ide in Figure 3-4B. E. A Globoside and GalNAc Carbohydrate.Globos ides are composed of ceramide bonded to a fatty acid via the NH group (green) and severa l carbohydrate molecules , including GalNAc(l ight blue square), bonded via the serine OH (upper panel ). The s tructure of N-acetyl -ga lactosamine or GalNAc i s shown in the lower panelwith the NH attachment highl ighted in green. F. A Ganglioside and NANA. The s tructure of N-acetylneuraminic (NANA), a type of s ia l i c acid, i sshown in the lower panel (N-acetyl group indicated in blue). [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

From the base molecule of a sphingol ipid (e.g., ceramide), carbohydrate molecules may a lso be attached to form a glycosphingolipid. Ingenera l , human glycosphingol ipids are grouped into four categories .

1. Cerebrosides—Ceramide (see above) attached to only a single glucose or ga lactose res idue, producing glucosylceramide orga lactosylceramide, respectively (Figure 3-4C). Cerebros ides are important in the membranes of muscle and nerve cel l s and are located inmyel in, which covers nerve axons and enables fast and efficient conduction of nerve impulses . Cerebros ides may a lso be involved in thebinding of morphine and other opiates .

2. Sulfatides—Galactose-based glycosphingol ipid molecules , which conta in a sul fur atom-conta ining sul fate group in place of thephosphorous atom (Figure 3-4D). Sul fatides are found mainly in the bra in and centra l and periphera l nervous systems but are seen in traceamounts in other ti s sues . Sul fatides are bel ieved to be involved in the regulation of cel l growth and s ignal ing and may serve to both helpform and, a l ternatively, break down blood clots poss ibly by affecting the transportation of sodium and potass ium in and out of cel l s such asplatelets . Sul fatides a lso may play roles as an adhes ion molecule, including the recrui tment of immune cel l s to inflamed ti ssue and thebinding and repl ication of influenza vi ruses . Changes in the production of sul fatides have been noted as one of the earl ies t indicators ofAlzheimer’s disease.

3. Globosides—Glycosphingol ipid molecules with the carbohydrate molecule N-acetyl-galactosamine (a .k.a . Ga lNAc) a long with two or more othercarbohydrate molecules (Figure 3-4E). Globos ides are found in severa l organs including red blood cel l s , serum, l iver, and spleen. Al thoughthe functions of globos ides are not wel l understood, they are bel ieved to play an important role in cel l receptors . Interestingly, the bindingof Escherichia coli, a bacterium common in urinary tract infections , to cel l s in the urinary tract i s bel ieved to occur through globos ides .

4. Gangliosides—Glycosphingol ipid molecules with one or more attached sialic acid molecules , most often N-acetylneuraminic acid (a.k.a. “NANA”),a complex, nine-carbon carbohydrate molecule (Figure 3-4F). There are a large number of di fferent gangl ios ides , which di ffer in thei rs tructure depending on the number and location of the carbohydrate and NANA molecules . Gangl ios ides are involved in binding,recognition, and s ignal ing between cel l s . Al though gangl ios ides are abundant in the nervous system, they are a lso bel ieved to play animportant role in binding immune cel l s . Gangl ios ides are a lso found in less abundance in other cel l types . Gangl ios ides are a lso bel ievedto play a role in the binding and entrance into cel l s of the influenza vi rus and the toxin that causes cholera .

EICOSANOIDS

Eicosanoid i s the genera l term for molecules that are a l l composed of 20-carbon fatty acids and involved in s igna l ing. The four major groups ofeicosanoids include the prostaglandins (PGs), the prostacyclins (PGIs), the thromboxanes (TXs), and the leukotrienes (LTs). Functions of eicosanoidsinclude promotion of inflammation (usual ly ω-6 derived), immune response, neurologica l s igna l ing, regulation of blood pressure, control ofplatelet aggregation/ disaggregation, and modulation of levels of triacylglycerols . They a lso have di rect/indirect effects on a variety ofdiseases , including cardiovascular and rheumatoid pathologies , among other roles . The s ignal ing actions of eicosanoids are mainlytransmitted via G-protein receptors (Chapter 8).

Al l eicosanoids are produced from ei ther ω-6 acids (dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), or an ω-3 acid [eicosapentaenoicacid (EPA)]. The predominant synthetic pathway for eicosanoids i s via the ω-6, AA pathway whose products are denoted by the subscript “2”(Figure 3-5). LTs are produced from AA via a di fferent pathway i l lus trated in the figure. Importantly, eicosanoids derived from ω-6/DGLA and theω-3/EPA precursors are much less inflammatory/anti -inflammatory in nature. Increased intake of DGLA (dietary supplements ), especia l ly EPA(“ω-3” fi sh oi l s ), resul ts in lowered associated diseases via di rect competi tion with the AA pathway. Medications such as aspi rin,nonsteroida l anti -inflammatory drugs , and cyclooxygenase (COX)-2 inhibi tors a lso decrease the inflammatory effects of PG, PGI, and TX(col lectively known as prostanoids) by di rect inhibi tion of COX-1 or -2, which are key enzymes in prostanoid synthes is (Figure 3-5).Corticosteroids (e.g., prednisolone) inhibi t phosphol ipase A2 (Figure 3-5).

Figure 3-5. Overview of Eicosanoid Synthetic Pathway. Phosphol ipids generated as noted above (see a lso Figure 3-3A) provide the bas ic bui ldingblocks for the formation of arachidonic acid (AA), the source of a l l eicosanoids . The varying synthetic pathways coming from AA are shown. Seealso Table 3-2 for a review of the major known eicosanoids and their functions . [Adapted with permiss ion from Naik P: Biochemistry, 3rdedition, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

TABLE 3-2. Overview of Eicosanoids

More speci fica l ly, PGs are eicosanoids whose major functions include regulation of inflammation, smooth muscle contraction (bloodvessels , gastrointestina l , bronchia l , and uterus), neurologica l pa in, platelet function, hormone activi ty, and cel l growth. PGIs are deriveddirectly from a prostaglandin precursor (PGH2) and are important in decreas ing platelet function and, therefore, blood clot formation as wel las di lation of blood vessels . TXs are a lso derived from PGH2 and, oppos i te of PGIs , act as vasoconstrictors to promote platelet aggregation andblood clot formation. LTs , the fourth class of eicosanoids , are produced via a pathway that deviates from the other eicosanoids ’ production atAA (Figure 3-5). LTs are mainly seen in inflammatory processes of the lung, including asthmatic reactions and the inflammation associatedwith bronchi ti s . Speci fic functions of the major eicosanoids and their role in disease and treatments are reviewed in Table 3-2 and wi l l becovered further in Section II I .

ABO Blood Groups: The human blood groups O, A, B, and AB are determined by speci fic glycosphingol ipids found in the red cel l membrane.The bas ic glycosphingol ipid, ca l led “H-substance,” i s composed of a sphingol ipid connected to three carbohydrate molecules and amembrane-bound protein. The addition of speci fic, extra carbohydrate molecule to a ga lactose res idue on H-substance produces the fourcommon blood types as fol lows:

Eicosanoids and Inflammation: Inflammation i s often characterized by the four s igns of ca lor, dolor, tumor, and rubor. Each of these features

resul ts , at least in part, from the action of one or more eicosanoids . Calor (warmth) i s caused by the prostaglandin PGE2. Dolor (pa in) i sheightened via the action of PGE2, which a lso increases the sens i tivi ty of neurons . Tumor (swel l ing) resul ts from the leakage of plasma fromblood vessels whose permeabi l i ty i s increased by the leukotriene LTB4. Inflammatory rubor (redness ) resul ts from the action ofthromboxane TXA2, ini tia l ly released after injury, which subsequently increases the concentration and, therefore, the blood vessel di lationactivi ty of PGE2 and LTB4, resul ting in engorged vessels and reddening.

CHOLESTEROL

Cholesterol i s an extremely important molecule found only in eukaryotic organisms with a variety of functions in the human body. Al thoughhumans can make cholesterol , they have to rely on a complex mechanism of binding and modi fications by other molecules so that cholesterolmay be el iminated from the body (see below). As a resul t, properly control l ing the day-today amount of cholesterol in the body—a balancebetween production, el imination, and the external influence of dietary intake—can play a key role in heal th and i l lness . The functions ofcholesterol are l i s ted below.

1. Cholesterol i s one of the essentia l l ipid components of biologica l membranes where i t i s a modulator of the fluidi ty of membranes . Theabi l i ty of membranes to modi fy thei r s tructure and/or the abi l i ty for other molecules to be able to move within the membrane i s cri ti ca l forcel l s igna l ing, binding, wound heal ing, immune response, and so on.

2. Cholesterol serves as the primary source for the production of s teroid hormones (see next section below), bi le sa l ts , and even vi tamin D.

3. Cholesterol metabol i sm is a lso important in the regulation of the transportation of l ipids throughout the body, and dis turbance of thissystem leads to the depos i tion of cholesterol in the artery wal l caus ing atheroscleros is , which ul timately leads to heart attacks and s trokes .An understanding of this regulation has led to important treatments for decreas ing the ri sk of these diseases .

The cholesterol molecule has four rings made of carbon atoms—three rings have s ix s ides and one has five s ides—with a s ix-carbon ringta i l (Figure 3-6A). Most of the carbons are s ingle bonded and, therefore, have their ful l complement of hydrogen atoms. The three-dimens ionals tructure of cholesterol i s approximately a flat plane, expos ing a l l of the hydrophobic parts of the cholesterol molecule to the envi ronment.Only one hydroxyl group with i ts hydrophi l i c nature creates any charged qual i ty to the cholesterol molecule. As a resul t, cholesterol does notl ike to be exposed to water envi ronments , preferring to be shielded by other hydrophobic molecules such as l ipids or hydrophobic parts ofproteins . The impact of cholesterol ’s unique s tructure and i ts role in plasma membrane s tructure and fluidi ty wi l l be examined further inChapter 8.

Figure 3-6. Cholesterol Molecule (A) Unesterified and (B) Esterified. A. Unesteri fied cholesterol molecule with free end hydroxyl group creating ahydrophi l i c charge important in biologica l membrane packing. B. Es teri fied cholesterol molecule with R group bonded by ester bond to oxygenfrom end hydroxyl group. The R group i s usual ly a fatty acid bonded to cholesterol by the enzyme leci thin cholesterol acyl transferase (LCAT).[Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

However, some cholesterol i s a l so found outs ide of biologica l membranes in adrenal glands , blood, and other ti s sues where i t i s oftenbonded via the hydroxyl group to a long fatty acid as a cholesterol es ter (Figure 3-6B). These cholesterol es ters are highly hydrophobic andinsoluble and can form fatty les ions or “plaques” in the artery wal l that can lead to heart attacks or s trokes . Fortunately, the human body hasdeveloped a unique way of address ing this problem, namely by forming l ipoproteins (see below and Figure 3-7).

Figure 3-7. Basic Lipoprotein Structure. Lipoprotein complexes are composed of a centra l l ipid core (cholesterol es ter and triacylglycerolmolecules ), a charged l ipid outer shel l (phosphol ipid and cholesterol molecules ), and surrounding apoproteins that help to transporthydrophobic l ipid molecules throughout the body. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28thedition, McGraw-Hi l l , 2009.]

Cholesterol, Phospholipids, Gangliosides, and Cancer: Emerging research has shown that some cancer cel l s (e.g., meningosarcoma and certa inleukemias) have s igni ficant changes in thei r cel l membrane cholesterol , phosphol ipid, and gangl ios ide content that a l ters the membranefluidi ty as wel l as l ipid s igna l ing that produces unregulated growth or carcinogenes is . These changes can a lso cause res is tance to agentsused to treat cancer. Understanding how changing membrane compos i tion leads to these findings may help to develop new and di fferentcancer-fighting agents with a unique mechanism of l ipid attack.

LIPOPROTEINS

Lipoproteins, as the name impl ies , are formed from the complexes of l ipid and protein molecules . Unl ike glycol ipids , in which s imple bondsconnect the principa l component molecules , l ipoproteins are far more complex and form large particles conta ining severa l l ipid classes andprotein. Their complexi ty emerges from the primary function of l ipoproteins , namely the transportation and del ivery of fatty acids ,triacylglycerol , and cholesterol to and from target cel l s in a variety of organs (see a lso Chapter 7 for further discuss ion). Thus , a l thoughglycol ipids once produced and in thei r fina l location remain there for relatively long periods of time, l ipo-proteins are more trans ient. Thebonding and s tructure of l ipoproteins reflect this characteris tic.

A s impl i fied model of a l ipoprotein includes a center core composed of cholesterol es ter and triacylglycerol molecules surrounded by anouter shel l of phosphol ipids and cholesterol molecules with thei r hydrophobic areas inward toward the l ipid core and their charged,hydrophi l i c areas facing outward toward the aqueous envi ronment (Figure 3-7). Specia l i zed proteins , known as apoproteins, wrap around theouter shel l of the l ipo-protein particle a lso involved in interactions with external water. The compos i tion of l ipoprotein particles varies butthey can be broadly categorized depending on their dens i ty as shown in Table 3-3. Lipoproteins , thei r components , function, metabol i sm, andmedica l impl ications wi l l be discussed in much more deta i l in Chapters 11 and 16.

TABLE 3-3. Bas ic Lipoprotein Characteris tics

BILE SALTS

Bile salts, composed of bi le acids conjugated with glycine or taurine (Figure 3-8A), are produced in the l iver di rectly from cholesterol and areimportant in solubi l i zing dietary fats in the mainly watery envi ronment of the smal l intestine. After production in the l iver and prior tosecretion into the ga l l bladder and/or digestive system, they are often bonded to the amino acid glycine (Chapter 1) or taurine, a derivative ofthe common amino acid cystine (Chapter 1), to increase their water solubi l i ty (Figure 3-8B). Glyco- and tauro-bi le acids are a lso ca l led asconjugated bi le acids .

Figure 3-8. A-B. Major Bile Acids and Bile Salts Found in Humans. A. Bi le sa l ts , including the primary forms chol ic acid and chenodeoxychol ic acid,are produced by the l iver and commonly found in humans . Removal of hydroxyl groups (OH) by intestina l bacteria produces the secondary bi leacids deoxychol ic acid and l i thochol ic acid. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1stedi tion, McGraw-Hi l l , 2009.] B. The addition of the amino acid glycine (left) or the cystine-related taurine (bottom right) increases thesolubi l i ty of the bi le sa l ts . [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Bile Salt Sequestrants: Medicines that bind with or “sequester” and help excrete bi le sa l ts are sometimes used in patients with highcholesterol levels in thei r blood. The removal of these bi le sa l ts causes further production of replacement bi le acids from the body’scholesterol s tore to a l low fat digestion, thereby lowering the tota l cholesterol in these patients . However, such treatments can interferewith normal l ipid absorption affecting dietary requirements as wel l as resul ting in the unpleasant s ide effect of fatty, foul -smel l ing bowelmovements .

LIPID-DERIVED HORMONES/VITAMIN DSteroid hormones , which are a l l produced from cholesterol (Figure 3-9), perform a variety of di fferent functions in the human body as l i s tedbelow.

Figure 3-9. Major Steroid Hormones Found in Humans. Steroid hormones , derived from cholesterol (upper left corner), control severa l importantbodi ly functions essentia l to l i fe. Their synthes is involves multiple, interrelated s teps , which occur in the adrenal cortex (minera locorticoidsand glucocorticoids ), testes (androgens), and ovaries (estrogens). The synthes is of the various s teroid-derived hormones can a lso becategorized by the speci fic enzyme (yel low boxes) involved and/or the number of carbon molecules in the substrate or product. Blue highl ightindicates pos i tion of chemica l change. Severa l diseases related to s teroid production involve these enzymes. [Adapted with permiss ion fromBarrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

CORTICOSTEROIDS (ADRENAL GLAND)

Mineralocorticoids. The primary minera locorticoid in humans i s aldosterone, which i s produced in the outer layer of cel l s of the adrenal cortex.Synthes is and secretion of a ldosterone i s control led by the renin–angiotens in system (see Chapter 18). In certa in disease s tates , a minorminera locorticoid, 11-deoxycorticosterone, may instead be produced in excess in the adrenal cortex and substi tute for a ldosterone. The role ofthe minera locorticoids i s to mainta in blood volume and blood pressure by increas ing the reabsorption of Na + from the urine to the blood. Theincrease of Na + reabsorption i s accompanied by the excretion of K+ and protons (H+) and leads to reabsorption of water by the action ofantidiuretic hormone (see Chapter 18).

Glucocorticoids. The primary glucocorticoid in humans i s cortisol, which i s produced in the middle and innermost layer of cel l s of the adrenalcortex. Glucocorticoids have a variety of unique functions . They play an important role in fuel homeostas is during s tarvation and other s tress -related conditions by promoting the mobi l i zation of fatty acids from triacylglycerols (Figure 3-2B) s tored in fat cel l s , promote the breakdown ofmuscle protein, and increase l iver glucose synthes is (gluconeogenes is ) us ing the amino acids derived from muscle and lactic acid (seeChapter 10). Additional ly, they diminish inflammatory responses by decreas ing production of PGs (see Figure 3-5).

Androgens. The cel l s in the innermost layer of the adrenal cortex produce androgens , primari ly androstenedione, as a major product. The adrenalcortex i s the sole source of androgens in females but a minor source in males who produce large amounts of androgens in the testes (seeChapter 20). Adrenal androgens , particularly in females , contribute to certa in secondary sexual characteris tics such as the growth of axi l laryand pubic ha i r, as wel l as contribute to l ibido.

Progestogens. The primary progestogen in humans i s progesterone. Progesterone produced in the adrenal cortex i s primari ly an intermediate inthe production of the other adrenal s teroid hormones . Its phys iologica l role i s implantation of a ferti l i zed egg in the uterine l ining andmaintenance of pregnancy. Its primary production occurs in the corpus luteum of the ovaries and the placenta fol lowing ferti l i zation.

ANDROGENS (TESTES) AND ESTROGENS (OVARIES)

Respons ible for sexual functions such as puberty (onset, development, and changes in secondary sexual characteris tics ), menopause,ovulation, and sperm formation.

VITAMIN D

Vitamin D, one of the fat-soluble vi tamins , i s required for ca lcium metabol i sm. A smal l amount of vi tamin D i s obta ined from dietary sourcesbut a majori ty i s produced by the convers ion of cholesterol through a unique process involving sunl ight and three separate organs (Figure 3-10). Cholesterol , now dehydrocholesterol , having had a second carbon–carbon double bond formed in the l iver, i s transported to the skinwhere ul traviolet rays break open the second and thi rd rings . Travel ing back to the l iver, a second hydroxyl group i s added to the formerlyhydrophobic “ta i l .” Fina l ly, the molecule travels to the kidney where another hydroxyl group i s added near the s i te of the lone cholesterolhydroxyl group. The resul ting molecule, “ca lci trol ,” i s the active form of vi tamin D whose functions wi l l be discussed more ful ly in Chapters 10and 13.

Figure 3-10. Production of Vitamin D from Cholesterol. Active vi tamin D (ca lci trol ) i s produced from cholesterol by means of four s teps taking placein the l iver, skin, l i ver, and kidney, respectively. Dietary sources can a lso provide choleca lci ferol di rectly. Smal l , dark arrows indicate thelocation where molecular change takes place in each s tep. Added hydroxyl groups (OH) are indicated in green. [Adapted with permiss ion fromKibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

REVIEW QUESTIONS1. What are fatty acids , triacylglycerols , and phosphol ipids?2. What are l ipid head groups and the key characteris tics of common ones found in man?3. What are cerebros ides , sul fatides , globos ide, and gangl io-s ides and their key features?4. What are l ipoproteins , cholesterol , bi le sa l t (acid), s teroid hormones , and ca lci trol and their key features?5. What are the bas ic s tructures and characteris tics of saturated, unsaturated, ci s , trans fatty acids , glycerol , and phosphol ipid?6. What are the bas ic s tructures and characteris tics of the various glycol ipids (i .e., cerebros ides , sul fatides , globo-s ides , and gangl ios ides)?7. What are the bas ic s tructures and characteris tics of the l ipoproteins (high-dens i ty l ipoproteins , low-dens i ty l ipoprotein, intermediate-

dens i ty l ipoproteins , and very-low-dens i ty l ipoprotein)?8. What are the bas ic s tructures and characteris tics of bi le sa l ts , s teroid hormones , and vi tamin D?9. What are the bas ic functions of the four types of glycol ipids?

10. How do l ipoproteins transport cholesterol and l ipid molecules throughout the body?11. What are the bas ic functions of bi le sa l ts , s teroid hormones , and vi tamin D that are produced from cholesterol?

CHAPTER 4NUCLEOSIDES, NUCLEOTIDES, DNA, AND RNA

Nucleos ides and NucleotidesRNA and DNA—Bas ic Structure and FunctionReview Questions

OVERVIEWNucleos ides and nucleotides are the fourth and fina l major group of biochemica l molecules and are essentia l for numerous biologica lfunctions in humans , including mainta ining and transferring genetic information, playing a major role in energy s torage, and acting ass ignal ing molecules . These molecules can be divided into two major fami l ies—purines , which include adenos ine and guanine, andpyrimidines , which include cyto-s ine, thymidine, and uraci l . The unique s tructures and interactions of these molecules serve as thefundamenta l bui lding block of RNA and DNA molecules and a l low fundamenta l processes of gene repl ication and protein synthes is to occur.Many other functions of the various nucleos ides and nucleotides wi l l be explored in later chapters .

NUCLEOSIDES AND NUCLEOTIDESNucleosides and nucleotides are closely involved in the preservation and transmiss ion of the genetic information of a l l l i ving creatures . Inaddition, they play roles in biologica l energy s torage and transmiss ion, s igna l ing, regulation of various aspects of metabol i sm, and even animportant role as an antioxidant. Mistakes or deficiencies in thei r synthes is usual ly lead to death. Overproduction or decreased el iminationof nucleic acid derivates a lso lead di rectly to medica l conditions .

Nucleos ides have a ni trogenous base and a five-carbon carbohydrate group, usual ly a ribose molecule (see Chapter 2). Nucleotides ares imply a nucleos ide with one or more phosphate groups attached (Figure 4-1). The resul ting molecule i s found in ribonucleic acid or RNA. If onehydroxyl (OH) group has been removed from the ribose, the deoxy vers ions of the nucleos ide and nucleotide form the bui lding blocks ofdeoxyribonucleic acid or DNA (Figure 4-1). Each component of nucleos ides and nucleotides i s discussed below.

Figure 4-1. Basic Structure of Nucleosides and Nucleotides. Five major nucleos ide bases are common in human biology, including the purines (two-ring s tructure) adenine and guanine (top) and the pyrimidines (one-ring s tructure) cytos ine, uraci l , and thymine (middle). Nucleos ides(bottom) are made of a ni trogenous base, usual ly ei ther a purine or pyrimidine, and a five-carbon carbohydrate, usual ly ribose. A nucleotideis s imply a nucleos ide with an additional phosphate group or groups (blue); polynucleotides conta ining the carbohydrate ribose are known asribonucleotide or RNA. If 2′ hydroxyl group (OH) i s removed, the polynucleotide deoxy ribonucleic acid (DNA) resul ts . [Adapted with permiss ionfrom Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

COMPONENTS OF NUCLEOSIDES AND NUCLEOTIDES

1. Ni trogenous base—The ni trogenous base of a nucleos ide or nucleotide (named because of the ni trogen atoms found in i ts s tructure) maybe ei ther a purine or a pyrimidine. Purines, including inosine (I), adenine (A), and guanine (G), are two-ring s tructures and pyrimidines, includinguracil (U), cytosine (C), and thymine (T), have only one ring (Figure 4-1). Both purine and pyrimidine ni trogenous bases are made, in part, fromamino acids as shown in Figures 4-2 and 4-3, respectively.

2. Carbohydrate—The carbohydrate component of nucleos ides and nucleotides i s usual ly the sugar ribose. When the hydroxyl group i s removedfrom carbon 2 (normal ly removed after the addition of the ni trogenous base to the carbohydrate), deoxyribose, the sugar molecule found inDNA, resul ts .

3. Phosphate Group—One or more phosphate groups (PO4–3) may be attached to the carbon 5 of the carbohydrate molecule. The phosphate

molecules are important in energy s torage and s ignal ing functions of nucleos ides and nucleotides .

Figure 4-2. A. Constituents of the Purine Ring. The sources of carbon, ni trogen, and oxygen molecules that form the ni trogenous base of inos ine areindicated. Atoms from glycine are indicated in blue shade. B. Synthesis of Adenosine and Guanosine. The nucleos ides and nucleotides adenos ineand guanos ine are produced from the molecule IMP. Synthes is of adenos ine requires one additional ni trogen molecule from the amino acidaspartate (blue shade). Guanos ine synthes is uti l i zes one additional ni trogen molecule from the amino acid glutamine (blue shade).[Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

SYNTHESIS OF PURINE NUCLEOSIDES AND NUCLEOTIDES

The adenos ine- and guanine-based nucleos ides and nucleotides are formed by fi rs t producing another nucleotide ca l led inosinemonophosphate (IMP). IMP i s produced from a ribose, parts of one glycine, one aspartate, two glutamine amino acids , two tetrahydrofolatemolecules (a modi fied form of the vi tamin folate), and one carbon dioxide (CO2) molecule (Figure 4-2A). After IMP is formed, adenosinemonophosphate or guanosine monophosphate may be produced depending on the body’s needs (Figure 4-2B).

The synthes is of purines s tarts by transference of a phosphate group from an exis ting adenos ine triphosphate (ATP) molecule to a newribose molecule. As a resul t of this requirement, the concentrations of ATP must be high for purine synthes is to proceed—a method ofregulating when to produce or not produce more of these molecules . Thus , the body only produces more nucleos ides and nucleotides whenenergy levels (from food) are high. Purines can be produced as above, or can be obta ined from the diet or by breaking down and recycl ingexis ting nucleos ides and nucleotides . Mammals , including humans , preferentia l ly use the recycl ing pathway when appropriate s tartingmateria ls are ava i lable.

SYNTHESIS OF PYRIMIDINE NUCLEOSIDES AND NUCLEOTIDES

Unl ike purines , the pyrimidine ni trogenous bases , uraci l and cytos ine, are formed before bonding to the carbohydrate portion of thenucleotide. The process s tarts by producing the uraci l ring (Figure 4-3A) in a three-s tep process uti l i zing one each of the amino acidsglutamine and aspartate and one bicarbonate molecule (HCO3

–). Next, cytidine triphosphate (CTP) i s derived from the nucleotide uridinemonophosphate (UMP) after the convers ion of UMP to the triphosphate form (uridine triphosphate); an extra ni trogen i s ga ined from oneadditional glutamine amino acid (Figure 4-3B, far right). Cytidine monophosphate (CMP) and diphosphate (CDP) can be produced from (CTP) bythe subsequent loss of two or one phosphate groups , respectively. The fina l pyrimidine-derived nucleotide, thymidine, found only in DNA, i sproduced by a separate process involving the deoxy form of UMP (dUMP). This process wi l l be discussed below.

Figure 4-3. Synthesis of Pyrimidine-Derived Nucleosides Uridine and Cytidine. A. Constituents of the Pyrimidine Uracil Ring. The nucleos ide uridine i sproduced from one aspartate amino acid, one glutamine amino acid, and a bicarbonate molecule from CO2 respi ration. Atoms from aspartateare indicated in blue shade. B. Uridine and Cytidine Nucleoside Synthesis. Cytidine synthes is uti l i zes one additional ni trogen molecule from theamino acid glutamine (indicated by arrow) after the addition of two additional phosphate groups to the UMP molecule. [Adapted withpermiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Figure 4-4. Synthesis of Thymidine Deoxyribonucleotide. UMP is deoxygenated at the 2′ hydroxyl of the ribose ring (see Figure 4-1 above) andsubsequently converted to dTMP (TMP) by the addition of a methyl group (indicated by arrow) from methylene tetrahydrofolate. [Adapted withpermiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

FORMATION OF DEOXY NUCLEOSIDES AND NUCLEOTIDES

Adenos ine, guanos ine, and cytidine deoxyribonucleotides found in DNA are formed di rectly from their corresponding, double-phosphorylated(nucleos ide diphosphates ) ribonucleotides by removal of the hydroxyl (OH–) group from the carbon 2 (e.g., ADP → dADP, GDP → dGDP, CDP →dCDP). Thymidine deoxyriboncleotide (dTMP), found only in DNA, i s formed from dUMP (UMP → dUMP → dTMP) by the addition of a methyl (CH3)group (Figure 4-4). Because there i s no oxyribo-nucleotide form of dTMP, the “d” des ignation i s often not used (TMP). See summary in Table 4-1.

TABLE 4-1. Summary of Ni trogenous Bases , Nucleos ides , and Nucleotides

Nucleoside and Nucleotide Analogues as Chemotherapy Agents and Antibiotics: Nucleos ides and/or nucleotides are essentia l components ofgenetic materia l and are, therefore, intimately involved in the prol i feration of cel l s . One method of cancer treatment i s to s top thereproduction of cancerous cel l s by inhibi ting the production of nucleos ides and/or nucleotides . Examples include the pyrimidine analogueof dUMP, fluorodeoxyuridine (FdUMP), which blocks the production of dTDP. Because dTDP has i ts own specia l synthetic process , no othernucleotides and nucleos ides are affected by FdUMP. Another analogue, fluorocytosine, can be used as an antibiotic because i t i s convertedinto an active “nucleos ide or nucleotide” only in bacteria—human cel l s are not affected by i ts presence. Aminopterin and methotrexate area lso chemotherapy agents , which act by blocking folate addition to purine rings . Unl ike fluorocytos ine, though, these agents a lso affectrapidly dividing human cel l s such as ha i r and intestines , resul ting in the common chemotherapy s ide effects of ha i r loss and nausea/vomiting.

BREAKDOWN OF PURINES AND PYRIMIDINES

Nucleotides can be broken down into their purine or pyrimi-dine ni trogenous bases by removal of the phosphate groups to form the respectivenucleos ides and removal and transfer of the ribose carbohydrate back into carbohydrate metabol i sm. Once the free purine bases remain, theyare changed into a s l ightly di fferent purine ca l led xanthine, which i s subsequently converted into uric acid and excreted mainly in urine by thekidneys . Breakdown of pyrimidine ni trogenous bases i s s imply a reverse of the s teps of synthes is with the resul ting molecules being di rectedinto regular metabol i sm.

Gout and Lesch–Nyhan Syndrome: Gout i s a medica l condition typi fied by excess ive amounts of uric acid in the body. These high levels resul t

in the formation of needle-shaped uric acid crysta ls , which then become lodged in soft ti s sues , especia l ly joints such as those in the fi rs ttoe, resul ting in severe “gouty arthri ti s” pa in. Gout i s treated by severa l di fferent types of medications , including a l lopurinol , which di rectlydecreases the convers ion of purine ni trogenous bases to xanthine and uric acid. Uloric (febuxostat) has been approved for use in thetreatment of chronic hyperuricemia. This drug appears to be more effective than a l lopurinol in preventing acute attacks and reducing thes ize of the crysta l depos i ts . Lesch–Nyhan syndrome i s a genetic disease, a ffecting a lmost solely males , of excess ive synthes is of purinesbecause of defective recycl ing and, therefore, uric acid production from their breakdown. Lesch–Nyhan syndrome is characterized by goutyarthri ti s but, in addition, a ffects the bra in, resul ting in menta l retardation, loss of control of arm/leg/face movements , aggress ive behavior,and sel f-muti lation by bi ting and scratching. Success ful treatment of this disorder i s s ti l l being sought.

Nucleotide molecules are able to form RNA and DNA strands by the bonding of the phosphate group of one nucleotide and the ribose sugarmolecule of the next nucleotide (Figure 4-5). This l inkage i s ca l led a “phosphodiester bond” and helps to form the fundamenta l s tructureessentia l for the s torage, maintenance, and transmiss ion of the genetic code of l iving creatures . For purposes of nomenclature, RNA and DNAmolecules have a 5′ and 3′ end, depending on the number of the carbon bonded to the remaining phosphate group (Figure 4-5).

Figure 4-5. Structure of Phosphodiester Bond Found in RNA and DNA. Phosphodiester bond (bold arrows) formed in RNA and DNA between thehydroxyl on the thi rd carbon of ribose and the hydroxyl from the fi fth carbon. Success ive bonds form the backbone of these molecules and l inksuccess ive nucleotides to form the organism’s genetic code. Formation of the bond resul ts in the production of one molecule of water (notshown). Both RNA and DNA are wri tten by convention 5′ and 3′ ends (indicated). [Reproduced with permiss ion from Naik P: Biochemistry, 3rdedition, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

RNA AND DNA—BASIC STRUCTURE AND FUNCTION

RNA

RNA molecules are s ingle s trands containing the nucleotides : adenine (A), guanine (G), cytos ine (C), and uraci l (U). RNA molecules often formsecondary (2°) s tructures much l ike proteins and may interact with DNA, other RNA molecules , and proteins . These interactions help to definethe particular function of each type of RNA.

In genera l , there are four dis tinct types of RNA molecules , each with a particular function (Table 4-2). Messenger RNA (mRNA) molecules ,

which can be 100s–1000s of nucleotides long, function as the transmitter of genetic information from the DNA genetic code to the resul tingprotein. In this task, mRNA is often bound by proteins that help to protect and regulate these important genetic messages . Transfer RNA (tRNA)molecules , normal ly 65–110 nucleotides long, carry individual amino acids and match them with a speci fic mRNA sequence during proteinsynthes is . tRNA molecules have a very characteris tic “T” shape that i s optimized for this function. Ribosomal RNA (rRNA) i s associated withproteins and makes up the actua l working “machinery” respons ible for the synthes is of protein molecules . The exact mechanism of proteinsynthes is and the roles of each individual type of RNA wi l l be discussed in more deta i l in Section II . A fourth type of RNA, often referred to asregulatory RNA, i s involved in regulation of DNA express ion, posttranscriptional mRNA process ing, and the activi ty of the transcribed mRNAmessage (see Chapter 9).

TABLE 4-2. Characteris tics of mRNA, tRNA, and rRNA

DNA

DNA molecules are composed of two s ingle s trands of deoxynucleotides , adenine (A), guanine (G), thymidine (T), and cytosine (C), in which the twostrands are pa i red to form a ladder-type molecule referred to as a double helix (Figure 4-6A). This double hel ix forms when atoms in thenitrogenous bases of the nucleotides form hydrogen bonds (G bonding with C and A bonding with T) (Figure 4-6B) whi le the hydrophi l i cphosphate and hydroxyl groups of the sugar “backbone” are exposed to the water envi ronment.

Figure 4-6. Basic Structure of DNA. A. Double hel ix model (left figure), i l lus trating the winding “ladder” s tructure with the ins ide “rungs” createdby nucleotide pa i ring and the outs ide “runners” created by charged phosphate groups . B. Deta i l (top) of how the double hel ix s tructureprotects the inner hydrophobic area and a l lows hydrogen bonding between pa ired G–C and A–T nucleotides whi le expos ing the outerhydrophi l i c deoxyribose and phosphate groups to the water envi ronment. Bonding (bottom) in DNA only between T and A and G and C, whichhelps to dictate the genetic code and fidel i ty in i ts repl ication and express ion. [Reproduced with permiss ion from Naik P: Biochemistry, 3rdedition, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.] Deta i l (bottom) of hydrogen bonding (arrows) between purine and pyrimdine pa i rs .C. Mechanism of DNA Repl ication. Unwinding of DNA to a l low access to new nucleotides during DNA repl ication. [Reproduced with permiss ionfrom Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

The double-helix s tructure of DNA is essentia l for i ts function because these two bonded s trands can temporari ly separate at speci fic parts ofthe DNA molecule to a l low for DNA repl ication (shown in Figure 4-6C). mRNAs and associated proteins can a lso access the DNA strands to copythe conta ined genetic sequence as the fi rs t s tep, leading to protein synthes is (see Chapter 9). This process i s discussed in more deta i l inSection II . The unique double hel ix can then rebond to aga in protect the vi ta l DNA message. A separate type of DNA is found in mitochondria .This mitochondrial DNA (mtDNA) codes for only a few proteins (13 tota l proteins in human mitochondria) involved in the production of energywithin the mitochondria . Unl ike DNA found in the nucleus , mtDNA forms a smal l ci rcular s tructure with the same bonding and s trandseparation seen in l inear double-hel ix DNA molecules .

The speci fic sequence of A, G, C, and T nucleotides or “genome” defines a l l the functional molecules of a l iving creature and, as such, DNA isthe blueprint of l i fe. The genome of humans i s es timated to conta in approximately 20,000–25,000 di fferent genes . Conta ined within thechromosomes (Figure 4-7) are the nucleotide s trands , which conta in the message or “code” for every s ingle protein. The sequences of A’s , G’s ,C’s , and T’s or “genes” that code for mRNA molecules and, subsequently, these proteins are referred to as “expressed sequences” or “exons.”Sequences that do not code for a protein are ca l led “intervening sequences” or “introns.” In fact, introns comprise over 90% of the tota l DNAsequence found in humans and are bel ieved to be leftover, nonfunctional remnants of evolutionary changes or, perhaps , important regulatorysequences whose functions are yet to be determined. Introns are removed or “spl iced” from the sequence during the early s tages of proteinsynthes is (see Section II).

Figure 4-7. Relationship between Chromosomes (right) and a Gene. Each human chromosome (far left, whose DNA content i s graphica l ly i l lus tratedas far left bar) conta ins approximately 1000–2000 genes arranged in clusters of approximately 20 genes . Within the gene are “expressedsequences” (exons , dark orange) and “intervening sequences” (introns , l ight orange). The intervening sequences are removed duringexpress ion of the primary mRNA transcript and process ing to the fina l mRNA product. Introns do not appear to code for any protein and theirrole, i f any, i s s ti l l unknown. Length of molecules i s indicated as bp (base pa i rs ) or nt (nucleotides). [(Left part) Reproduced with permiss ionfrom Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010. (Right part) Adapted with permiss ion from MurrayRA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Ribozymes: Ribozymes (ribonucleic acid enzymes) represent a unique departure from the origina l thought that enzymes can only be proteins .Like thei r protein counterparts , particular RNA sequences possess secondary or tertiary s tructure that enables them to cata lyze a reaction.Most ribozymes act on ei ther themselves or another RNA molecule. However, some ribozymes , including those in ribosomes (see Chapter 9),cata lyze the transfer of amino groups to a growing protein sequence and ass is t new proteins to fold into thei r appropriate conformation.The development of potentia l scienti fi c and medica l appl ications of ribozymes i s ongoing, including poss ible treatments aga inst ini tia lHIV infection. Theories suggesting that RNA, not DNA, molecules were the origina l genetic code molecules a lso infer that ribozymes mayhave been some of the ini tia l enzymatic molecules that a l lowed propagation of early l i fe.

Knowing the exact sequence of an organism’s DNA genome would a l low one to dupl icate or “clone” that organism exactly. “Cloning” i ss imply the abi l i ty to copy the DNA sequence and repl icate i t to form the particular organism. Isolating speci fic sequences that are the code fora particular protein a l lows researchers to s tudy and manipulate these proteins (see Appendix I I), and i s vi ta l for scienti fi c and medica lpurposes .

Adenosine Deaminase Deficiency and Gene Therapy: Deficiencies in the breakdown of nucleotides and nucleos ides lead to often fata l diseasesearly in l i fe in which the immune response i s markedly decreased. Severe combined immunodeficiency syndromes encompass severa lexamples of these diseases but a lmost ha l f of patients have a deficiency in the breakdown of adenos ine by adenosine deaminase. Patientshave been treated by bone marrow transplant but a lso by the emerging technology of gene therapy with some success . Gene therapy offersthe potentia l for producing a “good” copy of a gene and uses i t to replace the defective copy. The impl ications of this treatment are vast.

REVIEW QUESTIONS1. What are nucleos ides , nucleotides , deoxynucleotides , purines , and pyrimidines , thei r key features and bas ic s tructure?2. How do you dis tinguish between adenine, guanine, cytos ine, thymidine, and uraci l s tructure?3. What are ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), thei r bas ic s tructure, and their roles in human biology?4. What are genes , exons , and introns and their key features?5. What i s the relationship between amino acids and the ni trogenous bases of purines and pyrimidines?6. What i s the role of folate in the synthes is of ni trogenous bases of purines?7. What i s the overa l l pathway of synthes is of the five major nucleos ides and nucleotides including the deoxy forms?

SECTION IINTEGRATED USMLE-STYLE QUESTIONS AND ANSWERS

QUESTIONSI-1. Severa l fami l ies were s tudied whose affected individuals have nephrogenic diabetes ins ipidus . This disease causes chi ldhood

symptoms of polyuria (frequent urination), polydips ia (constant thi rs t and frequent drinking), poor growth, and hypernatremia(increased serum sodium concentration). Adminis tration of antidiuretic hormone was not curative, focus ing attention on a renal waterloss due to a transport defect. A gene named aquaporin-2 was cloned from renal tubular epi thel ium, i ts amino acid sequence derived,and s tructura l domains hypothes ized to faci l i tate separation of mutations from benign variants . The hypothes ized s tructure conta inedsevera l transmembrane domains demarcated by β-turns , and these potentia l water channels were found to be mutated in affectedindividuals . Which of the fol lowing amino acids i s most suggestive of β-turns?A. Arginine and lys ineB. Aspartic acid and glutamic acidC. Glycine and prol ineD. Leucine and va l ineE. Tryptophan and tyros ine

I-2. A 7-year-old female presents with dehydration after 3 days of explos ive diarrhea after consuming a large amount of i ce cream. Analys isof her feca l matter reveals a large amount of lactose. Which of the fol lowing sugar pa i rs would be found in this disaccharide?A. Fructose and glucose joined by a α-1, 2 bondB. Galactose and fructose joined by a β-1, 2 bondC. Galactose and glucose joined by a β-1, 4 bondD. Two molecules of glucose joined by a α-1, 1 bondE. Two molecules of glucose joined by a α-1, 4 bond

I-3. A deficiency of which of the fol lowing vi tamins would a l ter metabol i sm of ca lcium?A. AB. CC. DD. EE. K

I-4. Which of the fol lowing characteris tics dis tinguish most RNA molecules from DNA?A. 3′-phosphate group l inked to a pentose sugarB. 5′-phosphate group l inked to a pentose sugarC. Adenine base on RNA, whereas a uraci l base on DNAD. Purine or pyrimidine base l inked to a pentose sugarE. Uridine base on RNA, whereas a thymidine base on DNA

I-5. A severe form of osteogenes is imperfecta (OI) was noted, in which newborn infants have extremely deformed l imbs and chest, caus ingthem to die shortly a fter bi rth because their chest wal l was not adequate for respi ration. This “type II” severe form was ini tia l ly thoughtto be autosomal recess ive, but was later shown to involve mutations in type I col lagen l ike other forms of OI. Col lagen i s a fibrousprotein cons is ting of three peptide chains entwined in a triple hel ix, formed by a repeating amino acid moti f (where X or Y can be anyamino acid). Which of the fol lowing shows that the repeating 3-amino acid moti f i s most compatible with col lagen triple hel ixformation and the mutation most l ikely to cause severe OI?A. Ala–X–Y mutated to Gly–X–Y in one repeatB. Ala–X–Y mutated to Leu–X–Y in one repeatC. Gly–X–Y mutated to Ala–X–Y in one repeatD. Gly–X–Y mutated to Pro–X–Y in one repeatE. Pro-X-Y mutated to Gly-X-Y in one repeat

I-6. A 60-year-old man i s brought to his phys ician from an insti tution for severe menta l deficiency. The phys ician reviews his fami ly his toryand finds he has an older s i s ter in the same insti tution. Their parents are deceased but reportedly had normal intel l igence and nochronic diseases . The man s i ts in an odd pos i tion as though he was sewing, prompting the phys ician to obta in a ferric chloride test onthe man’s urine. This test turns color with aromatic (ring) compounds , including certa in amino acids , and a green color confi rms thephys ician’s diagnos is . Which of the fol lowing amino acids was most l ikely detected in the man’s urine?A. GlutamineB. GlycineC. MethionineD. Phenyla lanineE. Serine

I-7. Chi ldren with severe vi tamin A deficiency develop accumulation of a glycosaminoglycan on the corneal epi thel ium and i s accompaniedby “dry eye.” Which of the fol lowing types of glycosaminoglycan would you expect to find accumulated on the surface of the cornea?A. Chondroi tinB. HeparanC. HeparinD. KeratinE. Perlecan

I-8. Which of the fol lowing i s an essentia l fatty acid?

A. Arachidonic acid (C-20:4-Δ5,8,11,14)

B. Eicosatetraenoic acid (C-20:3-Δ8,11,14)

C. Linoleic acid (C-18:2-Δ9,12)

D. Oleic acid (C-18:1-Δ9)E. Pa lmitic acid (C-16:0)

I-9. A 47-year-old man compla ins of pa in in the joints of his left big toe, which are obvious ly swol len and tender. The pa in has been chronicbut became intolerable the day after Thanksgiving when he had a large meal and severa l glasses of red wine. He i s obese, and hispast medica l his tory i s s igni ficant for removal of kidney s tones . Which of the fol lowing i s involved in the pathophys iology of thispatient’s condition?A. Deficiency of fol ic acidB. Elevated orotic acidC. Elevated uric acidD. Loss of red blood cel l sE. Low blood glucose

I-10. A chi ld presents with severe vomiting, dehydration, and fever. Ini tia l blood s tudies show acidos is with low bicarbonate. Prel iminaryresul ts from the blood amino acid screen show two elevated amino acids , both with nonpolar s ide cha ins . A ti tration curve performedon one of the elevated species shows only two ionizable groups : one that i s acidic and the other that i s bas ic (i .e., no charged s idechain). Which of the fol lowing pa i rs of elevated amino acids i s most l ikely elevated?A. Arginine and i soleucineB. Aspartic acid and glutamineC. Glutamic acid and threonineD. His tidine and va l ineE. Leucine and i soleucine

I-11. Certa in amino acids are not part of the primary s tructure of proteins but are modi fied after trans lation. In scurvy, which of the fol lowingamino acids that i s normal ly part of col lagen cannot be hydroxylated after trans lation?A. AlanineB. His tidineC. Prol ineD. TryptophanE. Tyros ine

I-12. A woman returns from a year long trip abroad with her 2-week-old infant, whom she i s breastfeeding. The chi ld soon s tarts to exhibi tlethargy, diarrhea, vomiting, jaundice, and an enlarged l iver. The pediatrician prescribed a switch from breast mi lk to infant formulaconta ining sucrose as the sole carbohydrate. The baby’s symptoms resolve within a few days . Which of the fol lowing was the mostl ikely diagnos is?A. Deficiency of an enzyme in the metabol i sm of pentose sugarsB. Deficiency of an enzyme in the pathway that metabol i zes glucoseC. GalactosemiaD. Intolerance to dietary fructoseE. Intolerance to lactase

I-13. Despi te the fact that trans fatty acids are unsaturated, thei r contributions to atheroscleros is are s imi lar to those of saturated fats . Thiss imi lari ty in phys iologica l action can be attributed to which of the fol lowing?A. Relatively l inear s tructuresB. Simi lar rates of metabol i smC. Simi lar ti s sue dis tributionsD. Solubi l i ties in waterE. Tendency to form triglycerides

I-14. Methotrexate i s a potent anticancer agent that s tarves dividing cel l s of deoxyribonucleotides through di rect inhibi tion of which of thefol lowing processes?A. Degradation of purine bases to uric acidB. Deoxygenation of the ribose ring to produce deoxyri -bose for DNAC. Metabol i sm of fol ic acidD. Synthes is of purine basesE. Synthes is of thymidine

I-15. An infant i s normal at bi rth but becomes lethargic after severa l feedings ; the medica l s tudent describes an unusual smel l to the urinebut i s ignored. Infection (seps is ) i s suspected, and blood tests show normal white blood cel l counts with a serum pH of 7.0. Eva luationfor an inborn error of metabol i sm shows an abnormal amino acid screen. The report s tates that branch-chain amino acids are s trikinglyelevated. Which of the fol lowing amino acids does the report refer to?A. ArginineB. Aspartic acidC. IsoleucineD. Lys ineE. Threonine

I-16. A 10-month-old white boy i s being eva luated for weakness , pa l lor, hemorrhages under the fingernai l s , and bleeding gums.Radiographs indicate that bone near the growth plates shows reduced osteoid formation and gross ly defective col lagen s tructure.What would be the most effective treatment for this patient’s condition?A. Exclus ion of da i ry products from the dietB. Growth hormone treatmentC. Ora l i ron supplementationD. Ora l vi tamin AE. Ora l vi tamin C

I-17. A 9-month-old gi rl i s suffering from vomiting, lethargy, and poor feeding behavior. Her mother reports that the symptoms began shortlyafter the baby was given a portion of a pops icle and mashed bananas by her grandparents . The baby’s discomfort seemed to resolveafter breastfeeding was resumed. Which of the fol lowing i s the most l ikely diagnos is?A. Deficiency of an enzyme in the metabol i sm of pentose sugarsB. Deficiency of an enzyme in the pathway that metabol i zes glucoseC. Galactosemia

D. Intolerance to dietary fructoseI-18. During s tarvation glucagon, a peptide hormone, i s important for activating glucose synthes is and mobi l i zing fats . Bes ides glucagon,

which of the fol lowing i s a l ipid-derived hormone that regulates sugar and fat metabol i sm in s tarvation and other s tress s tates?A. GlucocorticoidsB. Minera locorticoidsC. ProstagensD. Vi tamin AE. Vi tamin D

I-19. A 2-year-old boy’s mother i s concerned about his tendency to bi te himsel f to the point of bleeding. The boy’s fingers show scarring andsevera l scabs , and his l ips are swol len and bruised. He exhibi ts poor coordination, poor muscle tone, and frequent jerking movementsof his arms and legs . He i s s igni ficantly delayed in speech. His urine i s orange in color and “gri tty.” Which of the fol lowing i s the mostl ikely diagnos is?A. Adenos ine deaminase deficiencyB. Cerebra l pa lsyC. GoutD. Lesch–Nyhan syndromeE. Tay–Sachs disease

ANSWERSI-1. The answer is C. A β-turn s tructure cons is ts of four amino acids in which the fi rs t res idue i s hydrogen bonded to the fourth res idue of the

turn (see Figure 1-5C). Glycine res idues are smal l and flexible, whereas prol ine res idues assume a cis or flattened conformation, makingthese res idues amenable to tight turns . Transport proteins often have severa l membrane-spanning domains demarcated by β-turns thata l low them to exi t and return back into the membrane. These transmembrane domains form channels that regulate transport of ions andwater in organs such as lung, gut, and kidney. Nephrogenic diabetes ins ipidus resul ts when the kidney i s less respons ive to antidiuretichormone excreted by the posterior pi tui tary, caus ing abnormal water excretion, dehydration, and electrolyte dis turbances .

I-2. The answer is C. Disaccharides are carbohydrates composed of two sugar molecules . Lactose in this case i s composed of ga lactose andglucose joined by a β-1,4 glycos idic bond. Sucrose i s a disaccharide of a fructose and a glucose molecule joined by a α-1,2 glycos idicbond. Maltose i s a disaccharide of two glucose molecules joined by a α-1,4 glycos idic bond. There i s no disaccharide composed offructose and ga lactose (see Figure 2-3).

I-3. The answer is C. Vi tamin D i s a fat-soluble vi tamin essentia l for the absorption of dietary ca lcium and the reabsorption of ca lcium by thekidney. Vi tamin A i s a lso a fat-soluble vi tamin essentia l for vi s ion, reproduction, and development but does not speci fica l ly a ffect anyminera ls . Vi tamin C i s a water-soluble vi tamin important in two enzymes in col lagen formation and as an antioxidant. Vi tamin E i s a fat-soluble vi tamin important as an anti -oxidant. Vi tamin K i s fat-soluble and important in blood clotting.

I-4. The answer is E. RNA and DNA are composed of nucleos ide uni ts where a purine or pyrimidine base i s l inked to a pentose sugar. The 1′carbon of the pentose i s l inked to the ni trogen of the base. In DNA, 2′-deoxyribose sugars are used; in RNA, ribose sugars are used thatconta in 2′-and 3′-hydroxyls . The ni trogenous bases are adenine, thymine, guanine, and cytos ine in DNA, with thymine replaced byuridine in RNA. Nucleotide polymers are cha ins of nucleotides with s ingle phosphate groups , joined by bonds between the 3′-hydroxyl ofthe preceding pentose and the 5′-phosphate of the next pentose. Polymerization requires high-energy nucleotide triphosphateprecursors that l iberate pyrophosphate (broken down to phosphate) during joining. The polymerization reaction i s given speci fici ty bycomplementary RNA or DNA templates and rapidi ty by enzyme cata lys ts ca l led polymerases .

I-5. The answer is D. Col lagen i s the most abundant fibrous protein found in connective ti ssue, bone, carti lage, skin, l igaments , and tendonsas wel l as other ti s sues . Col lagen cons is ts of two s trands of α1 and one s trand of α2 col lagen intertwined in a triple hel ix. The triplehel ix has 3.3 res idues per turn. The triple hel ix i s s tabi l i zed by hydrogen bonds between res idues in di fferent s trands . The presence ofglycine in every thi rd res idue a l lows for very tight packing of each s trand in the triple hel ix because glycine res idues are smal l and conferflexibi l i ty. Mutations substi tuting a larger or di fferently configured amino acid for the glycine of one repeat would dis rupt the tightpacking at that pos i tion of the col lagen s trand and a l ter triple hel ix conformation; prol ine substi tutions for glycine (answer D) woulda l ter cha in di rection and be more dis ruptive than those with a lanine (answer C). Substi tutions of amino acids at the X–Y pos i tions of arepeat would have less influence on packing, better preserving the triple hel ix s tructure. Mutations at pos i tions in the α1 cha in woulda lso be more severe because two of these s trands are incorporated with each triple hel ix. Mi lder mutations a l ter bone col lagen todecrease bone thickness and cause susceptibi l i ty to fractures . More severe mutations interfere with bone formation duringdevelopment, caus ing severe deformities of l imbs and chest wal l that are incompatible with l i fe. These mutations a lso inhibi t col lagensynthes is in the sclerae (whites of the eyes), caus ing them to be thinner so the underlying blue choroid shows through.

I-6. The answer is D. Before phenylketonuria was recognized and before the advent of routine newborn metabol ic screening, chi ldren with thisautosomal recess ive disorder developed severe menta l retardation without other identi fying symptoms. Al though few chi ldren withdevelopmenta l disabi l i ty are being placed in insti tutions today, the movement for release into home or ha l fway house care in the 1960sand 1970s was compl icated by long-term res idents who had not developed l i fe ski l l s . Thus , some older individuals in insti tutions havedisorders that could have been detected by modern screening and would never have prompted insti tutional i zation with modern care.Phenyla lanine, with i ts benzene ring, i s an essentia l amino acid that i s converted to tyros ine by phenyla lanine hydroxylase, the enzymethat i s deficient in phenylketonuria . This disease can a lso be caused by a deficiency of the enzyme’s cofactor, biopterin.

I-7. The answer is D. Keratin i s a component of carti lage and cornea of the eye. In vi tamin A deficiency, keratinization of the skin and of themucous membranes in the respiratory, GI, and urinary tracts can a lso occur. Drying, sca l ing, and fol l i cular thickening of the skin andrespiratory infections can resul t from this deficiency. Chondroi tin i s a s tructura l component and attachment point to col lagen. Heparan i sfound attached to col lagen for various cel l s , including l iver cel l s . Heparin regulates the immune response and blood clot formation.Perlecan i s a s tructura l component of the kidney membrane.

I-8. The answer is C. The essentia l fatty acid l inoleic acid (C-18:2-Δ9,12), wi th 18 carbons and two double bonds at carbons 9 and 18, i sdesaturated to form β-l inolenic acid (C-18:3-Δ6,9,12), which i s sequentia l ly elongated and desaturated to form eicosatrienoic acid (C-20:3-Δ8,11,14) and arachidonic acid (C-20:4-Δ5,8,11,14). Many of the eicosanoids (20-carbon compounds)—prostaglandins , thromboxanes , andleukotrienes—are derived from arachidonic acid. Arachidonic acid can only be synthes ized from essentia l fatty acids obta ined from thediet. Pa lmitic acid (C-16:0) and oleic acid (C-18:1-Δ9) can be synthes ized by the ti ssues (Table 3-1).

I-9. The answer is C. This patient shows many symptoms cons is tent with an episode of gout. His joint pa in i s due to gouty arthri ti s , aninflammatory condition aris ing from depos i tion of sodium urate crysta ls . The swel l ing in the joints of his big toe (tophaceous gout) i sa lso a mani festation of this phenomenon. In his case, the episode seems to have been triggered by excess ive eating at Thanksgivingdinner a long with a lcohol consumption, leading to degradation of large quanti ties of purine nucleotides and consequent increased flux

through the pathway that produces uric acid. Whether his gout ari ses from impaired excretion of uric acid or i s due to a mutation ofphosphoribosylpyrophosphate synthetase cannot be determined from the data . Analys is of his blood may confi rm the gout i f highconcentrations of uric acid (hyperuricemia) are present.

I-10. The answer is E. Leucine and i soleucine have nonpolar methyl groups as s ide cha ins . As for any amino acid, ti tration curves obta ined bynoting the change in pH would show an acidic ionizable group for the primary carboxyl group and a bas ic ionizable group 5 for theprimary amino group; there would be no additional ionizable s ide cha in. Aspartic and glutamic acids (second carboxyl group), his tidine(bas ic imino group), glutamine (second amino group), and threonine (hydroxyl group) a l l have ionizable s ide cha ins that would show anadditional group on the ti tration curve. The l ikely diagnos is here i s maple syrup urine disease, which involves elevated i soleucine,leucine, and va l ine together with thei r ketoacid derivatives . The ketoacid derivatives cause the acidos is , and the fever suggests that themetabol ic imbalance was worsened by an infection.

I-11. The answer is C. Prol ine and lys ine are hydroxylated after the synthes is of new col lagen molecules . The hydroxylation of prol ine and lys ineres idues occurs in reactions cata lyzed by prolyl and lysyl hydroxylase enzymes that require the reducing agent ascorbic acid (vi tamin C).In scurvy, which resul ts from a deficiency of vi tamin C, insufficient hydroxylation of col lagen causes abnormal col lagen fibri l s . Theweakened col lagen in teeth, bone, and blood vessels causes tooth loss , bri ttle bones with fractures , and bleeding tendencies withbruis ing and bleeding gums.

I-12. The answer is C. The patient’s symptoms and course in response to a lactose-conta ining formula are cons is tent with a diagnos is ofga lactosemia. Because the chi ld can consume sucrose, that conta ins glucose and fructose, the problem cannot be in pathways thatmetabol i ze ei ther glucose or pentose sugars . Additional ly, the chi ld can tolerate dietary fructose as evidenced by the abi l i ty to consumesucrose. Fina l ly, whi le one can be intolerant to lactose, the sugar, the same is not true regarding lactase, the enzyme. Al though geneticscreening tests required in most s tates identi fy newborns with ga lactosemia, these tests may not have been performed on a chi ld bornouts ide the United States .

I-13. The answer is A. Saturated fatty acids and trans fatty acids are s tructura l ly s imi lar; thei r hydrocarbon ta i l s are relatively l inear. This a l lowsthem to pack tightly together in semicrysta l l ine arrays such as the membrane bi layer. Such arrays have s imi lar biochemica l properties interms of melting temperature (fluidi ty). Al though some of the other properties l i s ted are a lso shared by saturated and trans fats , they arenot thought to account for the tendency of these fats to contribute to atheroscleros is (Figure 3-1B).

I-14. The answer is C. Methotrexate i s an analog of fol ic acid that binds with very high affini ty to the substrate-binding s i te of dihydrofolatereductase, the enzyme that cata lyzes convers ion of dihydrofolate to tetrahydrofolate, which i s used in various forms by enzymes of boththe purine and pyrimidine de novo synthetic pathways . Thus , synthes is of deoxythymidine monophosphate from deoxyuridinemonophosphate cata lyzed by thymidylate synthetase and severa l s teps in purine synthes is cata lyzed by formyltransferase are indirectlyblocked by the action of methotrexate because both those enzymes require tetrahydrofuran coenzymes. Xanthine oxidase that cata lyzesdegradation of purine bases to uric acid i s unaffected.

I-15. The answer is C. Amino acids are class i fied as acidic, neutra l hydrophobic, neutra l hydrophi l i c, or bas ic, depending on the charge orpartia l charge on the R-group at pH 7. Hydrophobic (water-hating) groups are carbon–hydrogen chains l ike those of leucine, i soleucine,glycine, or va l ine. Bas ic R-groups , such as those of lys ine and arginine, carry a pos i tive charge at phys iologic pH owing to protonatedamide groups , whereas acidic R-groups , such as glutamic acid, carry a negative charge owing to ionized carboxyl groups . Threonine withi ts hydroxyl s ide cha in i s neutra l at phys iologic pH. Leucine, i soleucine, and va l ine are amino acids with branched s ide groups , and theyshare a pathway for degradation that i s deficient in chi ldren with maple syrup urine disease. Their amino groups can be removed, butthe resul ting carboxyl ic acids accumulate with resul ting acidos is , coma, and death unless a diet free of branch-chained amino acids i sinsti tuted.

I-16. The answer is E. The patient shows many s igns of vi tamin C deficiency or scurvy, which i s seen most frequently in infants , the elderly, andin a lcohol ic patients . Particularly indicative of vi tamin C deficiency are the multiple smal l hemorrhages that occur under the skin(petechiae) and na i l s and surrounding ha i r fol l i cles . Bleeding gums are a class ic indicator of scurvy.

I-17. The answer is D. The main sugar in mother’s mi lk i s lactose. When the baby was given the frui t and the arti fi cia l ly sweetened pops icle,she was exposed to fructose for the fi rs t time and apparently i s intolerant to dietary fructose. Because the chi ld can consume lactose,which conta ins glucose and ga lactose, the problem cannot be in pathways that metabol i ze ei ther glucose or pentose sugars . Thesymptoms are a lso cons is tent with ga lactosemia, but would be expected as a reaction to lactose intake. This diagnos is of fructoseintolerance should be confi rmed by genetic testing. Essentia l fructosuria i s a benign condition that would not have produced suchsevere symptoms.

I-18. The answer is A. Glucocorticoids are made in the adrenal cortex, the principa l one being corti sol in humans . Corti sol promotes breakdownof proteins in s tarvation to provide precursors for the synthes is of glucose (blood sugar) and a lso promotes the breakdown of s toredl ipids (triglycerides) to provide the energy for this process . Corti sol performs a s imi lar function in chronic s tress . Minera locorticoids , theprincipa l one being a ldosterone, controls sodium reuptake by the kidney in exchange for potass ium and thereby regulates and changesblood volume and pressure. Prostagens are required for implantation of a ferti l i zed egg in the uterine l ining and maintenance of apregnancy. Vi tamin A i s not a l ipid-derived hormone. It i s involved in vis ion, reproduction, and ti ssue development. Vi tamin D controlsthe blood ca lcium–phosphate matrix and thereby promotes bone formation.

I-19. The answer is D. This patient’s sel f-muti lation behavior, neurologic symptoms, and developmenta l delay are cons is tent with diagnos is ofLesch–Nyhan syndrome. This disorder i s due to the deficiency of hypoxanthine–guanine phosphoribosyl transferase, which preventssa lvage of hypoxanthine and guanine to thei r respective nucleotides , inos ine monophosphate and guanos ine monophosphate. Thisleads in turn to hyperactivi ty of the purine synthes is pathway, excess ive purine degradation, and overproduction of uric acid. The gri ttysubstance and orange color of the patient’s urine are because of excretion of both dissolved uric acid and precipi tated sodium urate.Gout might account for the excess ive uric acid production but not the neurologic symptoms. Sel f-muti lation i s not characteris tic of Tay–Sachs disease, cerebra l pa lsy, or adenos ine deaminase deficiency.

SECTION IIFUNCTIONAL BIOCHEMISTRY

CHAPTER 5ENZYMES AND AMINO ACID/PROTEIN METABOLISM

EnzymesAmino Acid Metabol i smThe Urea CycleReview Questions

OVERVIEWEnzymes are specia l i zed proteins , which accelerate or catalyze a biochemica l reaction. Each enzyme cata lyzes a speci fic reaction and i sregulated by competi tive and noncompeti tive inhibi tors and/or by a l losteric molecules . Multiple enzymes can cata lyze a series of consecutivereactions , known as pathways, to produce and/or break down complex biologica l molecules . Examples include amino acid synthes is anddegradation, the coordinated reactions involved in protein synthes is , and the urea cycle. Problems with enzyme pathways can not only lead todisease but a lso offer the opportuni ty for disease treatment via medications , which target speci fic points in these pathways .

ENZYMES

ENZYME REACTIONS

Enzymes bring together one or more molecules , ca l led substrates, to form a resul ting molecule ca l led a product (Figure 5-1). Most enzymescata lyze one speci fic reaction. However, some multipart enzyme complexes cata lyze a series of s tep-by-s tep reactions—the fi rs t enzymepasses i ts product, now a new substrate, to a second enzyme that i s part of the complex, the second passes i ts product to a thi rd and so on.Enzymes are respons ible for many essentia l reactions in the human body; in fact, there are from 20,000 to 25,000 tota l human genes , withabout 25% of them producing enzymes. It i s not surpris ing that problems with enzymes are caused by or resul t in diseases .

Figure 5-1. Illustration of Simple One and Two Substrate Enzyme Reaction. Components include enzyme (E), substances (S1, S2, S3), and product (P). Ifthe substance can bind to the enzyme’s substrate-binding s i te (e.g., S1, top figure, and S1 and S2, bottom figure), then i t acts as a substrate.Molecular shape as determined by secondary, tertiary, and quaternary s tructure as wel l as the hydrophobic/hydrophi l i c and neutra l or chargednature influences which substrate molecules can bind as substrates (represented graphica l ly by di ffering shapes of S1, S2, and S3 and thetriangular and ci rcular binding pockets ). The enzyme binds the substrate molecule(s ), cata lyzes the enzymatic reaction, and releases theproduct after which the enzyme is ready to cata lyze the same reaction aga in. The rate of the overa l l reaction i s influenced by each s tep in theprocess , including substrate binding, rate of the reaction, and product release as discussed in the text below. [Adapted with permiss ion fromNaik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

The concept of enzyme kinetics a l lows an exact description of the enzymatic reaction, including the influence of substrate and productmolecules and how fast the enzyme cata lyzes the reaction and the impact. More advanced enzyme kinetics a l lows the mathematica lexpress ion of how other molecules such as cofactors, inhibitors, and activators (see below) affect the enzyme reaction.

For example, as discussed in Chapter 1, speci fic amino acids form an enzyme’s primary s tructure and, therefore, secondary to quaternarystructure. These s tructures , in turn, form substrate-binding sites (a l so known as the “active site”) to accommodate the substrates , thei r chemica lreaction, and the departure of the product. Substrates and products can both be a hydrophobic, hydrophilic, charged, uncharged, or neutralmolecule, or a combination of the above. Mutations that resul t in the change of an amino acid or amino acids that make the substrate pocketcan drastica l ly change the enzyme’s activi ty. For example, a hydrophobic substrate wi l l eas i ly enter an enzyme pocket that i s l ined withhydrophobic amino acids . However, i f one of these hydrophobic amino acids i s changed to a highly charged amino acid, the substrate may notbe able to enter the pocket and the enzyme may no longer function. How wel l the substrate interacts with the substrate-binding s i terepresents the “affinity” of the enzyme for the substrate. The s tronger the affini ty, the less substrate i s needed to achieve a certa in rate ofreaction. This concept i s important i f a mutation changes the substrate-binding s i te and lowers the affini ty. This concept i s i l lus trated inFigure 5-1.

Substrate Concentration and Enzyme-based Diseases: Phenylketonuria (PKU) i s an important autosomal recess ive disease occurring in an averageof 1 in 15,000 bi rths . This genetic disease i s usual ly caused by the deficiency of the enzyme phenylalanine hydroxylase, required to convertphenyla lanine to tyros ine (see reaction below; hydroxyl group indicated by smal l arrowhead), important in the production ofneurotransmitters and skin pigment (melanin). However, the disease does not resul t only from the decrease of tyros ine levels ; rather, theresul ting high concentration of phenyla lanine leads to the activi ty of an otherwise minor enzymatic pathway, which produces phenylketones ,including phenylacetate, phenylpyruvate, and phenylethylamine. The presence of these phenylketones i s normal ly screened for in theblood as part of s tandard neonata l testing and, aga in, at 2 weeks of age.

[Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Patients with PKU usual ly express tra i ts of a lbinism, i .e., very fa i r skin; white-blond ha i r, and notable, pa le blue eyes ; a “musty” odor fromphenylacetate in thei r sweat, urine, skin, and ha i r; and sometimes develop eczema. Untreated PKU resul ts in decreased bra in development(microcephaly), hyperactivi ty, bra in damage, seizures , and severe learning disabi l i ties/menta l retardation. PKU is usual ly treated with adiet low in phenyla lanine content, a l though some feel that res idual phenyla lanine levels may s ti l l cause neurologica l damage. As a resul t,addi tional treatments are being developed, which further decrease phenyla lanine levels .

The number of molecules of substrate increases or decreases the l ikel ihood of the substrate interacting with the enzyme and, therefore,the rate of the reaction. When a l l of the enzyme molecules have substrate occupying the binding s i tes , the enzyme is sa id to be saturated bysubstrate and the rate of the reaction i s maximized. If lower amounts of substrate are present, the reaction would a lso be s lower thannormal , potentia l ly leading to cl inica l mani festations . If a disease s tate causes a molecule to abnormal ly increase in concentration, i t maybecome a substrate for an enzyme with which i t would normal ly not react.

The fina l factor that decides maximum rate of the reaction i s the efficiency at which an enzyme can cata lyze a s ingle reaction. Enzymemutations can diminish this efficiency, a l though, in rare instances , a mutation can increase activi ty. Likewise, the cel l has regulatorymolecules that can change the rate of the reaction by decreas ing or increas ing the efficiency of the enzyme.

COFACTORS

Enzyme reactions often require additional molecules ca l led cofactors (Figure 5-2). Examples of these cofactors include vi tamins , nicotinamideadenine dinucleotide (NADH), nicotin-amide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FADH2), and coenzyme A(CoA). For example, during an enzyme reaction, NADH or NADPH donates a hydrogen atom as wel l as electrons to the product and becomesNAD+ or NADP+, respectively, and are then released. FADH2 a l so donates electrons and two hydrogen atoms to produce FAD. Conversely, NAD+,

NADP+, and FAD accept electrons to become the respective reduced forms. Some inorganic meta l ions such as i ron (Fe), copper (Cu), ca lcium(Ca), manganese (Mn), magnes ium (Mg), or zinc (Zn) a lso serve as cofactors for reactions . Meta l ions may contribute or accept an electron (e.g.,Fe2+ → Fe3+) or thei r associated charge may s imply provide essentia l s tabi l i zation for the chemica l reaction without changing the meta l .

Figure 5-2. Enzymatic Reaction Involving a Cofactor. Components include enzyme (E), substrates (S1, S2), cofactor (Co), and product (P). The cofactoris necessary for the creation of the fina l product but i s unchanged at the end of the reaction. [Adapted with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Diseases of Copper Deficiency: The importance of proper levels of copper (Cu2+) to the human body i s often not appreciated. For example, theincorporation of copper into severa l enzymes in mitochondria i s essentia l for oxidative phosphorylation (Chapter 6), and low levels canadversely affect the production of ATP. The oppos i te, an excess of copper, leads to the important cl inica l conditions of Wilson’s disease andMenke’s “kinky hair” disease.

Wi lson’s disease resul ts from low activi ty of ATPase, Cu2+ transporting, beta polypeptide (ATP7B), which i s respons ible for copper transportout of cel l s . The transported copper i s carried by ceruloplasmin in the ci rculation. In Wi lson’s disease, copper ion concentrations areincreased in l iver, bra in, eyes , and other ti s sues . Liver depos i ts resul t in fatigue, confus ion from high ammonia levels (hepaticencephalopathy), heightened blood pressure (porta l hypertens ion), an increased ri sk of bleeding from blood vessels in the esophagus(esophageal varices ), and, eventual ly, l i ver fa i lure and/or cancer. Accumulation in the bra in can lead to deterioration of memory andthought processes , loss of muscle control and tone, seizures , migra ines , depress ion, anxiety, psychos is , and, often, the tremors and s lowmovement of Parkinson’s disease. Depos i tion in eyes resul ts in the diagnostic “Kayser–Fleischer ring,” a brownish-green ring evident in thecornea. Kidneys , heart, and parathyroid glands can a lso be adversely affected by copper depos i ts .

Menke’s “kinky ha i r” disease i s a very rare, X-l inked recess ive disorder that mutates ATPase, Cu2+ transporting, alpha polypeptide (ATP7A)and, therefore, a ffects males more often than females . The disease usual ly s trikes infants (a l though a chi ldhood vers ion i s a lso known),s lowly caus ing growth fa i lure, developmenta l delay, and menta l retardation. The subtle onset often leads to no treatment and death in thefi rs t decade. Excess copper accumulation mainly in the kidneys and the digestive tract leads to symptoms s imi lar to Wi lson’s disease.However, low levels of copper in bone, bra in, skin, blood vessels , and ha i r a ffect copper-conta ining enzymes. The ATP7A enzyme is absentin the l iver. Other symptoms particular to Menke’s disease include abnormal body temperature; rupture or blockage of arteries in the bra in;weakened bones and increased fractures ; and the sparse, coarse, fragi le, kinky, colorless , or s teel -colored ha i r that gives the disease i tsa l ternative name. The only treatment i s da i ly injection of copper as wel l as symptomatic and supportive care.

REGULATION

The speed or “rate” at which an enzyme cata lyzes a reaction depends on numerous factors , which can increase (activate) or decrease (inhibit)this rate. This effect i s ca l led regulation and i s essentia l not only for s ingle enzyme reactions but a lso for the overa l l coordination of multiple,and often competing, reactions in the human body. Like any compl icated process , resources need to be conserved and used in an efficientmanner. It i s this regulation that a l lows the body to use energy or s tore energy and/or produce certa in proteins or carbohydrates or l ipidmolecules when particular needs ari se.

Enzyme regulation takes many forms. The s implest i s by varying the concentration of the substrate. As discussed above, the increase ordecrease of the amount of ava i lable s tarting materia l increases or s lows the reaction, respectively, and forms the bas is of not only the s ingleenzyme regulation but a lso global regulation of metabol i sm as wel l . Examples wi l l be seen in other chapters in Sections II and II I .

Enzyme regulation can a lso occur as the resul t of a molecule other than substrate or product interacting separately with the enzyme tomodi fy i ts reaction rate. This molecule, whether an activator or inhibi tor, works in one of two ways : (1) competitive—binding to the same s i teas the substrate molecule(s ) or (2) noncompetitive—binding to a completely separate part of the enzyme, which changes the shape of theenzyme and, as a resul t, the efficiency of the reaction (Figure 5-3). A thi rd type of enzyme competi tion, uncompetitive, i s rare and wi l l not becovered here. The binding of a regulatory molecule may ei ther increase or s low the reaction by increas ing/decreas ing substrate bindingand/or the actua l chemica l reaction and/or product release. (Note: The terms “competi tive” and “noncompeti tive” are normal ly not used todescribe activation; however, for s impl ici ty, these terms are used here.) Both activators and inhibi tors of enzymatic reactions are important indisease processes . However, they are even more important in disease treatment because most medicines are des igned to increase theproduction of a beneficia l product or decrease/stop the formation of a harmful product.

Figure 5-3. Competitive and Noncompetitive Inhibition. Competi tive inhibi tor (upper panel ) binds to and blocks the enzyme’s substrate-binding s i teand hence the binding of the substrate (S1), thereby preventing production of the product. Noncompeti tive inhibi tor (bottom panel ) binds to as i te other than the substrate-binding s i te, leading to a conformational change of the enzyme, which a l ters the normal substrate-binding s i te.This change blocks substrate (S1) binding and production of the product. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion,Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

β-Lactam Antibiotics and Competitive Inhibition: Gram-pos i tive bacteria have cel l wal l s composed of up to 40 layers of peptidoglycans ,specia l i zed carbohydrate, and amino acid s tructures , which offer support and protection. Synthes is of the peptidoglycan cel l wal l involves afina l s tep, which l inks the individual peptidoglycans together and involves the cofactor penici l l in-binding protein (PBP). β-Lactam antibiotics(see the figure below), of which penici l l in i s the archetype, mimic the end amino acid sequence of the unl inked peptidoglycans andirrevers ibly bind to the substrate s i te to competi tively block the fina l cross -l inking reaction of the PBPs . The dis ruption of this fina l s tep ofcel l wal l synthes is leads to death of the bacteria . Many types of antibiotics are β-lactams, including cephalosporins , monobactams,carbapenems, and β-lactamase inhibi tors (e.g., clavulanic acid, sulbactam, and tazobactam).

The structure of penicillins (left) and cephalosporins (right) includes the β-lactam component shown in red. These antibiotics mimic the terminal end ofproteoglycans and block Gram-positive cell wall synthesis.[Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Feedback regulation i s a particular type of s imple regulation in which a product of a s ingle or a series of enzyme reactions can bind to andactivate or inhibi t i tsel f or an earl ier enzyme in the series (Figure 5-4). Higher or lower concentrations of this product offer “feed back”regarding levels of the product and the need to increase or decrease the rate of the reaction(s ) according to the needs of the body. Severa lexamples of feedback regulation are seen in metabolism—the breakdown of biochemica l molecules discussed in later chapters in this sectionand Chapter 10.

Figure 5-4. Feedback Inhibition and Activation. The product can bind to i ts own enzyme or another enzyme and ei ther inhibi t or activate theenzymatic reaction according to the body’s needs . Components include enzyme, substrate (S1), and products (P). [Adapted with permiss ionfrom Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

The activi ty of an enzyme can a lso be control led by the concept of precursor or proenzymes. Proenzymes are often, but not a lways , denoted bythe suffix “-ogen.” Proenzymes conta in the active enzyme and an additional amino acid sequence that keeps the activi ty “turned off.” Aseparate enzyme, speci fic for the proenzyme to enzyme transformation, removes this addi tional sequence to a l low the enzyme function tobegin. Many enzymes involved in the digestion of food are control led this way and are only turned on when speci fic s igna ls are present (e.g.,the presence of a recently ingested food source or a clot formation). Examples of proenzymes include peps inogen → peps in, fibrinogen →fibrin, and procarboxypeptidase → carboxypeptidase.

Isozymes (a l so known as i soenzymes) are two or more enzymes in the same individual that di ffer in amino acid sequence but which cata lyzethe exact same chemica l reaction. Isoenzymes are usual ly found in di fferent ti s sues as wel l as in di fferent cel l organel les , where they can bedi fferently regulated. Examples of proenzymes and i sozymes wi l l be seen in future chapters .

Isozymes: Isozymes a l low the body to di fferentia l ly regulate the same enzyme reaction in di fferent ti s sues of an individual . One of the bestexamples i s hexokinase and glucokinase (see Chapter 6). Hexokinase, found in a l l human cel l s , i s able to bind glucose even at very lowconcentrations . Glucokinase, found only in pancreas , l i ver, smal l intestine, and bra in, requires glucose concentrations 100 times higher forbinding and i s not inhibi ted by glucose-6-phosphate. Even though hexokinase and glucokinase cata lyze the same reaction, the di fferentbinding of glucose and regulation a l lows the body to properly regulate glucose use (e.g., release of insul in by beta cel l s of the pancreas)and s torage (e.g., l i ver cel l s ).

Fina l ly, allosteric regulation involves the binding of an effector molecule to a part of a s ingle subunit (“monomeric”) enzyme, or to one ofsevera l subunits , in the case of enzyme complexes with more than subunit (“multimeric”). For the more common a l losteric regulation ofmultimeric enzymes , the effector i s often the substrate for more than one active s i te conta ined within the complex. Binding of the effectorcauses a conformational change, which affects the active s i te of an enzyme (Figure 5-5). The binding ei ther activates (pos i tive cooperativi ty) orinhibi ts (negative cooperativi ty) addi tional substrate binding or enzyme activi ty—an effect known as a l losteric regulation. Al lostericregulation has been class ica l ly i l lus trated in the sequentia l ly increas ing binding of oxygen by the four subunits of hemoglobin as wel l as thedecreased binding of oxygen by the subunits by the molecule 2,3-bisphosphoglycerate (both a l low increased but regulated release of oxygennear ti s sues low in oxygen). An example of a l losteric regulation of monomeric enzymes includes the effect of lactate produced during periodsof prolonged phys ica l effort to decrease oxygen binding by myoglobin to help mainta in ATP concentration. Severa l drugs , including ibuprofenand acetaminophen, a lso a l losterica l ly a ffect the binding of the anti -anxiety class of drugs ca l led benzodiazepines to a lbumin, therebyinfluencing the actua l concentration of this medication in the blood.

Figure 5-5. Allosteric Regulation of Enzyme Activity. Binding of a pos i tive a l losteric modulator (upper panel ) leads to a conformational change ofthe enzyme (E), which enhances enzyme activi ty (bold arrows) by increased substrate binding (smal l curved arrow) and/or increased rate ofproduct (P) formation. Binding of a negative a l losteric modulator (lower panel ) leads to a conformational change, which decreases enzymeactivi ty (thin arrows) by decreas ing substrate binding (smal l curved arrow) and/or leading to reduced or no product formation. [Adapted withpermiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

AMINO ACID METABOLISM

AMINO ACID SYNTHESIS

The synthes is of amino acids i s of paramount importance to the human body. Once synthes ized or ingested, amino acids are used as bui ldingblocks not only for proteins but a lso for severa l other cri ti ca l biologica l molecules such as nucleic acids (both purines and pyrimidines),hormones , neurotransmitters , anti -oxidants , and various s ignal ing molecules (Figure 5-6; see a lso Chapter 1 and later chapters ).

Figure 5-6. Sources and Fates of Amino Acids. Amino acids , from endogenous or dietary proteins as wel l as de novo synthes is , are used forsynthes is of a variety of ni trogenous compounds as wel l as new proteins . Their carbon skeleton can make a variety of fuels , depending onenergy needs , or be burned as a fuel , whereas the toxic ni trogen waste i s largely excreted as urea. [Reproduced with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Al though individual amino acids are usual ly obta ined from the diet, especia l ly the essentia l amino acids , nonessentia l amino acids canalso be produced from other sources . The non-essentia l amino acids are a l l made from precursors obta ined from the metabol i sm ofcarbohydrates to be s tudied in the next chapter. These amino acids and their precursor molecule(s ) are shown in Figure 5-7.

Figure 5-7. Synthesis of Amino Acids. Precursors of the 20 amino acids found in humans are predominately from carbohydrate metabol i sm(glycolys is and ci tric acid cycle, see Chapter 6). Essentia l amino acids required from the diet are indicated by asterisks (*). His tidine i s derived

partly from the nucleic acid precursor phosphoribosyl pyrophosphate and the amino acid glutamate. Speci fic medica l conditions associatedwith the synthes is of amino acids are shown in Table 5-1.

TABLE 5-1. Diseases of Amino Acid Metabol i sm

AMINO ACID DEGRADATION

Protein and amino acid breakdown, known as “degradation,” i s essentia l as an energy source when carbohydrate and/or l ipid sources are low,as wel l as to produce atoms and molecules required for the synthes is of other molecules . The breakdown of particular amino acids a lsohelps the body to regulate how much ni trogen i t conta ins and to excrete excess ni trogen in the form of urea in urine. Each amino acidundergoes a speci fic series of enzymatic reactions , leading to molecules involved with and, therefore, leading into carbohydrate or l ipidmetabol i sm and energy production (Figure 5-8). As with amino acid synthes is , deficiencies and decreased activi ty of the enzymes involved inthese breakdown pathways lead to disease s tates . These diseases are usual ly present at bi rth and require adherence to speci fic dietaryregimens to control the i l lness .

Figure 5-8. Overview of Amino Acid Degradation Pathways. Amino acids enter into and are metabol i zed via the carbohydrate metabol ic pathways(ci tric acid cycle and glycolys is , see Chapter 6). [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

Severa l diseases involving the absence or inactivi ty of an enzyme involved in amino acid metabol i sm are l i s ted in Table 5-1. Thesedeficiencies in enzymes respons ible for the synthes is , degradation, absorption, and transport of the amino acids genera l ly cause fa i lure orabnormal development, neurologica l/psychiatric problems, some qui te unique (repeti tive sel f-hugging and “l i ck and fl ip” activi ty—l icking offingers and fl ipping of book and magazine pages), musculoskeleta l disorders , abnormal function or fa i lure of organ and organ systems,severa l unique odors [“maple syrup,” “mousy,” “cabbage-l ike,” “oasthouse” (bui lding for drying hops)], and colors of urine (black and blue).These conditions may a lso lead to death of the fetus or infant. A more complete description of each of these conditions i s found in AppendixI.

THE UREA CYCLEUrea provides a cri ti ca l means of ridding the body of ni trogen waste. The urea cycle i s the series of enzyme reactions in l iver cel l mitochondriaand cytoplasm respons ible for the removal of excess ni trogen and the potentia l ly toxic by-product ammonia via the production of urea. Moreimportantly, the urea cycle helps keep in ba lance ni trogen atoms in the human body. The urea cycle accepts ni trogen atoms in the form ofamino groups exclus ively from the amino acids glutamate and aspartate (Figure 5-9A). Other sources of excess ni trogen, including fromother amino acids , feed through the glutamate and aspartate pathways .

Figure 5-9A. Urea Cycle. A. The urea cycle converts two amino groups (NH3) from glutamate and aspartate into urea as part of the body’s ni trogenbalance. Glutamate donates the fi rs t ni trogen atom (1) via the molecule carbamoyl phosphate that i s synthes ized via the regulated enzymeCPS. This enzyme is activated by increased glutamate, N-acetylglutamic acid, and/or arginine concentrations [indicated by (+)]. Carbamoylphosphate then enters the urea cycle by combination with orni thine via orni thine transcarbamoylase. Aspartate donates the second ni trogen(3). Other amino acids contribute amino groups after convers ion into glutamate or aspartate. [Adapted with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.] B. α-ketoglutarate/Glutamate/Glutamine Ammonia BufferingSystem. The interconvers ion of α-ketoglutarate to/from glutamate to/from glutamine offers the body an important system to uti l i ze, generate,and/or s tore ammonia .

Fina l ly, the body a lso rel ies on an ammonia buffering system of α-ketoglutarate, glutamate, and glutamine for removal , production, and/orstorage of ammonia groups (Figure 5-9B).

Regulation of the urea cycle i s via the enzyme carbamoyl phosphate synthetase I (CPS-I), located within mitochondria . This enzyme cata lyzesthe reaction that converts ammonia from glutamate into carbamoyl phosphate, which then combines with orni thine to form ci trul l ine. CPS i sregulated by increased concentrations of N-acetylglutamic acid, produced from the combination of acetyl -CoA and glutamate, as wel l asincreased concentration of arginine. Both of these amino acids are di rect or indi rect participants in the urea cycle, and increasedconcentrations of ei ther reflect higher amino acid concentrations .

REVIEW QUESTIONS1. What i s an enzyme and how do the terms cata lyze, substrate, and product relate to enzymes?2. What i s s imple feedback and a l losteric regulation of enzymes?3. What i s meant by amino acid synthes is and amino acid degradation?4. What are the relationships between amino acid synthes is and degradation and other metabol ic pathways?5. For each bas ic enzyme reaction what i s the role of cofactors and regulation?6. What i s the function of the urea cycle and how would you diagram this pathway?

CHAPTER 6CARBOHYDRATE METABOLISM

Glycolys isCi tric Acid CycleOxidative PhosphorylationGluconeogenes isThe Pentose Phosphate PathwayGlycogen Synthes isGlycogen BreakdownModified Carbohydrates (Glycoproteins , Gags)Review Questions

OVERVIEWThe breakdown (catabolism) and synthes is (anabolism) of carbohydrate molecules represent the primary means for the human body to s tore anduti l i ze energy and to provide bui lding blocks for molecules such as nucleotides (Figure 6-1). The enzyme reactions that form the metabol icpathways for monosaccharide carbohydrates (Chapter 2) include glycolysis, the citric acid cycle, and oxidative phosphorylation as the main meansto produce the energy molecule adenos ine triphosphate (ATP). Gluconeogenesis and the pentose phosphate pathway represent the two mainanabol ic pathways to produce new carbohydrate molecules . Glycogen has i ts own metabol ic pathway for lengthening, shortening, and/oradding branch points in the carbohydrate cha in(s ). Not surpris ingly, a l l of these processes are highly regulated at multiple points to a l low thehuman body to efficiently uti l i ze these important biomolecules . Fina l ly, many modi fied carbohydrates are part of a variety of surface andcytosol ic s igna l ing molecules , including glycoproteins and glycosaminoglycans (GAGs) (Chapter 2). These important carbohydrate moleculesand the control points in carbohydrate and glycoprotein metabol i sm, therefore, present cl inicians with opportuni ties to modi fy these manyreactions to improve heal th or to fight disease.

Figure 6-1. Overview of Carbohydrate Metabolism. Glucose from the diet can be metabol i zed via glycolys is or glycogenes is . Resul ting metabol ic

products can return to glucose via gluconeogenes is or glycogenolys is , respectively, or proceed further a long carbohydrate metabol i sm to theci tric acid cycle. Al ternatively, glucose products can be shunted off to fat or amino acid metabol i sm as indicated. Deta i l s are discussed in thetext and other chapters . [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

GLYCOLYSISGlycolys is i s the metabol ic pathway that breaks down (catabol i sm) hexose (s ix-carbon) monosaccharides such as glucose, fructose, andgalactose into two molecules of pyruvate, two molecules of ATP, two molecules of NADH, two water (H2O) molecules , and two hydrogen ions

(H+) (Figure 6-2). Glycolys is involves 10 enzyme-mediated s teps and i s best envis ioned in two phases—phosphorylation and energy production—al l of which occur in the cytoplasm. The phosphorylation phase (sometimes referred to as the preparatory phase) s tarts with the s ix-carboncarbohydrate glucose and involves two phosphorylations from ATP and the cleavage into two molecules of the tri saccharide (three-carbonsugar) glycera ldehyde-3-phosphate. The energy production phase involves the next five s teps during which the two molecules ofglycera ldehyde-3-phosphate are converted to two pyruvate molecules with the production of two NADH molecules and four ATP molecules .Glucose-6-phosphate, the fi rs t intermediate of glycolys is , cannot exi t the cel l -l ike glucose, so i t a l so traps the glucose molecule in the cel l forenergy production via glycolys is or glycogen synthes is (see below). NADH represents an a l ternative energy s torage form than ATP, which maybe uti l i zed by the oxidative phosphorylation pathway.

Figure 6-2. Glycolysis. The pathway of glycolys is includes 10 enzyme steps , which break down one molecule of glucose to two molecules ofpyruvate, yielding two ATP and two NADH molecules . Pyruvate subsequently enters the mitochondria and the ci tric acid cycle (see below).Glycolys is can be divided into phosphorylation (Steps 1–5) and energy production (Steps 6–10) phases for better comprehens ion. Enzymes are

indicated by yel low-shaded boxes . [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P)Ltd., 2009.]

Not surpris ingly, a l l three regulatory s teps involve ei ther an investment or production of ATP molecules , a committing process in thebalance of energy production and s torage. The fi rs t reaction of glycolys is (cata lyzed by hexokinase) resul ts in the addition of a phosphate groupto form the molecule glucose-6-phosphate. In the l iver and pancreas , hexokinase i s replaced by the enzyme glucokinase. Phosphofructokinase-1adds a second phosphate group to produce fructose-1,6-bisphosphate. Pyruvate kinase, the fina l reaction in glycolys is , produces an ATPmolecule. Al though not regulated at the enzyme level such as the above enzymes , phosphoglycerate kinase i s involved in the ini tia l productionof ATP from glycolys is and represents the “break even” point when ATP invested in the preparation phase i s ga ined back. Al though regulatedin many di fferent ways and by many di fferent molecules , a theme emerges that, when energy s tores are low, glycolys is i s activated to producemore ATP. When energy levels are high, ei ther the glycolytic pathway i s inhibi ted or a l ternative pathways are promoted for s torage of theglucose molecule for later use. Regulation of carbohydrate metabol i sm and these important regulatory enzymes wi l l be discussed in furtherdeta i l in later chapters (e.g., Chapter 10).

Phosphofructokinase Deficiency: Because of i ts importance in energy production, diseases involving enzymes from glycolys is are rare. Oneexample, though, i s the deficiency of phosphofructokinase, a lso known as glycogen s torage disease type VII or Tarui’s disease. It i s inheri tedin an autosomal recess ive manner. This disease leads to a bui ldup of glycogen in cel l s and impairs various red blood and muscle cel l sfrom us ing glucose and many other monosaccharides to produce ATP. There are three di fferent variations of the disease, depending on theseveri ty of impairment. The fi rs t, termed “infanti le,” presents in the fi rs t year of l i fe usual ly s tarting with bl indness/cataracts andretardation with death by the age of 4 years . A second “class ic” variant presents in chi ldhood with muscle cramping and weakness afterexercise and death/breakdown of muscle cel l s as wel l as low red blood cel l count (anemia). The thi rd “late onset” variant presents in earlyadulthood with progress ive weakness of arms and legs but without muscle cramping/breakdown as wel l as anemia. Diagnos is i s by notinghigh levels of glycogen in muscle samples and measuring the activi ty of phosphofructokinase. The only treatment i s avoidance of highcarbohydrate meals and any vigorous exercise.

ATP i s the primary energy molecule used by the human body (Figure 6-3). Three enzymes involved in glycolys is , hexokinase,phosphofructokinase-1, and pyruvate kinase, are regulated to a l low the body to decide both whether to use i ts carbohydrate resources for ATPproduction and how much to produce. At each s tep, the cel l decides to continue energy production or to channel the resul ting intermediatesinto other molecules such as glycogen, l ipids , and/or proteins . The abi l i ty of the human body to sense i ts biochemica l surroundings and thenrespond to that envi ronment enables intel l igent and efficient use of i ts l imited resources .

Figure 6-3. ATP Structure with Its Magnesium Cofactor. ATP serves as the primary energy molecule of the human body uti l i zing the energy conta inedin the phosphate bonds . Magnes ium (Mg2+) s tabi l i zes these bonds and hence i s necessary for ATP to be biologica l ly functional . [Reproducedwith permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

CITRIC ACID CYCLEThe citric acid cycle i s the second metabol ic pathway involved in the catabol i sm of carbohydrates into energy and involves eight enzyme steps ,s tarting and ending with the molecule oxaloace-tate (Figure 6-4). In humans , a l l of the s teps of the ci tric acid cycle, including the l inkingreaction of pyruvate convers ion to acetyl -CoA, take place within the mitochondria l matrix, the area within the inner mitochondria l membrane.Pyruvate enters the ci tric acid cycle fol lowing i ts convers ion to acetyl -CoA; the reaction i s cata lyzed by pyruvate dehydrogenase that l inksglycolys is to the ci tric acid cycle and produces one molecule of NADH. By means of acetyl -CoA and intermediates in this cycle, the body canalso divert fats and amino acids into energy production (discussed later in this chapter). In addition, the ci tric acid cycle provides precursormolecules for the synthes is of amino acids and/or receives these amino acids when proteins are used for energy production (see Chapter 5,Figure 5-7).

Figure 6-4. Citric Acid Cycle. The ci tric acid cycle includes eight enzymatic s teps (tan-shaded boxes), which receive pyruvate from glycolys is via theacetyl -CoA molecule. The cycle produces one GTP, three NADH, and one FADH2 as i t progresses to oxa loacetate at which point the cyclerepeats . [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Acetyl-CoA, a two-carbon molecule bonds to the four-carbon molecule oxa loacetate to form ci trate, the namesake of this pathway. Thispathway i s a lso known as the tricarboxyl ic acid cycle, because ci trate conta ins three carboxyl ic acid groups , or the Krebs cycle after thephys ician/biochemist Si r Hans Krebs , who earned a Nobel Prize for his work on elucidating this sequence of reactions . Further enzymereactions remove carbon molecules in the form of carbon dioxide (CO2) and H2O. By doing so, the cycle produces three molecules of NADH, onemolecule of FADH2, and one molecule of guanos ine triphosphate (GTP), a l l of which may be used in energy production and s torage byoxidative phosphorylation (see below) and convers ion to ATP.

The regulation of the ci tric acid cycle i s mainly via the ava i labi l i ty of each enzyme’s substrate as wel l as inhibi tion when the concentrationof any enzyme’s product gets too high. One prime example i s ci trate, the product of the fi rs t reaction, which inhibi ts not only the cycle but a lsophosphofructokinase activi ty in glycolys is . That ci trate i s the fi rs t intermediate of the cycle and i s important in the control of the body’scarbohydrate resources . As with glycolys is , the same genera l rules a lso apply: i f energy levels (in this case NADH or FADH2, which areconverted to ATP via the later oxidative phosphorylation pathway) are high, the ci tric acid cycle i s s lowed or intermediates are diverted toother purposes .

Red Blood Cells (Erythrocytes) and Glycolysis: Red blood cel l s are unique in the human body in that they lose their nuclei and organel les ,including mitochondria , early in thei r development (Chapter 14). As a resul t, red blood cel l s are unable to uti l i ze the ci tric acid cycle oroxidative phosphorylation and are solely rel iant on ATP production from glycolys is . NADH produced from glycolys is i s used to producelactate from pyruvate; the resul ting lactate di ffuses from the red blood cel l s and i s transported into and used by ti ssues such as heartmuscle and l iver. In addition, red blood cel l s do not respond to insul in l ike other ti s sues in the human body and are, therefore, notregulated by this important hormonal s igna l that controls carbohydrate metabol i sm.

OXIDATIVE PHOSPHORYLATIONThe thi rd and fina l pathway of convers ion of carbohydrates to energy i s oxidative phosphorylation (Figure 6-5). This metabol ic pathway takesplace exclus ively ins ide the inner mitochondria l membrane, taking advantage of di fferences in concentration of the products of the ci tric acidcycle to drive the formation of ATP.

Figure 6-5. Overview of Oxidative Phosphorylation. Electrons are accepted from NADH (Complex I), succinate (Complex II), reduced Q protein viaComplex II-generated FADH2 (Complex II I), and from cytochrome c (Complex IV). The resul ting excess of hydrogen drives ATPase synthase(Complex V) to produce ATP. The process uses molecular oxygen (O2) and the fina l product of this series of reactions i s water (H2O). [Adaptedwith permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Speci fica l ly, oxidative phosphorylation includes both the respiratory cha in (i .e., oxidative) and ATP synthes is via ATP synthase [i .e.,phosphorylation of adenos ine diphosphate (ADP)]. The respiratory cha in portion involves four complexes (complexes I–IV) and a Q proteinthat help to transport H+ from the ins ide of the mitochondria to the intermembrane space between the inner and outer membranes . These H+

ions can then readi ly di ffuse to the cytoplasm so that the pH in the intermembrane space and the cytoplasm become equiva lent. Complex II i sactua l ly the same enzyme seen in the ci tric acid cycle (succinate dehydrogenase; Figure 6-4) and i s respons ible for the generation of oneFADH2 and a molecule of fumarate for each molecule of succinate. Complex I accepts electrons from NADH, complex II accepts electrons fromsuccinate, and complex III accepts electrons from reduced Q protein that i tsel f receives electrons from FADH2 generated from complex II . ComplexIV accepts electrons from cytochrome c molecules (see Figure 6-5). The process uses molecular oxygen (O2) and the fina l product of this seriesof reactions i s H2O.

The four complexes are thought to be associated into a “super” complex, a l lowing the H+ to be channeled from one complex to another; i fthis concept i s correct, oxidative phosphorylation i l lus trates how association of severa l enzymes together can increase their efficiency, atheme that wi l l be repeated severa l times in human biochemistry. ATP synthase (a lso ca l led complex V) then transports the H+ back throughthe inner membrane into the matrix, uti l i zing the energy from the H+ concentration gradient to form ATP (Figure 6-5).

Toxins and Poisons—Blocking Oxidative Phosphorylation: Because oxidative phosphorylation represents a key s tep in the production ofbiologica l energy, i t i s a l so the target of various chemica l agents . Cyanide and carbon monoxide, both toxic substances , interfere withhemoglobin molecules but a lso speci fica l ly inhibi t complex IV, blocking the respiratory cha in. Tissues highly dependent on energyproduction (e.g., nervous and heart muscles ) are particularly affected, resul ting in headache, dizziness , seizures , coma, and cardiac arrestleading to death. Antidotes (e.g., ni tri tes and sodium thiosul fate) work by caus ing the release of complex IV from the cyanide moleculefol lowed by metabol i sm of the nontoxic product. Some insecticides (e.g., rotenone) a lso work via blocking speci fic complexes of oxidativephosphorylation.

ATP Synthase: The enzyme respons ible for production of ATP offers unique ins ights into protein conformational changes in a membrane-bound protein. ATP synthase i s composed of two subunits , a cyl indrica l -shaped F0 protein with a hol low centra l channel and a s ta lk andbal l -shaped F1 headpiece subunit (see the figure below). The enti re complex appears s imi lar to and, in fact, works much l ike an electrica l

generator with H+ replacing the flow of H2O. The biochemica l generator i s complete with a thi rd rod-shaped “stator” subunit, which holdsthe F0 and F1 subunits together (see the figure).

The F0 subunit i s composed of severa l smal ler proteins , with thei r hydrophi l i c amino acids on the interior and hydrophobic amino acids

adjacent to the membrane. When H+ flow back into the interior mitochondria l membrane space, they cause rotation of the F0 subunit within

the surrounding membrane. Studies indicate that the H+ may influence particular amino acid R-groups of the internal F0 s tructure to createthe rotation. The F1 headpiece s ta lk, connected di rectly to the F0 subunit, spins ins ide the s tationary external ba l l portion (held in place bythe s tator protein subunit), producing a conformational change that binds ADP, cata lyzes the reaction to form ATP, and then releases ATP. Atthe end of the rotating cycle, the ba l l -shaped subunit i s ready for a repeat round of ATP synthes is .*

[Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

*Controversy surrounds the actual number of ATP molecules generated by oxidative phosphorylation with suggestions ranging from two to three ATP per NADH (and numbers in between). FADH2 produces

only about two ATP molecules because of its later entry at complex III into oxidative phosphorylation. Some scientists feel that the number may actually change depending on cellular conditions. This book hasadopted the convention of exactly three ATP per NADH and exactly two ATP per FADH2.

NADH is often produced outs ide the mitochondrial matrix (e.g., glycolys is , convers ion of lactate to pyruvate) but cannot cross the innermitochondria l membrane for convers ion to ATP. To address this problem, the body uti l i zes two molecular “shuttle” mechanisms. The fi rs trel ies on the enzyme glycerol-3-phosphate dehydrogenase, which has both cytoplasmic and inner mitochondria l membrane i soforms. Thecytosol ic enzyme converts glycerol -3-phosphate into dihydroxyacetone phosphate by oxidizing NADH to NAD+. The mitochondria l enzymecaptures the energy by cata lyzing the oxidation of dihydroxyacetone phosphate via the convers ion of flavin adenine dinucleotide to FADH2.FADH2 then continues through the oxidative phosphorylation pathway as above to yield two ATP molecules . A more efficient shuttle involvesconvers ion of oxa loacetate to malate by reduction with NADH in the cytosol ; this reaction i s cata lyzed by an i sozyme of malate dehydrogenasefrom the ci tric acid cycle except in reverse (Figure 6-4). The malate i s transported into the mitochondria l matrix where the reverse reactiontakes place, resul ting in NADH that yields three ATP molecules . Malate i s ul timately converted to aspartate, which returns the carbons to thecytosol .

GLUCONEOGENESISGluconeogenesis, the creation of new glucose, cons is ts of 11 s teps (some s imply the reverse of reactions found in glycolys is ) and i s the fi rs tanabol ic or “synthetic” carbohydrate pathway to be discussed. The process takes place a lmost exclus ively in the l iver, a l though someproduction of new glucose occurs in the kidney. Other ti s sues (e.g., muscles ) conta in many of the gluconeogenic enzymes with the exception ofthe fina l s tep that actua l ly produces glucose. In muscles , these reactions a l low for the convers ion of pyruvate to glycogen.

Gluconeogenes is occurs when the body has low energy s tores (e.g., s tarvation, lengthy periods of exercise) and elects to divert non-carbohydrate molecules [e.g., lactic acid, glycerol , and amino acids (except lys ine and leucine)] into ATP molecules (see Chapter 10). Points ofentrance by these molecules are shown in Figure 6-6. Any of the intermediates of the ci tric acid cycle and molecules that feed into the cyclecan a lso be diverted to gluconeogenes is via oxa loacetate. The bas ic gluconeogenes is pathway (Figure 6-6) shows the convers ion of pyruvateto glucose. Referring back to the glycolys is pathway above (Figure 6-2), two pyruvate molecules are formed by the enzymatic breakdown of eachglucose molecule. Therefore, two pyruvate molecules are required by gluconeogenes is to form one glucose molecule.

Figure 6-6. Gluconeogenesis. The pathway of gluconeogenes is includes 11 enzymatic s teps , which form one molecule of glucose from twomolecules of pyruvate. Reactions and enzymes speci fic only to gluconeogenes is are shown in red. Irrevers ible reactions speci fic for glycolys isare shown in green. Additional substrates for gluconeogenes is are shown in blue and may a lso enter gluconeogenes is as denoted by theassociated arrows. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

However, gluconeogenes is i s not s imply a reversa l of glycolys is . Fi rs t, gluconeogenes is i s a far more energy-expens ive pathway, uti l i zingfour ATP (two at each of two s teps), two GTP, and two NADH (the equiva lent of three additional ATP molecules per NADH) per glucoseproduced; a s imple reversa l of glycolys is would use two fewer ATP and GTP molecules each. This energy investment does not include that usedby the substrate molecule to enter gluconeogenes is . Second, four instead of three di fferent enzymes are seen in gluconeogenes is at reactionsimportant to drive formation and regulation of glucose production. In order to drive the energetica l ly favorable glycolys is pathway in reverse,the convers ion of each pyruvate to phosphoenolpyruvate requires one ATP and one GTP molecule per pyruvate (i .e., four tota l high-energyphosphate molecules per glucose molecule produced) as wel l as two enzymes speci fic to the gluconeogenic pathway, the enzymes pyruvatecarboxylase and phosphoenolpyruvate carboxykinase. Thi rd, the next s teps to fructose-1,6-bisphosphate are glycolys is in reverse and, hence,

occur in the cytoplasm. Al though glycolys is generates NADH and ATP within this portion of the pathway, gluconeogenes is requires the netconsumption of two ATP and two NADH molecules per glucose molecule produced. Fourth, because gluconeogenes is cannot regenerate theATP used in the reverse di rection (phosphofructokinase-1) during glycolys is , the convers ion of fructose-1,6-bisphosphate back to fructose-6-phosphate requires a thi rd new gluconeogenic enzyme, fructose-1,6-bisphosphatase. This reaction i s driven by the energy from cleavage andrelease of an inorganic phosphate (Pi ). The next to las t s tep i s a s imple reversa l of the glycolys is reaction. Lastly, the fina l convers ion ofglucose-6-phosphate to glucose occurs in the lumen of the endoplasmic reticulum, with glucose being transported into the cytoplasm and,subsequently, into the blood to mainta in i ts concentration during s tarvation or s tress . Aga in, energy release of Pi drives the reaction forward.

As the body does not want to “waste” energy-converting molecules into glucose; gluconeogenes is i s highly regulated, in particular withrespect to glycolys is (see Chapter 10). Not surpris ingly, gluconeogenes is i s regulated at the exact points important for glycolys is but bydi fferent enzymes. Pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase replace theglycolys is enzymes pyruvate kinase, phosphofructokinase-1, and hexokinase or glucokinase. High acetyl -CoA and ATP, representative of high-energy s tores in the body, activate pyruvate carboxylase, the fi rs t s tep in gluconeogenes is , whereas the analogous glycolytic s tep, pyruvatekinase, the fina l s tep in glycolys is i s inhibi ted, insuring that only one pathway—glycolys is or gluconeogenes is—is functional at any one time.Primary regulation of gluconeogenes is in humans occurs at phosphoenolpyruvate carboxykinase via corti sol and glucagon effects on DNAtranscription levels of the enzyme. High concentrations of ci trate, evidence of plenti ful ATP production and carbon ava i labi l i ty, and lowamounts of fructose-2,6 bisphosphate cause increased activi ty of the enzyme, fructose-1,6-bisphosphatase, helping to regulategluconeogenes is in mammals ; in non-mammal species , this enzyme is , in fact, the primary regulator of gluconeogenes is . Further deta i l s ofregulation are discussed in Chapter 10.

Gluconeogenesis and Metformin: Most patients with type 2 diabetes mellitus have gluconeogenes is rates in the l iver three times that of personswithout this disease. Metformin, one of the fami ly of biguanide medications , works in the l iver by speci fica l ly inhibi ting gluconeogenes is .Al though severa l mechanisms of biguanide action may be at work, evidence points to an increase in cytosol ic adenos ine monophosphate,indicating to the body a low ATP concentration and prompting use of glucose via glycolys is . Metformin a lso increases the body’s sens i tivi tyto insul in, increases cel lular uptake of ci rculating glucose, the breakdown of fatty acids (a l l which promote glucose use instead of newproduction), and decreases glucose absorption by the intestines .

THE PENTOSE PHOSPHATE PATHWAYThe second “synthetic” carbohydrate pathway to be discussed i s the pentose phosphate pathway, sometimes referred to as the pentosephosphate shunt. This pathway takes place in the cytoplasm and produces five-carbon, pentose carbohydrate molecules for nucleotide andnucleic acid synthes is . It i s a l so the major pathway for the production of the important reducing molecule nicotinamide adenine dinucleotidephosphate (NADPH), a s imi lar molecule to NADH but with a very di fferent function. NADPH has an additional phosphate group on the carbon 2pos i tion of the adenine ribose molecule. It i s the primary molecule indi rectly respons ible for el iminating toxic oxygen “radica ls” (e.g.,hydrogen peroxide)—highly reactive oxygen molecules produced by some reactions in the body, which can cause s igni ficant damage to ti ssues .NADPH is a lso required for certa in reactions in the synthes is of nucleic acids (Chapter 3) as wel l as fatty acids , cholesterol and s teroids , andnucleic acids (Chapter 7), which require a reducing molecule.

The pentose phosphate pathway occurs in two phases . The fi rs t, ca l led the oxidative phase, produces NADPH and ribu-lose-5-phosphate, apentose sugar molecule (Figure 6-7A). The second, ca l led the nonoxidative phase, produces products for glycolys is (Figure 6-7B) or for nucleotidesynthes is (Figure 6-7C). Regulation of this pathway i s via NADPH concentration—when NADP+ concentration i s high, the oxidative pathway i saccelerated; when NADPH concentration i s high, the oxidative pathway i s inhibi ted. Normal concentrations of NADPH in the cytoplasm are 100times that of NADP, so the pathway wi l l only be turned on when NADPH stores are used.

Figure 6-7. A. Pentose Phosphate Pathway, Oxidative Phase. Glucose-6-phosphate i s converted into ribulose-5-phosphate with generation of twomolecules of NADPH and one molecule of CO2 (blue shading). Various cations (Mg2+, Mn2+, and Ca 2+) play roles as cofactors in these reactions .

The overa l l reaction for this process i s : glucose-6-phosphate + 2 NADP+ + H2O → ribulose-5-phosphate + 2 NADPH + 2 H+ + CO2. B–C. PentosePhosphate Pathway, Nonoxidative Phase. Ribulose-5-phosphate i s further converted to 3-, 4-, 5-, 6-, and 7-carbon carbohydrates , which can be usedfor glycolys is (B), or in a reversa l of reactions , glycolytic intermediates can be used for nucleotide synthes is (C). Enzymes involved in thereactions are indicated. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Glucose-6-phosphate Dehydrogenase (G6PDH) Deficiency: Abnormal ly low levels of G6PDH i s the most common inheri ted disease of enzymedeficiency, a ffecting an estimated 400 mi l l ion persons worldwide, mainly those of African, Middle Eastern, or South As ian descent. Thedisease i s inheri ted via the X chromosome in a recess ive manner and, therefore, a ffects males much more often than females . However,females with two affected X chromosomes suffer from an immune disorder ca l led chronic granulomatous disease, which blocks the action ofimmune cel l s that uti l i ze the oxygen radica ls to ki l l bacteria , often leading to death due to infection. Multiple mutations can affect G6PDHactivi ty with five di fferent classes of the disease cl inica l ly defined.

This pathway i s the only way that red blood cel l s may el iminate reactive oxygen molecules , which can serious ly damage the red bloodcel l membrane and cel l wal l and lead to red blood cel l death. As a resul t, any problems with the pentose phosphate pathway, includingG6PDH deficiency, can be detrimenta l to red blood cel l s and the body. Al though most individuals with G6PDH deficiency are asymptomatic,severely affected patients display symptoms of red blood cel l breakdown resul ting in l iver and kidney problems. In newborn babies , thismay resul t in damage to the bra in, termed kernicterus, and death.

Diagnos is i s by s igns of red blood cel l breakdown and l iver problems and i s confi rmed by a di rect assay of G6PDH activi ty. Individualsdiagnosed with the deficiency must avoid use of a large number of sulfa drugs, including various antibiotics ; a burn medication; severa lanticonvulsants ; thiazide and loop diuretics (used to treat high blood pressure), a class of medications used to treat diabetes ; someglaucoma medications ; certa in pa in medications ; and, fina l ly, severa l antimalaria l medications . Treatment of G6PDH deficiency i savoidance of agents , which cause red blood cel l destruction and, in some cases , blood transfus ions and/or removal of the spleen, theorgan respons ible for removal of damaged and destroyed red blood cel l s .

However, one advantage of moderate G6PDH deficiency has been noted in res is tance to malaria l infection by Plasmodium falciparum. Incerta in individuals , usual ly of African or Mediterranean descent, a decreased level of G6PDH activi ty leads to weakening of the red bloodcel l membrane, which does not affect the person but does s top reproduction and further infection by the malaria paras i te. Persons withs ickle cel l disease a lso enjoy a heightened res is tance to malaria infections . A suggested mechanism is the selected clearance by thespleen of the red blood cel l s partia l ly damaged by the malaria l paras i te, whereas uninfected red blood cel l s are preserved.

Arrows indicate ring phase of Plasmodium falciparum in infected cel l s .[Reproduced with permiss ion from Chamberla in NR: The Big Picture: Medica l Microbiology, 1st edi tion, McGraw-Hi l l , 2009.]

GLYCOGEN SYNTHESISStep 1 of glycolys is , production of glucose-6-phosphate cata lyzed by hexokinase, involves an investment of one ATP molecule and i s , therefore, acommitting s tep toward uti l i zation of the glucose molecule. If energy (ATP) s tores are high, the body may decide to s tore the glucose moleculein the form of glycogen (Chapters 2 and 10; Figure 6-8). The fi rs t s tep of this process i s the convers ion of glucose-6-phosphate to glucose-1-phosphate. Glucose-1-phosphate reacts with the nucleotide uridine triphosphate (one of the nucleotides in RNA) to form uridine diphosphate

glucose (UDP glucose), the reaction depends on the energy released from the two phosphate bonds hydrolyzed during this reaction (Figure 6-8A). Glucose from UDP glucose i s then attached to a preexis ting glycogen molecule (Figure 6-8B), which includes a protein ca l led glycogenin asthe core of the growing glycogen chain. The new bond i s formed between carbon 1 of the glycogen molecule and carbon 4 of the new glucose. Aseparate “branching” enzyme forms bonds between carbons 1 and 6, approximately every 10 glucose molecules .

Figure 6-8. A–B. Glycogen Synthesis. A. The synthes is of glycogen begins with the convers ion of glucose to glucose-1-phosphate and subsequentbonding to uridine triphosphate (UTP) to form uridine diphosphate–glucose (UDP glucose) and two phosphate groups . B. UDP glucose servesas the source for the addition of new glucose molecules to an exis ting glycogen molecule ei ther via a 1,4-bond or a 1,6-bond with a resul tinguridine monophosphate (UMP) molecule. Enzymes are shown in blue-shaded boxes . [Adapted with permiss ion from Naik P: Biochemistry, 3rdedition, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Glycosyltransferase Inhibitors: The enzymes that l ink carbohydrate molecules together to form glycogen and s imi lar complex sugar moleculesare col lectively known as glycosyltransferases. This fami ly of enzymes i s essentia l to the efficient s torage of carbohydrate energy, as wel l asthe synthes is of important s tructura l proteins . As a resul t, inhibi tors of glycosyl transferases have been developed for many medica l andother uses . The drug caspofungin ki l l s fungi by inhibi ting bonds between carbons 1 and 3 of glucose molecules in fungal cel l wal l s , leadingto their death. Ethambutol blocks growth of the bacteria that cause tuberculos is by blocking carbon 5 on the carbohydrates of the bacteria ’scel l wal l . Other glycosyl transferase inhibi tors are a lso being developed as agents aga inst cancer and vi ra l infections , including aga inst thehuman immunodeficiency vi rus (HIV).

GLYCOGEN BREAKDOWNGlycogenolysis, the breakdown of glycogen to release glucose, i s an important pathway in energy use and reactions that uti l i ze carbohydrates .Glucose-6-phosphate i s the entry point of sugars l iberated from glycogen catabol i sm into glycolys is , thus bypass ing the fi rs t regulatory s tep ofhexokinase. Glucose from the digestion of s tarch molecules , a major part of the human diet, a l so enters glycolys is at this point.

Glycogenolys is involves three s teps (Figure 6-9). Fi rs t, the repeti tive removal of glucose res idues by phosphorylase breaking bonds betweencarbons 1 and 4, producing a glucose-1-phosphate molecule. However, this process s tops when four glucose res idues are left before thecarbons 1 and 6 branch points . Second, a glucan transferase enzyme moves three res idues to the l inear part of the glycogen chain, leaving onlythe fina l branched glucose res idue. Thi rd, a debranching enzyme cleaves the carbons 1 and 6 bond. A fina l enzyme converts the resul tingglucose-1-phosphate molecules into glucose-6-phosphate (not shown) for entry into glycolys is .

Figure 6-9. Glycogen Breakdown. The breakdown of glycogen proceeds by glycogen phosphorylase cleavage of the α 1,4-bonds unti l four res iduesremain before the α 1,6-bond. Glucan transferase then moves three res idues to the other cha in. Fina l ly, the remaining 1,6-bound glucose i sreleased as free glucose by a debranching enzyme. Resul ting glucose-1-phosphate molecules are subsequently converted to glucose-6-phosphate for reentry into glycolys is (not shown). [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

Removing glucose res idues from the l inear cha in and branch point res idues continues as long as the body requires them. The process i scontrol led by the addition of a phosphate to (leading to activation) or removal of a phosphate from (leading to inhibi tion) the fi rs t enzyme inthe process . Phosphorylation of this enzyme is , in turn, under the control of the hormones epinephrine and glucagon, both of which promoteactivation of glycogenolys is to produce new glucose in l iver. The l iver i s the primary organ involved in glucose uti l i zation or s torage for thebody (see Chapters 10 and 11 for further discuss ion). As a resul t, the l iver conta ins an enzyme, glucose-6-phosphatase, which removes thephosphate from glucose, a l lowing i t to leave the l iver cel l and be transported to other ti s sues . Muscles , too, can s tore glucose in the form ofglycogen but do not conta in glucose-6-phosphatase. Glucose released during glycogenolys is i s , therefore, used by the muscle cel l s under

control by epinephrine but not by glucagon.

Glycogenolysis Inhibitors: As with glycosyl transferases , the enzymes respons ible for breakdown of glycogen and other complex carbohydratemolecules are a lso the targets of severa l medications . Acarbose and miglitol, two medications used in the treatment of diabetes , inhibi t therelease of new glucose res idues in the smal l intestine and pancreas , thereby reducing the ava i labi l i ty of these glucose molecules to thebody. Zanamivir (trade name Relenza) and oseltamivir (trade name Tamiflu) are both inhibi tors of a reaction involving cleavage ofcarbohydrate bonds that a l lows new influenza A and B vi rus particles to be released from an infected cel l . As a resul t of these medications ,the new vi ra l particles are trapped and soon die. Because of the mechanism involved, these medications must be taken at the verybeginning of a suspected influenza infection before repl ication and release of new vi ruses takes place.

Because of the importance to the proper use of carbohydrate and other energy sources , defects in the pathways of glycogen synthes is andbreakdown within the l iver and muscles , col lectively known as glycogen storage diseases (GSDs), can cause major medica l problems. As furtherresearch has identi fied speci fic enzyme defici ts , severa l GSDs have been combined. Ten GSDs have been identi fied and are l i s ted in Table 6-1.

TABLE 6-1. Glycogen Storage Diseases

MODIFIED CARBOHYDRATES (GLYCOPROTEINS, GAGS)Glycoproteins and GAGs, discussed in Chapter 2, are proteins with a cha in of carbohydrates , known as an oligosaccharide, a ttached to a coreprotein after protein synthes is . These carbohydrate molecules can be attached to the amino (NH3) group of an asparagine amino acid (knownas “N-glycosylation”) or to the hydroxyl (OH) group of amino acids serine, threonine, or specia l ly modi fied hydroxylys ine or hydroxyprol ineres idues (known as “O-glycosylation”). Enzymes from the fami ly of glycosyl transferases , a l so seen in glycogen synthes is (see above), cata lyzethe additions of carbohydrates to the protein molecules . Only eight carbohydrate molecules are usual ly seen in glycoproteins—glucose,

galactose, mannose, fructose, xylose, N-acetylga lactosamine, N-acetylglucosamine, and N-acetylneuraminic acid. GAG carbohydrates can varywidely but usual ly include ga lactose, glucosamine, glucuronic acid, and/or iduronic acid. Speci fic glycosyl transferases are respons ible foraddition of a particular carbohydrate molecule to a speci fic amino acid res idue or to the growing ol igosaccharide cha in.

The resul ting glycoproteins/GAGs perform important functions both in the cytoplasm and in the membrane (glycosylated portions of theprotein usual ly point out into the extracel lular envi ronment), the latter i s important as cel l–cel l s igna l ing molecules . Functions of GAGs arel i s ted in Table 2-2; glycoprotein functions are reviewed in Table 6-2.

TABLE 6-2. Biochemica l Roles of Glycoproteins

Glucosamine/Chondroitin and Osteoarthritis: Certa in GAGs serve s tructura l roles in joint carti lage, poss ibly acting as molecular “shockabsorbers .” Osteoarthritis i s a disease characterized by decreased carti lage in weight-bearing joints such as knees , hips , and back due to“wear-and-tear” on these joints . One therapy for osteoarthri ti s involves the ora l replacement of glucosamine and chondroitin by tablets .Al though evidence i s lacking for true effectiveness , s tudies have shown that these tablets pose no heal th threat to patients and many notemarked improvement in osteoarthri ti s pa in.

REVIEW QUESTIONS1. What are the key features of glycolys is and gluconeogenes is and how are they s imi lar and di fferent?2. What are the key features of the ci tric acid cycle?3. What are the key features of oxidative phosphorylation and i ts relationship to the ci tric acid cycle and glycolys is?4. What are the key features of the pentose phosphate pathway?5. What are the key features of glycogen metabol i sm and how does i t relate to glycolys is and gluconeogenes is?6. How is carbohydrate metabol i sm regulated and how does this regulation relate to synthes is and degradation of the various carbohydrates

important to humans and choices between other metabol ic pathways?

CHAPTER 7LIPID METABOLISM

Fatty Acid Metabol i smMetabol i sm of Complex LipidsReview Questions

OVERVIEWLipids perform severa l essentia l functions , including forming biologica l membranes , efficient s torage of energy, and as components of severa limportant s tructura l and functional molecules . Lipid metabol i sm includes both the synthes is and degradation of fatty acids and/or morecomplex l ipid molecules . The choice between synthes is and degradation represents an important regulatory s tep in human biology andreflects the level of food and, therefore, energy s tores ava i lable to the body. Severa l processes are involved in this decis ion of producing fattyacids/ l ipids or, ins tead, di recting thei r precursors to energy production via carbohydrate metabol ic pathways . Separate but s ti l l dependent onthis process , the production of cholesterol and severa l l ipid-derived hormones and s ignal ing molecules i s essentia l for multiple functions inthe human body. As in most metabol ic pathways , deficiencies or problems can lead to serious disease s tates . As seen in other chapters ,understanding of the exact mechanism of these problematic metabol ic s teps a lso a l lows treatment of these diseases .

FATTY ACID METABOLISM

FATTY ACID SYNTHESIS

Although most fatty acids needed by humans are suppl ied in the diet, fatty acid synthes is plays a role in certa in ti s sues to convert any excessof sugar molecules into the more efficient s torage form of fatty acid/l ipid molecules and/or to produce specia l i zed l ipid molecules . The l inkbetween sugar and fatty acid/l ipid metabol i sm is seen at acetyl coenzyme A (CoA), the intermediate between glycolys is and the ci tric acidcycle. As wi l l be seen below, acetyl -CoA i s a lso the ini tia l molecule of fatty acid metabol i sm. Therefore, acetyl -CoA i s an important branchpoint of human metabol i sm and i ts use i s highly control led to reflect the body’s nutri tional s tate and needs . As an i l lus tration of this fact, thefi rs t and committing s tep of fatty acid synthes is i s the addition of a carboxyl (CO2

−) group, donated by HCO3−, to acetyl -CoA to produce malonyl-

CoA.

Al though the reaction i s s imple, the choice to commit the potentia l energy production from carbohydrates to produce l ipid moleculesreflects severa l important biologica l principles in human metabol i sm. Fi rs t, the enzyme that cata lyzes this reaction, acetyl-CoA carboxylase, i sproduced as smal ler, inactive protein complexes . When the concentration of citrate i s high (the fi rs t intermediate of the ci tric acid cycle and as ign of abundant sugar resources), these smal ler complexes join to form active enzymatic polymers (Figure 7-1). Therefore, ci trate increasesfatty acid synthes is when the body has plenti ful energy and needs to s tore this energy in an efficient fashion. If l ipid s tores are high, though,increased concentration of palmitoyl-CoA (the fina l product of fatty acid synthes is ) decreases fatty acid synthes is by depolymerizing the activeenzyme complex into the origina l smal ler, inactive complexes . The ba lance between ci trate and pa lmitoyl -CoA or, more s imply, a measure ofsugar versus fat levels (e.g., high sugar/fat indicates plenti ful food suppl ies ; low sugar/fat indicates low food and/or s tarvation conditions)controls the activi ty of this committing s tep and, therefore, uti l i zes ava i lable energy suppl ies most efficiently.

Figure 7-1. Regulation of the Acetyl-CoA Carboxylase and Production of Malonyl-CoA by Hormones. Pa lmitate and ci trate regulate malonyl -CoAproduction a l losterica l ly by enhancing the formation of the low-activi ty monomer or the highly active polymer form of acetyl -CoA carboxylase,

respectively. Both forms are unphosphorylated . Insul in, epinephrine, and glucagon regulate acetyl -coA carboxylase activi ty by influencing

the phosphorylation (inactivation, ) or dephosphorylation (activation, ) of the monomer. [Adapted with permiss ion from Naik P:

Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

There i s a second, longer term, hormonal regulation that a l lows the human body even further control of energy sources . Glucagon, thehormone that promotes the breakdown of glycogen to glucose, and/or epinephrine, released in times of s tress when energy requirements arehigher, activate phosphor-ylation of acetyl -CoA carboxylase, caus ing inhibi tion of fatty acid synthes is (Figure 7-1). Al ternatively, insulin, thehormone that promotes uptake and s torage of sugars (Chapter 10), causes dephosphorylation, which increases production of malonyl -CoA and,therefore, fatty acid synthes is . Therefore, glucagon and epinephrine drive acetyl -CoA molecules away from l ipid s torage and toward theproduction of energy via the ci tric acid cycle. Additional ly, food deprivation or chronic high-fat diet decreases the amount of the enzyme thatproduces malonyl -CoA.

Fol lowing the committing s tep of malonyl -CoA production, fatty acid synthes is i s a relatively s imple pathway that takes place on amultienzyme–protein complex in the cytoplasm. This enzyme complex, ca l led fatty acid synthase, conta ins seven separate subunits , cata lyzingl inked enzymatic reactions , and an acyl carrier protein “arm,” which carries the growing fatty acid cha in through to pa lmitate, the 16-carbon-long fatty acid with s ingle carbon–carbon bonds . The overa l l reaction for production of one pa lmitate molecule, including the sevenadenos ine triphosphate (ATP) molecules to generate malonyl -CoA via acetyl -CoA carboxylase, i s as fol lows:

Other fatty acids are derived from palmitate by speci fic enzymatic reactions . Production of fatty acids with an odd number of carbon atomsrel ies on the substi tution of the three-carbon, propionyl-CoA, molecule for acetyl -CoA to ini tiate the fatty acid synthase reaction. The bas icprocess of fatty acid production, which rel ies on the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) to drive the reaction, i sshown in Figure 7-2. Simi lar to the malonyl -CoA reaction, the quanti ty of the enzyme complex i s pos i tively regulated by higher sugar levels andnegatively regulated by high fatty acid/ fat levels in the diet.

Figure 7-2. Fatty Acid Synthase. The human fatty acid synthase enzyme complex i s composed of two identica l monomers (I and II , “subunitdivis ion”), which are, themselves , spl i t into condens ing and reducing enzyme halves (“functional divis ion”). The enzymes respons ible for thegrowing fatty acid cha in are l ined up (a l though not in the l inear order of reactions), s tarting at ketoacyl synthase. As one reaction concludes ,the product i s carried to the next enzyme by an “acyl carrier protein” (ACP), a section of the enzyme complex, which can fl ip back and forthbetween the success ive enzyme reactions . Bui lding blocks of acyl or malonyl subunits are attached to the ACP by acetyl transacylase ormalonyl transacylase, respectively. Reducing enzymes creates the fina l carbon–carbon bond of the growing fatty acid cha in. The new fatty acidproduct ei ther returns to the s tart of the process or, once a 16-carbon, pa lmitate molecule i s achieved, i s released by thioesterase. The exacttertiary and quaternary s tructure of fatty acid synthase i s s ti l l in question but a l l models agree with the success ive enzymatic processes andthe role of the ACP. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Pa lmitate i s used to produce longer fatty acid cha ins by the addition of more two-carbon uni ts from malonyl -CoA as indicated by theequation below. This reaction mainly occurs in the endoplasmic reticulum by a four-s tep process including the enzyme fatty acid elongase wi thcontinued use of NADPH as a source of both energy and reducing power. Fatty acid elongation can a lso occur in the mitochondria (NADHdependent) and in selected ti ssues (see Sidebar) where particular fatty acid types are required.

Fina l ly, the production of carbon–carbon double bonds in fatty acids takes place via a desaturase enzyme, uti l i zing NADH, in theendoplasmic reticulum.

[Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.

[Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Lipid Synthesis in Selected Tissues: New synthes is of fatty acids in the human body takes place in l iver, adipose (fat) ti s sue, kidney, bra in, andthe mammary glands . The l iver i s intimately involved in mainta ining the ba lance of l ipids , including cholesterol (Chapter 11). Adiposetissue, commonly referred to as fat cel l s , plays a key role in the s torage of l ipids in the form of triacylglycerol molecules . Triacylglycerol canstore 9 ki loca lories of energy per gram (kca l/g) versus only 4 kca l/g for carbohydrates, making triacylglycerol the most efficient form of energystorage in the body. Fatty acid, triacylglycerol , and cholesterol syntheses are a lso found in kidneys , and increased levels of l ipids are seenin various kidney diseases (Chapter 18). The bra in i s one of only a few ti ssues that use ketone bodies (reviewed below) as an energysource. Because the bra in uses approximately 25% of the body’s tota l energy production, this a l ternative energy source to carbohydratesmost certa inly i l lus trates an essentia l adaptation for surviva l during periods of reduced food intake. The bra in a lso synthes izes l ipidmolecules essentia l in the growth and maturation of the nervous system from infancy to adulthood. Lack of these synthetic functions leadsto a number of neurologica l diseases . Mammary glands a lso respond during pregnancy by producing triacylglycerol conta ining medium-chain fatty acids found in human mi lk. These l ipids , which make up 3%–5% of human breast mi lk, are essentia l for the development of thenewborn.

Essential Unsaturated Fatty Acids: Humans cannot produce desaturated bonds beyond the ninth and tenth carbons and, therefore, need toacquire fatty acids with more dis ta l double bonds from dietary sources (Chapter 3). These essentia l , unsaturated fatty acids are required forproduction of molecules such as prostaglandins (important in inflammatory reactions , various aspects of pregnancy, the spread of somecancers , control of blood flow to the kidney, regulation of ion flow across membranes , the conduction of nerve impulses , and modulation ofs leep), thromboxanes (required for blood clotting and poss ibly involved in constriction of blood vessels , e.g., Prinzmeta l ’s angina), andleukotrienes (important molecules in immune reactions including asthmatic and a l lergic reactions as wel l as certa in cardiovascular andneuropsychiatric diseases).

FATTY ACID DEGRADATION

The process of the breakdown or degradation of fatty acids s tarts in the cytoplasm with the l inkage of a fatty acid to CoA forming a fatty acyl-CoA molecule. Fol lowing this l inkage, fatty acids less than 12 carbons in length cross the mitochondria l membranes without ass is tance.However, fatty acids longer than 12 carbons are transported into the mitochondria via a unique process involving the molecule carni tine. Inthe fi rs t of three s teps , the fatty acyl -CoA l inks to carni tine by the enzyme carnitine palmitoyltransferase I (CPT I), located on the outermitochondria l membrane (Figure 7-3). The new molecule moves across the mitochondria l inner membrane via a speci fic membrane proteinca l led trans locase. Ins ide the mitochondria , CPT II, located on the ins ide of the inner mitochondria l membrane (Figure 7-3), removes thecarni tine molecule. This carni tine molecule moving out of the mitochondria exchanges with a new molecule of pa lmitoylcarni tine moving intothe mitochondria . High concentration of malonyl -CoA, the molecule representing commitment to the synthes is of fatty acids , prevents thetransport of fatty acids into mitochondria by inhibi ting CPT I. Increased amounts of fatty acyl -CoA reverse this inhibi tion and s timulates fattyacid degradation.

Figure 7-3. Transport of Fatty Acids Longer than 12 Carbons Cross the Mitochondrial Membrane Using Carnitine Palmitoyltransferase I and II. [Adapted withpermiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

CPT Diseases: Not surpris ingly, deficiencies in CPT I or II lead to severe disease s tates . CPT I deficiency i s an autosomal recess ive disorderthat usual ly s trikes in infancy or early chi ldhood and causes low sugar, low ketone levels , and increased ammonia levels in associationwith fasting or i l lness (i .e., when the body attempts to use fatty acids as an energy source). Patients with CPT I deficiency can developdamaged and enlarged l ivers (partly due to the bui ldup of fatty acids ), kidney problems, muscle breakdown, seizures , coma, and impairedgrowth. Without a diet of only medium-chain triglycerides (whose fatty acids can cross the mitochondria l membranes independent ofcarni tine) and the avoidance of fas ting, death i s common.

CPT II deficiency occurs in three di fferent forms and i s the most common inheri ted disorder of mitochondria l long-chain fatty acidoxidation. Inheri tance can be autosomal recess ive or due to multiple forms of heterozygous mutations that affect CPT II activi ty. Overa l l , CPTII deficiency occurs more often in males than in females . The three forms include a letha l neonata l form, a severe infanti lehepatocardiomuscular form, and a mi lder form, which may occur from infancy to adulthood. The fi rs t two forms cause low sugar, low ketonelevels , l i ver fa i lure, heart damage, fatigue, seizures , and, often, early death. The second and thi rd forms involve breakdown of muscleduring periods of exercise, fas ting, or infection; reddish-brown urine usual ly resul ts from the resul ting muscle breakdown products .Treatment of CPT II deficiency includes a high-carbohydrate/low-fat diet to encourage only carbohydrate metabol i sm (including infus ion ofglucose during infections , use of medium-chain fatty acids in the diet, mainta ining constant feeding so as to avoid fasting s tates , andrestricting lengthy periods of exercise), adequate hydration to help flush breakdown products through the kidneys without damage, and theaddition of carni tine to the diet to convert potentia l ly toxic, long-chain fatty acids to molecules , which can be el iminated via othermetabol ic pathways .

Once ins ide the mitochondria , fatty acid breakdown continues in a four-s tep process ca l led the β-oxidation cycle, which removes two carbonsfrom the fatty acid with each cycle (Figure 7-4). The fi rs t s tep involves the acyl-CoA dehydrogenase enzyme, which removes electrons from the twoend carbons to form a carbon–carbon double bond. The electrons are transferred to a flavin adenine dinucleotide (FAD) cofactor moleculeassociated with the enzyme that later produces ATP via the electron transport cha in. In the second s tep, enoyl-CoA hydratase resaturates thedouble bond with hydrogen (H+) and a hydroxyl (OH−) group from a water molecule. Next, the bond i s oxidized to a ketone by L-3-hydroxyacyl-CoAdehydrogenase, wi th the transfer of another electron to the cofactor NAD+, which later produces ATP via the electron transport cha in. In thefourth and fina l s tep, the potentia l ly reactive ketone and another CoA molecule react via the enzyme β-ketothiolase (sometimes ca l led acetyl -CoA acyl transferase) to form a two-carbon acetyl -CoA fragment and a new fatty acyl -CoA that i s now two carbons shorter. The acetyl -CoAfragment i s then used for other metabol ic pathways for energy production or synthes is of new molecules . The four-s tep cycle repeats unti l theenti re fatty acid cha in i s degraded into acetyl -CoA uni ts . In the fina l s tep for an even-chain fatty acid, two acetyl -CoA molecules are formedfrom the remaining four-carbon fragment.

Figure 7-4. Fatty Acid Degradation (β-Oxidation, see the text for details). [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, JaypeeBrothers Medica l Publ i shers (P) Ltd., 2009.]

Fatty acid cha ins with an odd number of carbon atoms are broken down s imi larly (Figure 7-5), a l though the fina l cycle produces a three-carbon propionyl -CoA molecule and an acetyl -CoA molecule. A carboxyl (CO2

−) group i s then added to the propionyl -CoA molecule in a two-stepprocess to produce succinyl -CoA, one of the major molecules of the ci tric acid cycle (Chapter 6).

Figure 7-5. Odd Number Fatty Acid Degradation (β-Oxidation, see the text for details). [Adapted with permiss ion from Naik P: Biochemistry, 3rdedition, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Mitochondrial Trifunctional Protein (MTP): Three of the enzymes involved in β-oxidation, namely 2,3-enoyl-CoA hydrase, 3-hydroxyacyl-CoAdehydrogenase, and 3-ketoacyl-CoA thiolase (see ins ide box in figure), are part of the MTP. This enzyme complex cata lyzes the fina l three s tepsof fatty acid degradation (see the figure below).

[Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

MTP deficiency resul ts when a l l three enzyme activi ties are deficient. Severa l mutations have been found to cause this disease, whichstops the convers ion of l ipids/fats to energy. Symptoms include low sugar, progress ive destruction of nerves in the l imbs and digi ts ,breakdown of muscle ti s sue, and l iver or heart damage, which can resul t in early death.

Mutations a lso affect i solated enzyme activi ties of the MTP complex. Low levels of the fi rs t enzyme enoyl -CoA hydrase resul t inabnormal ly smal l body, arms, fingers , and head. Deficiencies of the second enzyme 3-hydroxyacyl -CoA dehydrogenase actua l ly involve twoseparate enzymes , which degrade fatty acids of a particular length. Medium- and short-cha in 3-hydroxyacyl -CoA dehydrogenase deficiency orlong-chain 3-hydroxyacyl -CoA dehydrogenase (LCHAD) deficiency involves loss of β-oxidation of medium- and short-cha in fatty acids or long-chain fatty acids , respectively. As a resul t, the body cannot convert these particular fatty acid cha ins to energy and increased levels bui ld upin particular ti s sues and organs caus ing damage. These diseases are present in infancy or later in l i fe. Symptoms of the infant diseaseinclude poor feeding, vomiting/diarrhea, fatigue, low blood sugar, muscle weakness , heart and lung problems, changes in the retina, coma,and death. The disease that presents in later l i fe i s usual ly less severe with symptoms including poor muscle tone and weakness ,increased breakdown of muscles , and problems with nerves in the arms and legs . Mothers carrying a fetus with LCHAD deficiency may a lsohave l iver disease as wel l as HELLP syndrome, which includes red blood cel l destruction (Hemolys is ), Elevated L i ver enzymes (indicative ofl iver injury), and Low Pla telets . Diseases involving i solated deficiencies of 3-ketoacyl -CoA thiolase have not been described.

Speci fic attacks of a l l these disease s tates can be brought on by decreased food intake and s tresses such as vi ra l infections . Therefore,treatment involves diet restrictions to avoid the affected fatty acid cha in(s ), addi tion of carni tine to the diet to promote a l ternativemetabol ic pathways , and the avoidance of fas ting and infective s tresses .

Degradation of unsaturated fatty acids with carbon–carbon double bonds depends on the exact conformation of the double bond. Because

part of the process of producing the acetyl -CoA fragment involves the creation of a double bond (Step 1), some unsaturated fatty acids cans imply be metabol i zed by the same enzymatic process . However, i f the double bond i s not in the correct conformation or at the wrong carbonfor the acyl -CoA dehydrogenase or the enoyl -CoA hydratase, two other enzymes provide ass is tance (Figure 7-6). If the double bond i s not in theproper conformation, enoyl-CoA isomerase converts i t to the correct s tructure, fol lowed by the normal reaction cata lyzed by enoyl-CoA hydratase. Ifthe double bond i s in the wrong location, 2,4-dienoyl-CoA reductase changes the pos i tion of the double bond uti l i zing NADPH and, i f needed,the enoyl -CoA i somerase insures i t i s the correct conformation.

Figure 7-6. Alteration of Carbon–Carbon Double Bonds. Enoyl -CoA i somerase changes a trans-double bond at carbon 3 to a cis-double bond at carbon2, and 2,4-dienoyl reductase changes the double bond at carbon 4 to a s ingle bond. These a l terations a l low normal fatty acid degradation byenoyl -CoA hydratase to continue. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd.,2009.]

Two points are worth emphas izing about the breakdown of fatty acids . Fi rs t, for every two-carbon acetyl -CoA fragment produced, one FADH2

and one NADH are produced, resul ting in the later production of two and three ATP molecules , respectively. Thus , one molecule of pa lmitateconverted to eight acetyl -CoA molecules can be used to produce 35 ATP molecules . Each acetyl -CoA molecule can then potentia l ly generate anadditional 12 ATP molecules via oxidation by the ci tric acid cycle. Second, this process i l lus trates the essentia l roles of FAD+ and NAD+ andtheir associated vi tamins riboflavin (vi tamin B2) and nicotinic acid (niacin) in the temporary s torage and subsequent transfer of biologica lenergy.

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD): The fi rs t enzymatic s tep in the breakdown of fatty acids i s actua l ly via activi ty of one ofthree separate enzymes , depending on the ini tia l length of the molecule. Long-chain acyl-CoA dehydrogenase (LCAD) helps to degrade fattyacids longer than 12 carbons . Medium-chain acyl-CoA dehydrogenase (MCAD) degrades fatty acids between 6 and 12 carbons in length. Fina l ly,short-chain acyl-CoA dehydrogenase (SCAD) helps to break down fatty acids shorter than s ix carbons .

MADD i s the generic term referring to the deficiency of any of the three enzymes (LCAD, MCAD, or SCAD) cata lyzing the fi rs t s tep in thedegradation of fatty acids . This i s an autosomal recess ive disease with a varying occurrence rate depending on the speci fic enzyme involved(MCAD deficiency i s the most common disorder of the three enzymes , occurring in approximately 1 in 17,000 people). An enzyme deficiencyleads to a ha l t in the breakdown of fatty acids and a requirement for energy production from carbohydrate s tores . In addition, ti s sues thatuti l i ze ketone bodies are unable to produce this a l ternative energy source. As a resul t, levels of sugar (hypoglycemia) and ketone(hypoketonuria) decrease and abnormal by-products of the “s tuck” fatty acid degradation increase. Some of these abnormal fatty acidsimpede normal ammonia metabol i sm, leading to toxic ammonia levels , and dramatica l ly decrease the body’s abi l i ty for gluconeogenes is—the production of new carbohydrate molecules from a l ternative sources such as amino acids .

Patients with MADD have a number of cl inica l symptoms including sweaty feet and s ta le breath odor (from the abnormal fatty acids ),nausea and vomiting, increased l iver s i ze (hepatomegaly) and l iver damage (sometimes resul ting in yel lowing of the skin a lso known asjaundice), dis tortions of the skul l and face (e.g., forehead, nose, l ips , and ears ), muscle weakness , breathing problems and lung damage,and defective kidneys . Death in infancy i s common.

Diseases of the Peroxisome: Peroxisomes are often-forgotten, intracel lular organel les that el iminate toxic substances , break down specia ltypes of fatty acids , synthes ize bi le acids and plasmalogen (an important type of phosphol ipid in the myel in membrane surrounding nervecel l s ), and perform posttrans lational process ing of proteins . Deficiencies in peroxisomes or thei r various functions lead to seriousdiseases . One such disorder, Zellweger syndrome, leads to decreased numbers of peroxisomes and/or deficiencies in any of a variety ofperoxisomal enzymes. The syndrome usual ly resul ts from mutations in genes coding for any of severa l peroxin proteins , which recognizespeci fic amino acid sequences on newly trans lated proteins and label them for transport to peroxisomes.

Two closely related diseases of peroxisome function, adrenoleukodystrophy (ALD) (a l so ca l led Addison–Schi lder Disease or Siemerl ing–Creutzfeldt Disease) and Refsum disease, combine to form a trio ca l led the “Zel lweger spectrum.” ALD i s caused by the deficiency of amembrane protein that transports VLCFA into the peroxisome. ALD i s usual ly recess ively inheri ted via the X chromosome and, therefore,s trikes only young males (symptoms usual ly become evident between the age of 4 years and 10 years ) at a rate of 1 in 20,000. However,women carrying a s ingle mutation may develop less serious symptoms in adulthood (ca l led adrenomyeloneuropathy). Refsum disease i scaused by a speci fic mutation on ei ther chromosome 6 or 10, which decreases the breakdown of phytanic acid, a 16-carbon fatty acid foundnormal ly in the human diet. Patients with Refsum disease usual ly present with symptoms in chi ldhood or early adolescence.

The three diseases of the Zel lweger spectrum share many s imi lar symptoms. Absent β-oxidation of VLCFA leads to damaging increases inlevels of 24- to 30-carbon fatty acids , which resul t in an enlarged l iver, jaundice, and intestina l bleeding. Without production ofplasmalogen, the insulating myel in membrane i s compromised, leading to progress ive bra in and nerve damage with seizures , loss ofvis ion and hearing, decreas ing muscle tone and s trength culminating in the inabi l i ty to move (aga in due to decreased production of themyel in sheath), and, in infants , poor or absent suckl ing or swal lowing abi l i ty. ALD patients often develop adrenal gland fa i lure secondaryto bui ldup of the VLCFAs in these organs . A variant of ALD predominately s trikes the spina l cord with symptoms including weakness andnumbness of the l imbs and problems with urination and defecation. Death usual ly occurs in chi ldhood or early adolescence due toproblems occurring in the affected organs .

Treatment for Zel lweger syndrome is mainly supportive, including prevention of infections ; however, death usual ly occurs before the fi rs tbi rthday. Some success in the treatment of ALD has been reported with bone marrow transplantation and a diet with low intake of VLCFAand inclus ion of Lorenzo’s oil, a mixture of 18- and 22-carbon triglycerides . Further research i s attempting to provide further support for thistreatment and to elucidate the mechanism of Lorenzo’s oi l . Fina l ly, patients with Refsum disease are mainta ined on a diet with nophytanic acid (found in beef, lamb, tuna, cod, and haddock); attempts to find a l ternative/natura l therapies are a lso ongoing.

Fina l ly, mitochondria are unable to degrade fatty acids greater than 22 carbons . In the case of very-long-chain fatty acids (VLCFA), breakdownoccurs in peroxisomes , organel les found in a l l eukaryotes that provide specia l i zed l ipid metabol i sm as wel l as process ing of toxicsubstances . Peroxisomes metabol i ze VLCFA down to an eight-carbon octanyl -CoA, which i s then further processed by mitochondria asdescribed above. Peroxisomal oxidation of fatty acids i s driven not by ATP but rather by the production of hydrogen peroxide (H2O2), a highlyenergized molecule, which i s converted to water and oxygen by the enzyme cata lase found only in peroxisomes. Simi larly, though, the fattyacid substrate i s transported into the peroxisome by a carni tine acyl transferase and the fina l s tep in the process i s via a s imi lar peroxisomalβ-ketothiolase.

METABOLISM OF COMPLEX LIPIDS

TRIACYLGLYCEROL SYNTHESIS

Triacylglycerols, commonly referred to as triglycerides , are the predominant s torage form of l ipids (Chapter 3, Figure 3-2B. In fact, the term “fat”is actua l ly a name for triacylglycerol s tores . As noted previous ly, triacylglycerol s tores 9 kca l/g versus only 4 kca l/g for carbohydrates . Synthes isof triacylglycerols mainly takes place on the smooth endoplasmic reticulum of the l iver but can a lso be generated in adipose (fat) cel l s .Regardless of the location of synthes is , the s tarting molecule i s glycerol -3-phosphate produced in l iver from glycerol s tores or in adipose cel l sfrom dihydroxyacetone phosphate, the product of the fourth s tep of glycolys is (Chapter 6). Triacylglycerol molecules form via a s imple series ofenzymatic reactions cata lyzed by a triacylglycerol synthase enzyme complex. This col lection of enzymes sequentia l ly attaches fatty acid cha ins toglycerol -3-phosphate to produce a triacylglycerol molecule. As a lways , the choice to divert s tored l iver glycerol or the potentia l energy fromcarbohydrate molecules depends on whether or not carbohydrate levels are high (fed) or low (fasting).

Once synthes ized, triacylglycerols produced in the l iver combine with cholesterol and phosphol ipids together with a l ipoprotein ca l ledapol ipoprotein B to form a complex referred to as very-low-density lipoprotein (VLDL), which i s released into the blood for transport to otherti ssues . VLDL plays an important role in triacylglycerol transport and low-dens i ty l ipoprotein (LDL) levels and i s a lso one of the l ipidsmeasured when assess ing a patient’s cholesterol and l ipid s tatus . VLDL wi l l be examined in much more deta i l in Chapters 11 and 16.

PHOSPHOGLYCERIDE SYNTHESIS

Phosphoglycerides are l ipid molecules with two fatty acid cha ins and a speci fic “head group,” which defines that particular phosphoglyceridemolecule. The various phosphoglycerides and their functions are discussed in Chapters 3 and 8. The synthes is of serine-, inos i tol -, andethanolamine-phosphoglycerides fol lows the same enzymatic pathway as triacylglycerols up to and including the addition of the second fattyacid molecule to form phosphatidic acid (Figure 7-7). Fol lowing this reaction, an activated cytidine diphosphoglycerol intermediate formed fromthe nucleotide cytidine triphosphate (CTP) binds at the phosphate group and then i s replaced by the serine or inos i tol head group (Figure 7-7A). Phosphatidyl ethanolamine i s formed by the loss of the COO− group from the serine (Figure 7-7B). Phosphatidyl chol ine i s from dietarysources by ATP-driven phosphorylation of the head group, attachment to CTP, and fina l attachment to the diacylglycerol molecule. This i s a l soa second and a l ternative synthetic pathway for phosphatidyl ethanolamine (Figure 7-7C).

Figure 7-7. A–C. Synthetic Pathways of Phosphatidyl Serine, Inositol, Ethanolamine, and Choline. Phosphatidyl serine and inos i tol are produced fromphosphatidic acid, uti l i zing cytidine triphosphate (CTP). Loss of a carbon dioxide molecule from phosphatidyl serine produces phosphatidylethanolamine. Phosphatidyl chol ine and ethanolamine synthes is a lso uti l i ze CTP fol lowing ATP phosphorylation of the chol ine or

ethanolamine head group. CDP, cytidine 5′-diphosphate. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

KETONE BODY SYNTHESIS

If carbohydrate levels are low, the human body wi l l often revert to an a l ternative source of energy in the form of ketone bodies . Ketone bodies,produced in the l iver from acetyl -CoA, are a col lective term for the molecules acetoacetate and D-3-hydroxy-butyrate, as wel l as acetone that i sproduced by the spontaneous degradation of acetoacetate (Figure 7-8A).

Figure 7-8. A. Ketone Body Synthesis. B. Conversion of Ketone Bodies to Acetyl-CoA for Subsequent Entrance into the Citric Acid Cycle. [Adapted withpermiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

The body can convert s tored ketone bodies back into oxidative metabol i sm by a series of enzymatic reactions that mimic the reverse ofketone body synthes is . Acetoacetate and D-3-hydroxybutyrate are produced by the l iver from acetyl uni ts . High concentrations especia l ly of D-3-hydroxybutyrate and, therefore, acetyl uni ts ava i lable for the ci tric acid cycle decrease this release of adipose-derived fatty acids . In theuti l i zation process , which occurs in ti s sues other than the l iver and red cel l s , ketone bodies are converted back to two acetyl -CoA molecules bythe addition of succinyl -CoA and then di rected into the ci tric acid cycle (Figure 7-8B). In times of low carbohydrate levels and s tarvation, thisseries of reactions i s used as the primary source of energy by the heart, kidneys , muscles , and bra in.

Ketone body synthes is/degradation i s regulated by the concentration of oxa loacetate, the las t molecule of the ci tric acid cycle (Chapter 6),which combines with acetyl -CoA to continue the process of production of energy from carbohydrates . Therefore, i f oxa loacetate concentrationis low, indicative of low carbohydrate levels , the body can di rect i ts resources to the production of new glucose molecules (gluconeogenes is ,Chapter 6) and ketone bodies to produce energy. At the hormonal level , glucagon and epinephrine a lso increase breakdown of triacylglycerolsand the production of fatty acids , acetyl -CoA, and, as a resul t, ketone bodies . The hormone corti sol has the same but longer las ting effects . Ifoxa loacetate concentration i s high, entry into the ci tric acid cycle wi l l increase because the acetyl uni ts wi l l condense with the oxa loacetate.Insul in has the oppos i te effect, promoting s torage of fatty acids in the form of triacylglycerols and inhibi ting ketone body synthes is ; the effectof insul in i s decreased or negated by high corti sol levels seen in Cushing’s syndrome. Not surpris ingly, high levels of ketone bodies are seenin forms of poorly treated diabetes (Chapter 10), low-cholesterol/high-fat diets , and fasting/starvation.

Cushing’s syndrome: Uncontrol led and excess ive production of the hormone corti sol by the adrenal glands , a condition known as Cushing’ssyndrome, i s caused by a tumor in the cortex portion of the adrenal glands . Excess ive use of some medications can cause a s imi lar conditionknown as hyperadrenocortici sm or hypercortici sm. Patients with these diseases exhibi t a “Cushingoid” appearance from increased anduncontrol led depos i tion of fat, resul ting in rapid weight ga in mainly in the trunk of the body, rounding of the face known as “moon face,” alarge fat pad in the back of the neck known as a “buffa lo hump,” and s tretch marks in the skin from the rapid weight increase. Patients mayalso experience other hormonal symptoms including excess sweating, male-pattern ha i r growth in females (hi rsutism), impotence,inferti l i ty, and cessation of menses and various psychiatric problems such as depress ion, anxiety, and psychos is . Treatment i s usual ly bysurgica l removal of the tumor. Interestingly, other mammals a lso often suffer from Cushing’s syndrome, including dogs , horses , and ferrets .

CERAMIDE/SPHINGOLIPIDS SYNTHESIS

Sphingosine i s the precursor molecule for ceramide, which replaces glycerol as the “backbone” of sphingol ipid molecules , includingcerebros ides , sul fatides , globos ides , and gangl ios ides (Chapter 3), essentia l in many neurologica l and muscle functions and in cel l–cel lrecognition and interactions . Sphingos ine i s produced from a pa lmitoyl -CoA and a serine molecule in a reaction that involves NADPH and FAD(Figure 3-4A), aga in showing the importance of these molecules and their respective vi tamins in l ipid production. Sphingos ine, in turn, l inks toa long-chain fatty acid activated by CoA to form the molecule ceramide. Ceramide i s then uti l i zed by severa l independent enzymatic reactions

to produce a variety of sphingol ipids molecules of varying function.Gangl ios ides are the most complex sphingol ipids , being bui l t by the success ive addition of carbohydrate molecules via speci fic

glycosyl transferase enzymes. These enzymes add glucose, ga lactose, and other modi fied sugars in a defined order and precise pos i tions(Figure 3-4F) to create one of more than 15 gangl ios ide molecules . The function of these molecules i s reviewed in Chapter 3 and wi l l bediscussed in more deta i l in later chapters .

Tay–Sachs disease: As discussed in Chapter 3, gangl io-s ides are found in highest concentration in cel l s of the nervous system and arenormal ly broken down ins ide lysosomes by orderly removal of the sugar res idues , partly by the action of the enzyme β-N-acetylhexosaminidase. When this enzyme is miss ing or deficient, gangl ios ide GM2 cannot be broken down and Tay–Sachs disease resul ts .Symptoms of weakness and delayed development of muscle ski l l s are usual ly evident before the age of 1 year with subsequent bl indnessand death, usual ly by the age of 3 years . Neurons of a ffected patients are l i tera l ly swol len with lysosomes fi l led with GM2. Tay–Sachsdisease occurs 100 times more often in Ashkenazi (Eastern European) Jews.

CHOLESTEROL SYNTHESIS

Although about ha l f of the body’s requirement for cholesterol i s usual ly obta ined from the diet, the remainder must be synthes ized. Anydeficiencies in this synthes is are, therefore, important in normal human phys iology and disease. The main location of cholesterol synthes isin humans i s the l iver/ intestina l tract. Cholesterol receives a l l of i ts carbon molecules from the two-carbon molecule acetyl -CoA, once aga inshowing the importance of this intermediate in carbohydrate and l ipid metabol i sm (Figure 7-9). The fi rs t s tep involves the formation of 3-hydroxy-3-methyl-glutaryl-CoA (3-HMG-CoA) from one molecule of acetyl -CoA, one molecule of acetoacetyl -CoA, and a water molecule.Interestingly, any 3-HMG-CoA made in the mitochondria i s used to produce ketone bodies (see above), whereas 3-HMG-CoA made in thecytoplasm forms cholesterol . 3-HMG-CoA is converted to the s ix-carbon mevalonate in a reaction cata lyzed by the HMG-CoA reductase enzyme.This s tep in cholesterol synthes is i s i rrevers ible and i s the committing s tep in the pathway. This s tep i s a lso the primary target of medica ltherapy for high cholesterol diseases (see Sidebar). The s ix-carbon mevalonate i s converted to the five-carbon isopentenyl pyrophosphate througha series of three reactions that involve two molecules of NADPH and one of ATP. Isopentenyl pyrophosphate molecules then join success ivelytogether to form a 10-carbon, 15-carbon (farnesyl pyrophosphate), and fina l ly a 30-carbon squalene molecule. This s tep aga in involves NADPH. Atthis point, the growing molecule i s s ti l l a l inear cha in of carbon molecules . For the fina l s tage in cholesterol synthes is , termed “cycl i zation,”three separate s teps uti l i ze NADPH and one atom of molecular oxygen (O2) to form new s ingle- and double-carbon bonds , remove three methyl

(CH3) groups , and create a hydroxyl (OH−) group (Figure 7-9). The fi rs t cycl ic intermediate i s lanosterol , and this i s converted to cholesterol via acomplex series of reactions .

Figure 7-9. Cholesterol Synthesis. Summary of s teps in the synthes is of cholesterol (top). The molecular s tructures of mevalonic acid and the fina lcholesterol molecule are shown in the bottom panel . [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica lPhys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Regulation of new cholesterol synthes is and, therefore, levels in the human body rely on HMG-CoA reductase, the enzyme cata lyzing the fi rs tand committing s tep of producing mevalonate (Figure 7-9). Speci fica l ly, the activi ty of this enzyme is decreased by high concentrations ofmevalonate, cholesterol , and i ts products . Decreased HMG-CoA reductase activi ty resul ts from (a) decreased DNA to messenger RNA (mRNA)transcription, (b) lowered mRNA for protein trans lation, and (c) increased degradation of exis ting HMG-CoA reductase enzyme complexes . Theenzyme is a lso under di rect hormonal control with glucagon and epinephrine decreas ing activi ty via phosphorylation, whereas insul in increasesactivi ty by dephosphorylation. In relation to the hormonal control , low concentrations of ATP decrease cholesterol synthes is as the bodydirects i ts resources toward energy production rather than l ipid synthes is .

Statins: Statins are an important class of medications , which reduce cholesterol in patients with high cholesterol and heart disease. Statinswork by inhibi ting the cri ti ca l and committing s tep in cholesterol synthes is , the enzyme HMG-CoA reductase, respons ible for the convers ionof HMG-CoA into mevalonate. Statins , whose s tructure i s s imi lar to HMG-CoA, mimic the substrate and competi tively inhibi t the enzyme.Decreased production of mevalonate and, therefore, cholesterol a lso causes increased production of receptors for LDL cholesterol , the mostharmful cholesterol -transporting l ipo-protein, increas ing clearance of LDL from the bloodstream.

REVIEW QUESTIONS1. What are the key features of fatty acid synthes is and how is i t regulated?2. What i s the role of fatty acid synthase in fatty acid synthes is?

3. How are fatty acids with an odd number of carbons synthes ized?4. How are unsaturated fatty acids produced?5. What are the functions of carni tine pa lmitoyl transferase I and II (CPT I and II) and mitochondria l tri functional protein (MTP) in the

degradation of fatty acids?6. What are the key features of ketone body synthes is and how do ketone bodies contribute to meeting energy demands in the body?7. What are the key features and regulation of triacylglycerol , phosphoglycerides , and ceramide synthes is?8. What are the key features and regulation of cholesterol synthes is?9. What role do HMG-CoA and HMG-CoA reductase play in cholesterol synthes is?

CHAPTER 8MEMBRANES

Membrane StructureMembrane FunctionsMembrane ChannelsMembrane Signal ingReview Questions

OVERVIEWLipids , driven by their hydrophobic and hydrophi l i c portions , form the biologica l membranes found in a l l l i ving creatures . These include thecel l and nuclear membranes as wel l as membranes that are part of organel les such as mitochondria and the endoplasmic reticulum (ER).Membranes provide separation of di fferent envi ronments to permit a variety of biologica l functions . Membranes are not s tatic s tructures ,though, rather they are dynamic and fluid and a l low selective movement of ions , energy sources , vi tamins and cofactors , and waste. Complexl ipids such as cholesterol and sphingol ipids both affect the s tructure of membranes where they are found and are a lso involved in speci ficfunctions .

The variety of l ipid and protein molecules , which make up membranes , are respons ible for essentia l functions such as channels andtransport across the membrane as wel l as s igna l ing. Membrane receptors , a long with thei r cytoplasmic partners , transmit s igna ls via s teroidhormones . An important type of membrane receptors are G-proteins with intrins ic enzyme activi ty. The resul ting secondary messengermolecules ampl i fy and transmit this s igna l to various parts of the cytoplasm and nucleus . This abi l i ty to transmit a message across amembrane i s paramount to not only the normal functions of cel l s but a lso thei r abi l i ty to perform specia l i zed functions , which define thehuman body.

Not surpris ingly, l ipids and membrane s tructure and function are important in disease processes as wel l as treatments . Any deficiencies orproblems with l ipid synthes is or breakdown lead to a serious disease s tate and/or death. Modulation of membrane fluidi ty i s , i tsel f,important in membrane functions and, as a resul t, a variety of diseases . For example, multiple bacteria and vi ruses are infective and severa lmedications work s imply because their hydrophobic nature affects and di rectly targets l ipids and membranes .

MEMBRANE STRUCTURE

LIPIDS

Membranes are composed of l ipids arranged in a lipid bilayer, wi th the hydrophi l i c glycerol and phosphate “head” groups of the l ipidmolecules forming the two outs ide layers and the hydrophobic “ta i l ” groups arranged ins ide (Figure 8-1).

Figure 8-1. Simplified Representation of Lipid Bilayer/Membrane. The l ipid bi layer i s composed of l ipid molecules , with hydrophi l i c head groups(e.g., phosphate) forming the outer surfaces and hydrophobic ta i l s grouped together in the hydrophobic center. See Chapter 3 for moredeta i led discuss ion of l ipid molecule s tructures . [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

A majori ty of membrane l ipids are phospholipids, usual ly with 16- or 18-carbon ta i l s , some saturated and some unsaturated. Other types ofl ipids , including cholesterol , modulate the s tructure and, therefore, the fluidi ty of the membrane and, in turn, a ffect some membranefunctions .

Particular l ipids are found in speci fic membranes and are respons ible for speci fic function as summarized below: Cardiolipin (Diphosphatidylglycerol)—Severa l locations including the inner mitochondria l membrane. It has a negative charge and may be

involved in (a) decreas ing blood clots , (b) s tabi l i zing important respi ratory cha in enzymes in the mitochondria , (c) moving proteins andcholesterol from the outer to the inner mitochondria l membrane, (d) ass is ting in the proper folding of mitochondria l proteins (Chapter 9) asa chaperone, and (e) poss ibly regulating deoxyribonucleic acid (DNA) synthes is .

Phosphatidylserine (PS)—Located in the inner platelet membrane and, during activation, the exterior platelet membrane. It carries a negativecharge and i s the primary phosphol ipid that promotes the anticoagulant protein C pathway, providing feedback inhibi tion of thrombinformation.

Phosphatidylethanolamine (PE)—Located in both the interior and exterior of cel l membranes . It carries a neutra l charge and promotes theanticoagulant protein C pathway, but to a lesser degree than PS.

Phosphatidylcholine (PC)—Located in the interior and exterior of cel l membranes . It carries a neutra l charge and promotes the anticoagulantprotein C pathway, but to a lesser degree than PS.

Phosphatidylinositol —Located in the interior and exterior of cel l membranes as wel l as the nuclear membrane. It carries a pos i tive charge andpromotes the anticoagulant protein C pathway, but to a lesser degree than PS, and i s important in s igna l ing processes for ini tiation of DNArepl ication.

Cardiolipin and Disease: Cardiolipin has been suggested to play severa l important roles in a variety of ti s sues , and deficiencies in theproduction and modi fication of this l ipid, not surpris ingly, lead to severa l human disorders . Barth syndrome i s an X-l inked disease resul tingfrom defects in an enzyme that remodels cardiol ipin. The disease resul ts in decreased phosphorylation of adenos ine diphosphatemolecules , presumably related to cardiol ipin’s proposed role in s tabi l i zing respiratory cha in enzymes. Changes in the amount orcompos i tion of cardiol ipin in heart and bra in mitochondria may a lso play a role in diseases such as heart failure, diabetes, Alzheimer’sdisease, and Parkinson’s disease. Fina l ly, Trepenoma pallidum, the bacterium respons ible for the disease syphilis, produces antibodies aga instcardiol ipin; the biologica l and disease impl ications of this are s ti l l not wel l understood.

The smal ler head groups of phosphol ipids such as PS and PE are preferred on the inner s ide of the membrane bi layer. On the other hand, PSis often located on the outs ide of the bi layer where i t can increase adherence to other cel l s and ti ssues . A membrane-bound enzyme ca l led“flippase” cata lyzes the process of moving particular phosphol ipid molecules from one s ide of the bi layer to the other when required. Otherphosphol ipids , because of thei r head group s ize and charges as wel l as the number and location of thei r ta i l double bonds , create a morefluid membrane more amenable to areas of cel l curvature or cel l–cel l connections (Figure 8-2).

Figure 8-2. Membrane Packing of Saturated and Unsaturated Lipid Tails. The l ipid bi layer i s composed of l ipid molecules whose ta i l groups can besaturated (“S”) or unsaturated (“U”). Saturated ta i l s pack close together, whereas unsaturated ta i l s have a looser packing s tructure, leading toa more fluid l ipid membrane. Increased amounts of saturated or unsaturated l ipids can markedly affect membrane fluidi ty and, therefore, thefunction of the membrane at that location. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion,McGraw-Hi l l , 2009.]

Compos i tion of the plasma membrane and, therefore, fluidi ty has been impl icated in severa l disease processes , including the fol lowing:

1. During s tages of infection with malaria , the compos i tion of infected red blood cel l s i s markedly changed to mimic that of the infectingparas i te. This may ass is t in the rupture of red blood cel l s and the subsequent spread of the paras i tes that i s a ha l lmark of this disease.

2. Various bacteria and vi ruses are bel ieved to change the plasma membrane of target cel l s to a l low the bacteria/vi rus to fuse and, therefore,infect the modi fied cel l s .

3. Human immunodeficiency vi rus (HIV) fuses with speci fic parts of an infected cel l ’s membrane s imi lar to i ts own vi ra l membrane incompos i tion and fluidi ty, a l lowing new vi ra l particles to emerge and spread to other cel l s . Research has shown that changing membranecompos i tion of target cel l s may a l low protection from HIV infection.

4. The paras i te Entamoeba histolytica produces proteins that bind to and form holes in human cel l membranes , ki l l ing these target cel l s .However, the paras i te membrane has a markedly di fferent membrane compos i tion, including di fferent phosphol ipids and a high level ofcholesterol , which protects i tsel f from these deadly proteins .

5. Membrane compos i tion i s markedly changed in the platelets of patients with frequent migra ines . Al though the mechanism is not known,these changes may be part of the cause of migra ine headaches and a better understanding could, therefore, lead to a novel treatment.

6. Vi rgin ol ive oi l a l ters the compos i tion of plasma membranes and affects regulatory proteins in the l ipid bi layer, which resul ts in beneficia lchanges in carbohydrate and l ipid metabol i sm.

The presence and amount of cholesterol a lso influence the fluidi ty of membranes . Cholesterol ’s hydroxyl group readi ly interacts withphosphol ipid head groups or exterior water molecules and the remaining hydrophobic tetracycl ic s tructure and “ta i l ” a lso natura l ly insertins ide the l ipid bi layer (Figure 8-3). Hydrogen bonding s tabi l i zes these interactions . Depending on the adjacent l ipids , cholesterol ei therincreases or decreases the l ipid ta i l packing. Loca l i zed membrane “rafts” conta ining high levels of cholesterol have been noted byresearchers and are fel t to di rectly influence not only membrane functions but a lso the functions of the membrane proteins within them.

Polyene Antifungals: Anti fungal medications including Nystatin and Amphotericin B* (see the figures below) share the s tructura l bas is of thepolyene molecule (i .e., multiple carbon=carbon double bonds) oppos i te an additional carbon chain conta ining multiple hydroxyl groups(OH). This s tructure enhances binding to ergosterol, a cholesterol -l ike, complex l ipid found in fungal cel l membranes . Binding of theseanti fungals to ergosterol decreases membrane fluidi ty and leads to leakage of the inner contents and fungal cel l death. Human cel lplasma membranes are not affected beacuse they do not conta in ergosterol , and the anti fungal s tructure does not bind to cholesterol .

[Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

[Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

*Amphotericin B is well known for a variety of serious and even fatal side effects, which are believed to be due to mechanisms other than its effects on fungal membrane fluidity.

Figure 8-3. Cholesterol Interaction with Membrane Lipids. I l lus tration of the pos i tioning of cholesterol within the l ipid membrane. The hydroxylgroup on one end of the molecule prefers to be near the polar, l ipid head groups of the external aqueous envi ronment, whereas theremainder of the cholesterol molecule prefers to be within the hydrophobic l ipid ta i l s . [Reproduced with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

PROTEINS

Proteins are the second major part of biologica l membranes and make up approximately 20%–80% of both the s tructura l and functionalcomponents of these membranes . Membrane proteins are class i fied as peripheral—predominately on the inner or the outer surface of themembrane bi layer—and integral—predominately within the membrane (Figure 8-4). Periphera l membrane proteins are bel ieved to mainly actas anchor points for attachment to external s tructures (e.g., the extracel lular matrix) and internal points (e.g., the actin, microtubule, andintermediate fi lament cytomatrix; Chapter 12). Integra l proteins are usual ly composed of uncharged, hydrophobic amino acids so they canenter and s tay in the hydrophobic envi ronment of the l ipid bi layer. Integra l proteins serve severa l functions including channels , “carrierproteins” (transporting molecules through the membrane), or as a s igna l ing protein (changing a portion of thei r internal cytoplasmic s tructurein response to a s igna l at an exposed external part of thei r s tructure to activate additional , closely associated molecules , leading to a changeof function, e.g., turning on a gene).

Figure 8-4. Peripheral and Integral Membrane Proteins. The relative pos i tions of periphera l and integra l membrane proteins in the l ipid bi layer arei l lus trated. Membrane proteins can a lso conta in carbohydrates resul ting in membrane glycoproteins important in membrane s ignal ing andfunctions . The presence and pos i tioning of cholesterol are a lso i l lus trated. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion,Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

As a l ready i l lus trated, the membrane content of cholesterol and saturated/unsaturated l ipids impacts membrane fluidi ty. Membraneproteins form connections to the extracel lular matrix, the internal cytomatrix, and even other cel l s that can a l low particular proteins and l ipidsto col lect at specia l areas of a biologica l membrane. Thus , a cel l can orchestrate a particular “area” or “s ide” where i t can receive s ignalsand/or transport particular molecules both in and out and/ or other essentia l functions . These specia l i zed “domains” within membranes areca l led “l ipid rafts” (Figure 8-5). Various examples of these membrane protein functions are discussed below.

Figure 8-5. Diagram of Lipid Raft. Lipid rafts are somewhat thicker and conta in higher amounts of specia l ty l ipids [e.g., sphingomyel in,gangl ios ides (not shown), saturated phosphol ipids (non zig-zag ta i l s ), and cholesterol ]. Membrane proteins can a lso conta in carbohydratesresul ting in membrane glycoproteins important in membrane s ignal ing and functions . [Adapted with permiss ion from Murray RA, et a l .:Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Hemoglobinopathies: Diseases of red blood cel l s , known col lectively as “hemoglobinopathies,” often resul t from changes in l ipid compos i tionand/or defects in proteins associated with the red cel l membrane. Elliptocytosis and spherocytosis (see the Figure) are two such diseases thatresul t from defects in spectrin and ankyrin and other red cel l membrane proteins important in the s tabi l i zation of the normal biconcaveshape of red blood cel l s .

Normal erythrocytes surrounding elliptocyte/spherocyte shown in the center [Courtesy of Dr. Walter Kemp, Montana State Department of Justice]Hemoglobinopathies involving changes in l ipid compos i tion of the red cel l membrane are a lso common. Red blood cel l s careful ly

mainta in a speci fic, asymmetric dis tribution of hundreds of phosphol ipids and proteins in the l ipid bi layer. Speci fic defects in ei thernumber or bi layer compos i tion of PS, PC, sphingomyel in, and cholesterol are seen in diseases such as s ickle cel l , tha lassemias , anemias ,and l iver disease. These changes a lso lead to destruction or removal of these blood cel l s as part of the disease processes .

MEMBRANE FUNCTIONSA primary function of biologica l membranes i s to mainta in the s tructura l integri ty and the individual functions of cel l s , the nucleus , andorganel les . Separation of envi ronments a l lows di fferences in concentration of ions and molecules that l i tera l ly a l low l i fe to exis t. Asexamples , i f biologica l molecules such as carbohydrates passed eas i ly through membranes , cel l s could not perform any s igni ficant biologica lfunctions . If cel l s were unable to selectively increase and mainta in the concentration of certa in ions on the ins ide and outs ide of themembrane, they could not mainta in thei r required internal envi ronment or perform functions such as conduction of a nerve impulse. However,the fact that molecules such as oxygen, carbon dioxide, ni trogen, and urea are able to move through the l ipid bi layer without any specia l i zedtransport proteins i s actua l ly beneficia l to the human body (Figure 8-6A). Specia l ci rcumstances of nonselective transport of molecules havealso been developed. For example, because of i ts rel iance on ketones as an energy source in times of low carbohydrate levels , bra inmembranes a l low free flow of ketone bodies without the need of a transport protein.

Figure 8-6. A-D. Illustration of Membrane Transport Proteins. Simple di ffus ion through (A) l ipid bi layer or (B) s imple channel . Faci l i tated di ffus ionvia a (C) channel and (D) active transport ei ther into or out of the cel l . See the text for further description. E. Na+–K+ ATPase Membrane Channel.Three sodium ions (Na +) ins ide the cel l bind to the Na +–K+ ATPase pump, which promotes phosphorylation of the protein by a boundadenos ine triphosphate (ATP) molecule. This phosphorylation causes a conformational change of the pump, which carries and releases theNa + to the outs ide of the cel l . Adenos ine diphosphate (ADP) i s released from the protein and two potass ium ions (K+) bind to the newconformation of the pump. Binding of K+ promote dephosphorylation of the protein, caus ing a reverse conformational change, which carriesand releases the K+ to the ins ide of the cel l . ATP rebinds to the pump, which a lso helps accelerate release of the K+, returning the pump to i tsorigina l s tate. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

MEMBRANE CHANNELSProteins that create membrane channels often have multiple α-hel ica l and/or β-s trand secondary s tructures that form a tube-l ike channelthrough the membrane. Hydrophobic amino acids on the outs ide of the protein’s tertiary and quaternary s tructure (Chapter 1) form an exteriorthat readi ly inserts i tsel f into the membrane and s tays within the hydrophobic l ipid ta i l s . Hydrophi l i c or charged amino acids on the ins ide ofthe channel form a sui table passageway that a l lows charged and/or uncharged molecules to move through the previous ly impeding l ipidmembrane.

Simple channels (Figure 8-6B) have no active control over the molecules that can enter through them except by the diameter of the tube and,sometimes , the exact hydrophobic/ hydrophi l i c envi ronment in the interior of the tube. Simple channels , therefore, only work via aconcentration gradient that provides the drive for molecules to cross the membrane. An example of a s imple channel i s the gap junction thatcreates a passageway in the gap between two cel l s a l lowing movement of ions , sugars , amino acids , and nucleotides between the cel l s . Gapjunctions are important in organs such as the heart, bone and eye lens , and in developing ti ssues .

Facilitated protein channels (Figure 8-6C) are s imi lar to s imple channels (no energy i s used, driven by a concentration gradient) but thetransport i s sped up or faci l i tated from particular amino acids that help to form the channel . These channels can a lso have wel l -pos i tionedamino acids (often charged) in the tube that serve as a “gate” to regulate the movement of speci fic molecules through the channel . Thesegated channels (not shown) have amino acids that block transport but a s igna l (e.g., binding of a speci fic molecule or a change in vol tage)

causes the protein to change conformation, moving the gatekeeper amino acid(s ) out of the way. These amino acids can be speci fica l lyhydrophobic or hydrophi l i c or even charged (Chapter 1), depending on the molecule to be transported.

Ionophore Antibiotics/Antifungals: The important function of membrane channels in mainta ining the correct ba lance of various ions ins ide andouts ide of cel l s has been uti l i zed in the development of an important class of antibacteria l and anti fungal medications—the “ionophores.”This class of drugs includes peptide- or l ipid-based molecules that insert into biologica l membranes . Al though mechanisms di ffer s l ightly,a l l dis rupt the ionic ba lance of the target cel l by a l lowing unregulated or abnormal movement or permeabi l i ty of ions , leading to cel ldeath. The s tructure of certa in ionophores lends i tsel f to selective insertion into only bacteria l or yeast cel l s with l imited or no effect onhuman cel l s , thereby a l lowing treatment of the infection without adverse effects on the human patient.

Active transport (Figure 8-6D) involves a carrier protein that undergoes a conformational change via the release of energy or phosphorylationfrom nucleotide molecules . Active transport can move molecules into or out of a cel l . A prime example of active transport i s the sodium–potassium ATPase (Na+–K+-ATPase) pump (Figure 8-6E). The Na +–K+ ATPase pump helps to establ i sh a high concentration of Na + outs ide and a highconcentration of K+ ins ide a cel l . This Na +–K+ gradient i s required for nerve and muscle function as wel l as to drive other channels to transportcarbohydrates and amino acids and nutrients into cel l s . Proper Na + and K+ cel lular concentrations a lso help to mainta in the correct cel lvolume because these ions affect the movement of water molecules into and out of cel l s . Other molecular pumps wi l l be discussed in laterchapters .

Membrane Channels and Channel Blockers: Membrane channels of a l l types are found throughout the human body performing a wide variety offunctions . These channels are prime targets for the development of various medications to treat diseases associated with membranechannel function and/or dys function.

One example i s ca lcium (Ca 2+) channels , which are important in the rate and force of heart contraction as wel l as contraction/relaxationof smooth muscle in blood vessels . Patients with high blood pressure are often treated by a class of medications known as calcium channelblockers (CCBs), eas i ly identi fied by the suffix “-dipine.” CCBs decrease the tota l transport of Ca 2+ into muscle cel l s , thereby decreas ing heartcontraction (rate and force) and increas ing the diameter of blood vessels leading to lower blood pressure.

Histamine (H2) receptors and the H+–K+ ATPase proton pump are both respons ible for the transport of acidic hydrogen ions (H+) into thestomach to a id digestion. In diseases such as heartburn, gastri ti s , peptic ulcer disease, reflux disease, Barrett’s esophagi ti s , gastrinomas ,and Zol l inger–El l i son syndrome, i t i s helpful to lower the amount of acid content in the s tomach. In each of these cases , speci fic H2 blockers

with the suffix “tidine” and proton pump inhibitors, ending with the suffix “prazole,” block H+ transport.

A “carrier protein” can a lso faci l i tate the movement of large or charged molecules through l ipid membranes . In this instance, the chargedmolecule to be transported binds to the membrane protein and, via a conformational change, i s surrounded by the carrier protein’shydrophobic exterior. This carrier protein can then cross from one s ide of the l ipid bi layer to the other with i ts molecular passenger safelyshielded from the l ipid ta i l groups only to emerge on the other s ide where i t can release the molecule into a friendl ier hydrophi l i cenvi ronment.

Aquaporins: Aquaporins are a fami ly of integra l membrane proteins found in the kidney and other organs , which help to regulate water flowinto and out of cel l s . Defects of one of these channel proteins aquaporin-2 (AQP2) cause excess ive loss of water in some patients withnephrogenic diabetes insipidus or increased water retention in pregnancy and congestive heart failure. Use of the psychiatric drug l i thium caninduce problems by decreas ing the amount of AQP2, whereas the peptide hormone vasopress in works via increas ing AQP2 concentrations inmembranes .

MEMBRANE SIGNALINGMany integra l membrane proteins do not form a channel or phys ica l ly move through the membrane but, ins tead, transmit a message (s igna l )from one s ide of the l ipid bi layer to the other. The signaling function of membrane proteins i s seen frequently and i s vastly important inhuman biology in multiple cel l types . These membrane proteins function by a change in thei r own conformation from external binding of amolecule, phosphor-ylation of one of i ts amino acids , and/or then interaction with other proteins , and so on. This conformational changeaffects the internal portion of the s ignal ing protein and can then activate internal proteins , leading to a selected function(s ). There aresevera l di fferent types of membrane protein s igna l ing, summarized in Table 8-1.

TABLE 8-1. Summary of Membrane Signal ing Receptors

The fi rs t example of s igna l ing molecules in Table 8-1 are the steroid hormones (see Chapter 3) and can be further divided into five categories ,depending on their particular receptor—androgens, estrogens, glucocorticoids, mineralocorticoids, and progestagens (precursors of a variety ofprogesterones). Vi tamin D, a l though not technica l ly a s teroid, transduces s igna ls such as s teroid hormones and i s , therefore, usual ly includedwith this group. The s teroid hormones work in two di fferent ways (Figure 8-7). Fi rs t, the hormone may bind to a membrane surface receptor,which then ei ther activates other s igna l ing pathways ins ide the cel l or moves from the membrane into the nucleus to activate DNAtranscription factors . Second, and more commonly, s teroids are l ipid soluble and can, therefore, pass eas i ly through the cel l membranewithout the a id of a membrane receptor. Once ins ide the cel l , the hormone can bind with cytoplasmic receptors to activate s ignal ing or maycontinue to receptors in the nucleus to activate transcription factors and DNA synthes is . Steroid hormones that affect DNA synthes is are ca l ledgenomic (i .e., a ffect genes), whereas those that do not affect transcription are referred to as nongenomic.

Figure 8-7. Activation by Steroid Hormone Receptor. A l ipid-derived s teroid hormone traverses the membrane to bind to a cytoplasmic receptor(bottom left), and the hormone–receptor complex enters the nucleus as a homodimer (upper). Al ternatively, the unbound hormone maytraverse through the nuclear membrane with subsequent binding to a hormone receptor to form a homodimer in the nucleus . Ei ther

mechanism leads to binding to speci fic parts of deoxyribonucleic acid (DNA) known as s teroid response elements and activation oftranscription of selected DNA. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion,McGraw-Hi l l , 2009.]

One of the most predominant and best understood membrane s ignal ing mechanisms i s via the G-protein fami ly (see Table 8-1). Al l of thesereceptors rely on the conformational change resul ting from the exchange/convers ion of the nucleotides guanos ine diphosphate (GDP) andguanos ine triphosphate (GTP) to convey an external s igna l to the ins ide of the cel l . G-proteins are a l l composed of three internal periphera lprotein subunits α-, β-, and γ-subunits , which associate with an integra l membrane protein receptor. The α- and β-subunits are closely boundand are represented as the dimer α/γ. G-proteins can be divided into five classes , Gs, Gi, Gq, G12/13, and Gt, depending on di fferences in the α-subunit, which affects interaction with the external s igna l ing molecule (Table 8-2).

Despi te the di fferences in the α-subunit and effects among the five classes , the genera l s igna l ing mechanism is a lmost identica l and i ssummarized in Figure 8-8 and as fol lows:

Figure 8-8. Function of G-proteins. The l igand ( ) binds to i ts receptor, which interacts with the inactive α/β/γ complex with bound GDP (left) andcauses an exchange of GTP for the GDP on the α-subunit with dissociation of the β- and γ-subunits (right). These subunits can then movewithin the membrane and serve as “effectors” to activate other protein s igna l ing pathways . GTP can be hydrolyzed back to GDP via a GTPasecaus ing re-association of the α–β–γ complex and termination of the s ignal . [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’sReview of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

TABLE 8-2. G-Protein Receptor Classes

1. The α-subunit with a bound GDP interacts with the internal portion of the receptor as wel l as the β/γ-subunits .

2. Upon binding of the s ignal ing protein to the exterior of the receptor, a GTP molecule displaces the GDP on the α-subunit, caus ing thedissociation of the β-subunit from the β/γ-subunits .

3. The freed α-subunit can then interact with other membrane-bound proteins (effectors), leading to thei r activation (Gs, Gq, G12/13, and Gt) orinhibi tion (Gi). Increas ing evidence a lso suggests that the β/γ-subunits can activate di fferent effector protein s igna l ing pathways (e.g., L-type

Ca 2+ channels involved in various CCB medications).

4. The α-subunit’s inherent GTPase activi ty, often enhanced by an accessory GTPase activating protein or sometimes by the effector proteins ,eventual ly converts GTP back to GDP, a l lowing this subunit to reassociate with the β- and γ-subunits , thereby turning the activation off.

The various G-proteins activate severa l important membrane proteins , which lead to the conveyance of the s ignal via second messengermolecules. Examples include cyclic adenosine monophosphate (cAMP) (Figure 8-9A), generated by adenyl cyclase (a l so known as adenylyl oradenylate cyclase), which leads to phosphorylation of any of severa l molecules by protein kinase A and (Figure 8-9B) cleavage of the membranel ipid phosphatidylinositol 4,5-bisphosphate by phospholipase C to form diacylglycerol and inositol triphosphate (IP3). IP3 subsequently leads to the

release of Ca 2+ generated from internal cel lular s tores (usual ly ins ide the ER) by subsequently activated ion channels and/or enzymes. Thesesecond messengers lead to multiple membrane, cytoplasmic, and nuclear effects . For example, the ca lmodul in kinases activate myos inmolecules caus ing muscle contraction, help to regulate secretion of neurotransmitter molecules , regulate a variety of transcription factors ,modulate carbohydrate s torage and use, and may be essentia l to bra in function. Protein kinase C i s known to change membrane s tructure,regulate transcription and cel l growth, ass is t in immune responses , and provide key activation of proteins involved in learning and memory.Importantly, the multi s tep, s igna l ing process a l lows selected ampl i fi cation of a smal l s igna l at the exterior of the cel l membrane into apotentia l ly large response within the cel l .

Figure 8-9. A. Cyclic adenosine monophosphate (cAMP) Signaling. Activation of adenylyl cyclase by the α-subunit resul ts in the convers ion ofadenos ine triphosphate (ATP) to cAMP which then activates protein kinase A and phosphorylation of various s ignal ing proteins that el i ci tphys iologica l effects such as the express ion of speci fic genes . Stimulatory l igands activate via Gs proteins , whereas inhibi tory l igands act viaGi proteins . B. Phospholipase C–Protein Kinase C (PKC) Signaling. Activation of phosphol ipase C, usual ly via the α-subunit of the G-protein Gq,resul ts in the cleavage of the membrane l ipid phosphatidyl inos i tol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inos i tol triphosphate

(IP3). Hydrophi l i c IP3 leaves the membrane and enters the cytoplasm to release ca lcium (Ca 2+) from the endoplasmic reticulum (ER). Ca 2+

subsequently activates Ca 2+ binding proteins (CaBP) leading to phys iologica l effects such as activation of enzymes and/or express ion ofspeci fic gene products . Hydrophobic DAG remains in the membrane and can activate PKC leading to separate phosphorylation of proteins andresul ting phys iologica l effects . Ca 2+ i s a l so required to synergis tica l ly maximize this effect of DAG on PKC. C. Janus Kinase and Signal Transducerand Activator of Transcription (JAK–STAT) Signaling. The l igand binds to the monomeric form of the receptor (1), leading to dimerization and cross -

l inking of the two receptors . Each JAK cross -phosphorylates (see arrows) the tyros ine res idue of other JAK, leading to thei r activation (2). Theactivated, dimeric JAKs cross -phosphorylate (see arrows) tyro-s ine res idues on the other receptor (3). The phosphorylated receptors nowconta in an appropriate s i te for binding with a number of other s igna l ing molecules such as STAT (note semici rcular binding pockets on STATmolecules ), leading to STAT phosphorylation (4). The phosphorylated, dimeric STAT molecules subsequently may travel to the nucleus andbind to response elements in deoxyribonucleic acid (DNA) with propagation of the ini tia l s igna l by the express ion of certa in genes (5).[Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

The fina l group of membrane receptors (see Table 8-1) i s the “soluble receptor-associated tyros ine kinases ,” including the receptor tyrosinekinases, Janus kinases, and integral guanyl cyclase (a l so known as guanylyl or guanylate cyclase) receptors (Figure 8-9C).

Al l three receptors have the abi l i ty, a fter activation by binding of the s ignal ing molecule, to carry out a speci fic enzymatic function (i .e.,phosphorylation of i ts own selected tyros ine res idues , production of cycl ic guanos ine monophosphate). These enzymatic changes then lead toactivation of a s igna l ing pathway speci fic for the s ignal ing molecule.

REVIEW QUESTIONS1. What are the bas ic s tructure, key features , and important components of the l ipid bi layer and biologica l membranes?2. How do speci fic phosphol ipids and cholesterol molecules affect the functioning of biologica l membranes?3. What i s meant by periphera l and integra l membrane proteins and what functions can they play?4. How would you describe s imple, gated, and ATPase-dependent membrane channels and what i s thei r s igni ficance?5. What i s the s igni ficance of s igna l ing proteins , carrier proteins , and second messenger molecules?6. What are the major classes of receptors and second messenger molecules?

CHAPTER 9DNA/RNA FUNCTION AND PROTEIN SYNTHESIS

Structure of the NucleusDNA Repl ication and TranscriptionProtein Synthes isPosttrans lational Trafficking/ModificationControl of Gene Express ionMutations and Repair MechanismsRegulation of Cel l Growth and Di fferentiationReview Questions

OVERVIEWThe nucleus i s often represented as a relatively empty s tructure, conta ining only deoxyribonucleic acid (DNA) being repl icated and transcribedalong with a few accessory molecules to help in the process . To the contrary, the nucleus i s actua l ly a highly organized, membrane-boundstructure that i s l i tera l ly fi l led with proteins , nucleotides , carbohydrates , and l ipids with multiple functions . Various proteins are involved,a long with the nuclear membrane, in the organization of chromosomes, which a lso helps to regulate the processes of DNA repl ication andtranscription, and, subsequently, protein synthes is . Other proteins di rectly influence the express ion of genes via di rect interactions withspeci fic nucleotide sequences . Posttrans lational modi fications affect both protein function and di rect particular proteins to intracel lularand/or extracel lular destinations .

STRUCTURE OF THE NUCLEUSThe nucleus i s surrounded by a double membrane ca l led the nuclear membrane (a l so known as the nuclear envelope), with the outer layerbeing continuous with the endoplasmic reticulum (ER) in the cytoplasm and, l ike the ER, conta ining ribosomes and newly synthes izedproteins . The inner and outer nuclear membranes are a lso continuous at the s i tes of nuclear pores. Approximately 2000 nuclear pores areconta ined within the nuclear membrane, and each pore can a l low movement of about 1000 molecules in and out of the nucleus per second.These nuclear pores , composed of proteins ca l led nucleoporins, are di rectly analogous to the membrane channels discussed in Chapter 8 andtransport severa l types of molecules , including ribonucleic acid (RNA) and ribosomes, proteins , carbohydrates , and l ipids . Smal ler moleculesand ions pass through the nuclear pore by s imple di ffus ion, but larger proteins and RNA molecules are blocked by a spoke-l ike gate ins idethe channel and must be actively ass is ted by carrier proteins ca l led “importins” or “exportins” by a process that requires two guanos inetriphosphate (GTP) molecules . Each type of RNA molecule [messenger RNA (mRNA) and transfer RNA (tRNA)] that must be transported into thecytoplasm has an exportin and a speci fic amino acid (AA), nuclear export sequence, which di rects them to a specia l i zed nuclear pore for thei rselected transport out of the nucleus .

Leptomycins: Leptomycins A and B, origina l ly developed as anti fungal drugs , speci fica l ly a lkylate an importin, which resul ts in inhibi tion ofthe nuclear export of severa l RNAs and transcription regulators in cel l cycle control . An additional leptomycin i s HIV-1 “regulator, which allowsHIV to take over host protein synthesis.” The leptomycins a lso s tabi l i ze p53, known to suppress tumor development/growth. Because of thei rcel l cycle effects (see below), leptomycins are now being cons idered for cancer therapy.

HISTONES

The most important s tructure ins ide the nucleus i s chromatin, cons is ting, in humans , of the 46 chromosomes and their associated proteins .These proteins enable not only efficient packing of over 12 bi l l ion nucleotides in human DNA, but a lso selective unwinding of thesechromosomes to expose genes for DNA repl ication, DNA to RNA transcription and mRNA process ing. Histones are one major example of theseassociated proteins , and are separated into s ix classes : H1, H2A, H2B, H3, H4, and H5. Two each of his tones H2A, H2B, H3, and H4 assembleinto an eight-subunit nucleosome and wrap 146 or 147 DNA base pa i rs of the s imple, double hel ix s tructure (Figure 9-1) around the complex.

Figure 9-1. Histone Structure. The his tone conta ins a core of his tone molecules , including pa i rs of H2A, H2B, H3, and H4, wrapped by double-hel ixDNA and held together by his tone H1. See text for further deta i l s . DNA, deoxyribonucleic acid. [Reproduced with permiss ion from Mescher AL:Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Severa l of these nucleosomes, l inked together by about 50 DNA base pa i r sequences (Figure 9-2A), create an approximately 11-nm “beadson a s tring” chromatin s tructure (Figure 9-2B). Addition of the H1 his tone (Figure 9-1) covers the entry and exi t points of the DNA molecule andal lows DNA to form coi led-coi l s tructures , including the 30-nm chromatin fiber (Figure 9-2C). The 30-nm fiber can readi ly unwind into i ts 11-nmcomponent to a l low DNA repl ication and transcription. Analogous proteins ca l led protamines are found in sperm and a l low even denserpacking of DNA in the sperm head than his tones .

Figure 9-2. A-F. DNA Structure from Chromosome to Double-Helix. Ini tia l DNA structure beyond the s imple, double hel ix (A) resul ts from his tonesH2A, H2B, H3, and H4 interactions with DNA to create (B) 11-nm “beads on a s tring” s tructure. (C) The subsequent addition of his tone H1 createsthe 30-nm fiber. Further tertiary and quaternary s tructures of DNA are created by the addition of various scaffolding proteins , which condenseDNA into varying compact chromosome structures . The level of s tructure varies depending on the cel l cycle s tage and, as a resul t, therequirement for DNA transcription or repl ication [e.g, (D) noncondensed, interphase s tructures (300 nm) and (E) condensed, metaphasestructure (700 nm), ending with (F) the highly compacted s tructure of the metaphase chromosome]. DNA, deoxyribonucleic acid. [Adapted withpermiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Important protein attributes (Chapter 1) include pos i tive/ negative/uncharged AAs (primary s tructure), a lpha hel ices (secondary s tructure),and grooves formed between separate his tone proteins that provide and optimize essentia l interaction points between the his tones and theDNA molecules . The properties of nucleotides a lso contribute to this binding because increased numbers of adenos ine (A) and thymine (T)res idues at the minor groove of DNA a l low improved nucleosome binding. Not surpris ingly, his tone AA sequences and, therefore, higher orderstructures are highly conserved in biology. However, his tones do much more than just wind DNA to a manageable s i ze. Severa l his tones aremodi fied on their N-terminal ta i l s and globular areas (see posttrans lational modi fications below). These modi fications include the additionof methyl , acetyl , phosphate, ubiqui tin, and adenos ine diphosphate groups among others . The modi fications change how the proteinsinteract with the DNA molecule, to expose (e.g., acetylation) and conceal (e.g., methylation) particular parts of the DNA strand as a part ofactivation and inhibi tion of gene transcription (gene regulation), as wel l as the repair of DNA errors from both DNA repl ication and ongoingprocesses that lead to nucleotide mutations .

NUCLEAR MATRIX/SCAFFOLD

Further compaction of the s tructure i s provided by the nuclear matrix or “scaffold” proteins , which help in mainta ining the s tructure and integri tyof the DNA molecules . The exact s tructure and function of this s tructura l element of the nucleus are s ti l l highly disputed. However, theexis tence i s genera l ly accepted of a supportive, highly dynamic network that produces both chromatin s tructure and, thereby, i ts functions byinfluencing the molecules with which i t interacts . In corroboration of the concept of a nuclear matrix, “scaffold/matrix attachment elements”(S/MARs) have been proposed/ identi fied in which speci fic DNA sequences would be able to connect to the scaffolding proteins . The exis tenceand function of S/MARs i s a lso now cons idered in biotechnology involved in gene therapy and modulation of DNA express ion to modi fydisease s tates .

The nuclear matrix or lamina i s a s tructure thought to be a three-dimens ional network of intermediate filaments, a thi rd type of s tructura lprotein (Chapter 12), as wel l as periphera l and integra l membrane proteins attached to the inner l ipid bi layer. Within the nucleus , addi tionalchromatin s tructure i s created from the scaffolding proteins by the formation of further super-coi led s tructures . These higher orders ofs tructure include a 30-nm zigzag formation and a 100-nm fiber (not shown), as wel l as a 300-nm fiber (Figure 9-2D) with noncondensed and,therefore, access ible loops and a highly condensed 700-nm fiber (Figure 9-2E) that i s part of the fina l metaphase chromatin s tructure (Figure 9-2F) found in cel l s . Other “scaffolding” proteins l ink the chromatin to the nuclear matrix (essentia l for chromosome movement during cel lrepl ication) and the cytoplasmic matrix to the nuclear membrane and internal nuclear s tructures . Of course, chromatin s tructure changesdramatica l ly from interphase throughout DNA repl ication.

Laminopathies: More than 2000 variants of medica l disorders of the nuclear membrane are known, many of which are caused by mutationsthat affect the proteins of the nuclear membrane or lamina. Each affects an otherwise important role of proteins involved in transport ors tructure of the membrane bi layer or i ts membrane proteins , which di rectly affect DNA repl ication and trans lation and the processes ofprotein trans lation and transport. These “laminopathies” have far-reaching effects , often resul ting in chi ldhood or adolescent disorders ofskeleta l and cardiac muscles , l ipid, skin, nerves , and white blood cel l s , as wel l as cancers , diabetes , premature aging, and even earlydeath.

The nuclear matrix i s not only actively involved in chromosome separation and cel l divis ion but a lso helps to mainta in the requiredstructure for each of these functions . The nuclear matrix creates a chromatin s tructure in the nucleus that i s ordered and constra ined indiscrete terri tories that reflect speci fic functions , but which, at times of need such as mitos is , can a l ter dramatica l ly whi le s ti l l mainta iningcontrol and organization. The resul t of the primary, secondary, tertiary, and quaternary folding of the DNA genome is the compaction of about1.8 m of human DNA about 40,000-fold so i t can fi t ins ide a microscopic nucleus . Of course, this level of compaction changes throughout thecel l cycle, being maximal ly compacted during mitos is and minimal ly compacted during interphase. Fina l ly, changes in the DNA structure,including the marked condensation and expans ion of chromatin, occur during the normal cel l cycle.

Changing Nuclear Matrix in Cancer: Recent research has shown that early changes in many human cancers include a notable and measurablechange in the proteins that make up the nuclear matrix. Al though eva luation of these changes i s s ti l l ongoing, the abi l i ty to detect thesechanges in the nuclear matrix may offer a rel iable way to detect cancers at very early s tages of the disease. Reversa l of these changes mayalso offer cl inicians , ins ights into prognos is and cures .

NUCLEOLUS AND RIBOSOME SYNTHESIS

Also found within the nucleus i s the nucleolus, made of proteins and nucleic acids , where ribosomal RNA (rRNA) i s produced and ribosomes areassembled for export into the cytoplasm. The protein components of ribosomes, often ca l led “r-proteins,” are made in the cytoplasm andtransported to the nucleolus via a connected network of nuclear membrane pores and nucleolar channels . rRNA is transcribed in the nucleusby RNA polymer-ases pol I, II, and III, and often methylated or shortened. The resul ting rRNA sequences are targeted to the nucleolus where theyjoin with the r-proteins to make the 40S and 60S subunits of mammal ian ribosome, which i s subsequently exported through a nuclear pore tothe cytoplasm.

DNA REPLICATION AND TRANSCRIPTIONThe convers ion of information in the genetic code to functioning proteins and nucleic acids rel ies on the abi l i ty of the DNA to repl icate i tsel fand to transcribe i ts code to mRNA for use in protein synthes is . DNA repl ication resul ts in very few mismatched nucleic acid pa i rs , wi th anapproximate error rate of only one mismatched nucleic acid per 10 mi l l ion nucleotides . The process of transcription a lso has mechanisms thatreduce errors , but these are far less efficient than that seen in DNA repl ication. Both processes involve severa l proteins essentia l to theprocess as functional or regulatory elements .

DNA REPLICATION

The process of uncoi l ing double-s tranded DNA, fa i thful ly copying each DNA strand and then separating the two, new, double-s tranded copies ,i s ca l led replication. The process s tarts at an origin of replication (ori), a particular sequence of nucleic acids at which a pre-replication complex(pre-RC) can bind and the repl ication can s tart. Approximately 100,000 origins of repl ication can be found in each human cel l , a l lowing thecopying of DNA to proceed in a para l lel fashion from a l l of these points , thereby speeding the process of DNA repl ication. The pre-RC i scomposed of the fol lowing four proteins : (a ) a s ix-subunit origin recognition complex binds fi rs t to the origin of repl ication; (b) two cel l cycleregulatory proteins , Cdc6 and Cdt1, ensure that the cel l i s prepared for DNA repl ication; and (c) the mini-chromosome maintenance complex, whichis bel ieved to conta in proteins essentia l for the establ i shment of a replication fork. The repl ication fork i s the point where two DNA strands ,one termed the leading strand and the other the lagging strand, are separated and DNA copying occurs . The coi led-coi l , double-hel ica l DNAstructure examined in Chapter 4 i s ini tia l ly unwound by the enzyme DNA helicase (poss ibly part of the minichromosome maintenance complex)by breaking the hydrogen bonds between complementary nucleic acids . Single-stranded binding proteins a ttach to the new DNA strands to keepthem separated. An enzyme termed primase then produces a short s trand of RNA (sometimes with DNA) to serve as a primer for the remainderof the process . The enzyme DNA polymerase repl icates each DNA strand in the 5′ to 3′ di rection by adding the correct, matching nucleotidetriphosphate to the 3′-hydroxyl end of the primer s trand. As each new nucleic acid i s added, a new phosphodiester bond i s formed, uti l i zingthe energy conta ined in the remaining diphosphate group. This process i s continuous on the leading s trand but, as DNA polymerase can onlyadd in the 5′ to 3′ di rection, short cha ins of newly added nucleic acids , ca l led Okazaki fragments, are generated on the lagging s trand. Theenzyme DNA ligase joins the Okazaki fragments together as lagging s trand repl ication proceeds . The process of repl ication a long the coi led-coi l s tructure of DNA soon leads to an unfavorable DNA conformation that i s wound about i tsel f. To rel ieve this problem, a DNA topoisomeraseefficiently cuts the phosphate backbone, “untangles” the DNA strands , and then repairs the cut, leaving the DNA otherwise unal tered. Thisenti re repl ication process i s depicted in Figure 9-3.

Figure 9-3. Overview of DNA Replication. Summary of DNA repl ication, i l lus trating the repl ication fork, leading and lagging s trand synthes is , andthe various proteins involved in repl ication and unwinding. A deta i led description i s offered in the text. DNA, deoxyribonucleic acid; RNA,ribonucleic acid. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

TRANSCRIPTION

Transcription i s the process whereby genetic information from a DNA sequence i s uti l i zed to create an equiva lent RNA—mRNA i f the gene codesfor a protein, tRNA for a tRNA, rRNA for assembly of a ribosome, or even cata lytic RNA, termed ribozyme. This DNA sequence conta ins not onlythe gene for the RNA (coding sequence) but a lso regulatory sequences that dictate when and how the particular RNA wi l l be produced. Again,the DNA is transcribed or “read” from 3′ to 5′ and occurs only on one of the DNA strands , known as the template strand.

Transcription s tarts with binding of the enzyme RNA polymerase to a promoter sequence on the DNA, usual ly located from 10 to 35 basesbefore the s tart of the actua l gene (Figure 9-4). RNA polymerase binding depends on a variety of protein transcription factors. Transcriptionfactors vary in type and activi ty, but a l l interact in some way with the DNA (ei ther di rectly or indi rectly) and the RNA polymerase to ei therenhance or block binding to the DNA molecule. The variation of transcription factors i s part of the regulatory mechanism that a l lowsdi fferentia l express ion of genes . Once bound, the RNA polymerase travels down the DNA from 3′ to 5′ whi le matching the appropriate RNA toi ts DNA counterpart, uti l i zing uraci l matched with adenine instead of thymine. As in DNA repl ication, energy for the formation of thephosphodiester bond i s derived from hydrolys is of the two terminal phosphate bonds of the nucleos ide triphosphate. Unl ike repl ication,multiple RNA polymerases can transcribe on a s ingle DNA gene sequence, a l lowing rapid production of the RNA product. The termination oftranscription i s s ti l l not wel l understood but i s fol lowed by the addition of severa l adenine uni ts to the 3′-end of the new RNA(polyadenylation, a l so known as a poly-A ta i l ). Next, the new RNA is usual ly spl iced and reannealed to remove noncoding regions , and a 7-methyl guanos ine nucleic acid i s added to the 5′-end (known as a 5′ cap) to protect the RNA from various enzymes (exonucleases) during i tssubsequent activi ties . If the RNA is destined for protein synthes is , the molecule i s exported from the nucleus to the cytoplasm or ER,depending on the fina l destination of the protein (see Protein Trafficking below). The 5′ cap a lso a ids in the recognition of mRNA by theribosomes.

Figure 9-4. mRNA Processing and Nuclear Export. Fol lowing transcription of DNA to produce the ini tia l complementary mRNA molecule,subsequent process ing occurs , including spl icing, polyadenylation, and addition of the 5 cap. The mRNA molecule bound for protein synthes isi s then exported through nuclear pores into the cytoplasm. See the text for further deta i l s . CTD, carboxy terminal domain; DNA,

deoxyribonucleic acid; mRNA, messenger ribonucleic acid. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry,28th edi tion, McGraw-Hi l l , 2009.]

Reverse Transcriptase and Retroviral Therapy: Severa l types of vi ruses , including those that cause human immunodeficiency vi rus(HIV)/acquired immune deficiency syndrome, have the abi l i ty to transcribe their RNA into DNA as part of the process of infecting a host cel land uti l i zing that cel l ’s repl ication machinery. The vi ra l enzyme that performs this RNA to DNA repl ication i s known as reverse transcriptase.Cl inicians can uti l i ze this unique abi l i ty of vi ruses aga inst them by us ing a class of medications known as reverse transcriptase inhibitors,commonly ca l led retrovirals. One such retrovi ra l i s Zidovudine or Azidothymidine (AZT), an analog of thymidine (see figure below). Becausereverse transcriptase enzymes are only found in these vi ruses and the thymi-dine analog i s 100 times less speci fic for the patient’s humanDNA polymerases , the medication preferentia l ly blocks vi rus repl ication. The azido group a lso has a hydrophobic qual i ty that a l lows AZT tocross through the plasma membrane for easy del ivery to infected cel l s , a fter which internal cel lular enzymes convert the AZT molecule intoan active 5′-triphosphate form that can incorporate into and terminate reverse transcription.

PROTEIN SYNTHESISProteins are cha ins of AAs connected by peptide bonds formed between the carboxyl ic acid (COOH) group of one AA and the amino group (NH3)of the second AA (Chapter 1, Figure 1-4A). Because each protein has a speci fic AA sequence, mRNA copied from the DNA gene template for thatspeci fic protein provides the nucleotide sequence/codon (Chapter 4) instructions to l ink the proper AAs together. Enzymes bring together theRNA and the necessary substrates a long with an energy source, ATP, for efficient production of proteins . This process , ca l led “translation,” i sbriefly described below and summarized in Figure 9-5.

Figure 9-5. A. Formation of the Ribosome Complex for “Translation.” The components of protein synthes is include the 60S and 40S ribosomalsubunits , mRNA, and tRNA–AA molecules , the fi rs t of which codes for methionine (AUG mRNA sequence with analogous UAC tRNA sequence).Severa l ini tiation factors (IFs ) and elongation factors (EFs ) are involved in the formation of the active 80S ribosomal complex. B. ProteinSynthesis (“Translation”). Simpl i fied model of protein synthes is . The fi rs t tRNA–AA is bound to the P-s i te fol lowed by binding of the secondtRNA–AA to the A-s i te [a ided by elongation factor 1α(eEF1α)]. A peptide bond i s formed between the two AAs , and the mRNA moves down theribosome [a ided by elongation factor 2 (eEF2)], releas ing the fi rs t tRNA molecule and freeing the A-s i te, so another tRNA–AA may bind. Thisprocess i s repeated unti l a nonsense codon i s reached in the mRNA sequence, s igna l ing the end of the protein. Termination i s fol lowed bydissociation of the ribosomal complex (a ided by eRF) and release of the new peptide. AA, amino acid; GTP, guanos ine triphosphate; mRNA,messenger ribonucleic acid; tRNA, transfer ribonucleic acid. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

Protein synthes is requires the concerted interaction of mRNA, tRNA, severa l accessory proteins , ca l led initiation factor (IF) and elongation (EF)factor, and ribosomes. The mRNA molecule i s copied from the DNA gene and conta ins the copy of the nucleotide sequence for the chosenprotein. Individual tRNA molecules can bind to only one speci fic type of AA. This tRNA–AA binds to the mRNA molecule via anticodons us ing thesame binding rules as DNA double s trands (e.g., a tRNA that binds the s tarting mRNA codon AUG has an anticodon sequence of UAC), insuringthe speci fic order of AAs required for proper production of the protein. The IF and EF accessory proteins serve a number of roles , includingenabl ing binding of the mRNA molecule to the ribosome, movement of the mRNA a long the ribosome to the s tart point of the synthes is ,docking of the tRNA–AA, joining of the 60S subunit to the 40S–mRNA complex (Figure 9-5A), and movement of the mRNA and growing peptidechain (Figure 9-5B), as wel l as accuracy assurance. IF and EF roles wi l l not be discussed further.

Elongation Factors, Bacteria, and Disease: Corynebacteria diptheriae causes the disease diphtheria, which, unti l effective immunization wasavai lable, caused up to 15,000 deaths a year, mainly in chi ldren and older adults . The bacteria Pseudomonas aeruginosa causes frequentinfections of burns , wounds , and medica l devices (catheters , venti lators , etc.), as wel l as serious problems in immunocompromisedpersons . Rotaviruses cause mi l l ions of chi ldhood deaths each year from serious diarrhea. Al l three of these infective agents work bymimicking human accessory proteins and binding to elongation factors . Al though the two bacteria inhibi t an elongation factor, rotavi rusesactual ly “hi jack” the factor and use the host’s protein synthes is machinery for reproduction and further infection.

The ribosome is a two-part s tructure composed of proteins and four s trands of rRNA. The fi rs t part of the ribosome is a 40S subunit (“S”stands for “Svedberg,” a uni t of molecular s i ze) a long with one rRNA, which helps in establ i shing the proper AUG codon s tart s i te on the mRNA.The 40S subunit a lso conta ins the binding s i tes for the mRNA strand and two binding s i tes for incoming AAs—the “P-site” (named for thepeptide cha in that grows at this s i te) and the “A-site” (named because new AAs bind here). The second part of the ribosome is a 60S subunitwith three rRNA molecules , which help in s tabi l i zation of the enti re ribosome, peptide bond formation, and movement of the ribo-some,a long the mRNA strand. The 60S subunit binds to the 40S subunit only after the mRNA is in place, the fi rs t tRNA–AA is bound at the P-s i te, andthe AUG start codon i s found and correctly a l igned. Assembly of this complex rel ies on the energy obta ined from GTP. Fol lowing the formationof the mRNA–fi rs t tRNA–AA–ribosome complex, a second tRNA with i ts speci fic, bound AA binds to the A-s i te. A peptide bond i s formedbetween the two AAs and the ribosome moves down the mRNA (a process that uti l i zes ATP energy), the P-s i te tRNA is released, and theprocess repeats i tsel f unti l the end of the protein i s reached.

Macrolides and the Machinery of Protein Synthesis: The class of drugs ca l led macrol ides includes the antibiotics azithromycin, clarithromycin,erythromycin, and roxithromycin (used for many respiratory, urinary, and soft-ti ssue infections), and the immunosuppressant drugs tacrolimusand sirolimus (used after organ transplants to reduce organ rejection). These medications block the movement of the ribosome a long themRNA molecule. Macrol ides take advantage of the di fferent 50S ribosomal subunit found in bacteria and do not affect the human 60Ssubunit. Macrol ides a lso tend to concentrate in white blood cel l s and are, therefore, conveniently transported to the speci fic s i tes ofinfection. As a resul t, macrol ides selectively and efficiently treat bacteria l infections without harm to the patient. Many other classes ofantibiotics take advantage of the di fferences in human and bacteria l protein trans lation for thei r selective actions .

POSTTRANSLATIONAL TRAFFICKING/ MODIFICATIONInherent in the process of protein synthes is/trans lation i s the production of a s tring of AAs , the primary protein s tructure, which must fold intothe correct secondary, tertiary, and quaternary s tructures to produce a functioning fina l protein and/ or protein complex (Chapter 1). Al thoughsome smal ler proteins are able to s imply fold as trans lation occurs and the AA sequence i s generated, most of the larger and/or morecomplex proteins require specia l i zed proteins , known as chaperones. These chaperones ass is t in the resul ting folding and unfolding of s ingle,sequence proteins (i .e., secondary and tertiary s tructures ) and the assembly of multiple proteins (quaternary s tructure).

Some chaperone proteins are known as “heat shock” proteins because they are sometimes preferentia l ly uti l i zed to s tabi l i ze protein foldingat times of cel lular s tresses such as an increase in temperature. In fact, a major role of chaperones i s to temporari ly s tabi l i ze the newlysynthes ized AA chain, especia l ly in a cytoplasm a l ready fi l led with other proteins , so i t does not randomly aggregate into a nonfunctionals tructure. Other chaperones are di rectly involved in the actua l folding, dissociating from their associated protein after the fina l s tructure i sachieved. Sti l l other chaperone proteins are involved in some membrane transport and even the breakdown of certa in proteins . Because oftheir close association with protein trans lation, chaperones are often found in or near the ER.

Protein trafficking or targeting di rects a protein made for a particular location (e.g., nucleus , cytoplasm, membranes , organel les , orextracel lular) to i ts correct destination. This process rel ies on signal sequences normal ly conta ined in the fi rs t 20–30 AAs of the protein.Exceptions include proteins bound for the nucleus , which have s ignals throughout thei r enti re sequence, some peroxisome-bound proteinswith a s igna l sequence in the las t three AAs , and mitochondria l proteins , which have two s ignal sequences to di rect them to ei ther themitochondria l matrix or the intermembrane space. In addition, protein synthes is for cytoplasmic-, nucleus-, and mitochondria-bound proteinsusual ly takes place on free ribosomes, whereas extracel lular-, peroxisome-, and membrane-bound proteins are usual ly synthes ized on ER-bound ribosomes. Accessory proteins ca l led sequence recognition particles (SRPs) and SRP docking protein a l so help to di rect proteins targeted forsecretion/ peroxisome/membranes to the ER and then the Golgi apparatus for further sorting and routing.

The fina l s tep in the synthes is of many proteins i s post-translational modification, the addition or removal of AAs (e.g., s igna l sequence andini tiation methionine), carbohydrates (e.g., speci fic carbohydrate sequences for glycoproteins/glycosaminoglycans), or chemica l modi fications(e.g., addi tion of l ipids , phosphate, hydroxyl or methyl groups , and/ or cysteine–cysteine disul fide bonding). These changes usual ly involveextracel lular or membrane-bound proteins and, thus , genera l ly occur in the ER and/or Golgi apparatus . Proenzymes (Chapter 5) can a lso beconverted to thei r active form at this time.

Protein Folding, Chaperones, and Mad Cow Disease: Mad cow disease/Bovine spongioform encephalopathy and i ts human equiva lent Creutzfeldt–Jakobdisease resul t from infectious , mis folded proteins known as prions (proteinaceous and infectious vi rions). Al though current research i s s ti l l

ongoing and i s often very controvers ia l , prions are fel t to infect thei r host as a normal ly folded protein, which then repl icates and mis foldsinto a tightly packed s tructure of β-sheets , referred to as an “amyloid fold.” These proteins aggregate into s tructures known as amyloidplaques , which create “holes” in the normal bra in ti s sue and a resul ting “spongioform” appearance that i s typica l of the disease (seepicture below). Prions affect the bra in/nervous ti ssue leading to detrimenta l and fata l neurologica l symptoms, including dementia ,memory/speech/movement/balance problems, marked personal i ty changes , ha l lucinations , and seizures . Some researchers are lookinginto the poss ible role of chaperone proteins in the prevention and/or treatment of this disease.

Courtesy of Dr. Dennis K. Burns , Department of Pathology, Univers i ty of Texas Southwestern Medica l Center

CONTROL OF GENE EXPRESSIONThe abi l i ty of the body to control the express ion of genes and their resul ting mRNAs and proteins a l lows the selective ini tiation and cessationof the various biologica l reactions that a l low l i fe. Because conservation of resources i s important, most regulation occurs at the level of genetranscription, a l though trans lational control as wel l as posttrans lational activation via protein modi fications a lso plays an important part inthe overa l l regulation.

Transcriptional control (DNA → RNA) rel ies on promoter regions of DNA, usual ly, but not a lways , located close to the s tarting point of thetranscription. These promoter regions a l low binding of the RNA polymerase and regulatory transcription factor and enhancer proteins . DNAsequences that appear in severa l di fferent promoter regions have been identi fied, such as the TATA box, an eight base-pa ir sequence oftenexclus ively cons is ting of adenine and thymine nucleotides . Many transcription factors a lso share conserved secondary and tertiary proteinstructures that a l low them to bind to particular parts of the DNA sequence; examples are shown in Table 9-1). The binding of di fferenttranscription factors and/ or enhancers to these sequences are essentia l for transcription and can increase or decrease the transcription of aspeci fic gene. Steroid hormones (Chapter 3) are speci fic type of transcription factors that bind often to unique receptors (cytoplasmic or nuclear).These s teroid receptors subsequently bind to speci fic DNA sequences ca l led steroid response elements for s teroid activation of particular genes .Steroid receptors a lways conta in an area for s teroid binding, a modi fied zinc finger s tructure for DNA binding, a sequence that activatestranscription, and an area that a l lows receptor dimerization. Examples of s teroids and their action wi l l be covered in Section II I .

TABLE 9-1. Examples of Some Common DNA Binding Moti fs

Iron Response Element (IRE): IRE i s a speci fic span of approximately 25–30 nucleotides found in the mRNA for proteins such as ferritin(respons ible for control led i ron s torage) and transferrin (respons ible for i ron transport). These nucleotides bind to form a secondary ha i rpinstructure, which can then bind proteins involved in regulation of i ron concentration in the human body. During times of low i ronconcentration, these proteins bind to the IRE of ferri tin and decrease i ts trans lation to increase the ava i lable i ron by decreas ing i ts s torage.These same proteins bind to response elements found in transferrin, which s tabi l i zes the mRNA leading to increased trans lation and,therefore, increased i ron acquis i tion. IREs , therefore, a l low regulation at the RNA level of concentrations of free and s tored i ron.

Gene express ion i s a lso control led by the revers ible acetylation of his tone, lys ine AA res idues in nucleosomes, carried out by speci ficenzymes known as histone acetyl transferases (HATs) and histone deacetylases. The addition of acetyl groups a l lows access of RNA polymerases tothe DNA, whereas the removal of the acetyl groups blocks this access . Some transcription factors or thei r associated proteins actua l ly conta inHAT activi ty to a l low them access to thei r target genes . In a s imi lar way, the addition of methyl groups to cytos ine nucleotide res idues usual lyresul ts in decreased transcriptional activi ty, and the removal of the methyl group i s usual ly required for express ion of that gene. Fina l ly,severa l a l ternative forms of gene express ion are a lso seen. In some instances , promoters only occur in particular ti s sue or cel l types .Al ternative spl icing/ removal/reanneal ing can a lso occur, a ffecting mRNA molecules that conta in introns that create severa l di fferent relatedmRNAs/ proteins with di ffering regulation (up to 30% of human genes may be affected by this process ). Deactivation of one a l lele of a gene oreven an enti re chromosome can a lso occur as i s seen by the inactivation of one of the X chromosomes in females during ini tia l embryodevelopment.

MUTATIONS AND REPAIR MECHANISMSAccidenta l changes or “mutations” in DNA or RNA sequence (Figure 9-6) are estimated to occur at an approximate rate of 1000–1,000,000 per

cel l per day in the human genome, and every new cel l i s bel ieved to conta in approximately 120 new mutations . One kind of mutation i s pointmutation, which includes two di fferent types (Figure 9-6B): transition occurs when a s ingle purine nucleotide i s changed to a di fferent purine (A↔ G) or a pyrimidine nucleotide i s changed to a di fferent pyrimidine nucleotide [C ↔ T(U)] and transversion occurs when the orientation of as ingle purine and pyrimidine nucleotide i s reversed [A/G ↔ C/T(U)]. Point mutations can be further class i fied as silent (when the same AA i scoded), mis-sense (when a di fferent AA i s coded), neutral (when an AA change occurs but does not affect the protein’s s tructure or function), ornonsense (when a s top codon resul ts , terminating trans lation and shortening the resul ting protein). Insertion and deletion mutations involvemore than one nucleotide and can not only change the trans lated protein, but, i f not occurring in multiples of three nucleotides , can a lsochange the enti re reading of the mRNA sequence (frameshift mutation). Chemica l -, radiation-, and even vi ra l -caused mutations can a lso causes ingle- or double-strand breaks and/or nucleotide–nucleotide or nucleotide–protein cross -l inking. A common example of cross -l inking occursbetween adjacent thymidine dimers whose purine bases can form a cyclobutane ring when exposed to ul traviolet l ight (Figure 9-6A–C).

Figure 9-6. A-C. DNA Mutations. Overview of mutations and some of thei r cl inica l impl ications (A and B) and example thymidine dimer (C). Seetext for further discuss ion. DNA, deoxyribonucleic acid. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

Al though some of these mutations are beneficia l , offering res is tance to disease or improved s tructure and/or function, they can often leadto disease and/or death of the cel l or organism. The human body has , therefore, developed a variety of repair mechanisms to attempt torestore the a l tered nucleotide sequence. In the case of s ingle-nucleotide mutations , particular endonuclease enzymes sense and remove(excision) the incorrect base and the correct nucleotide i s replaced by a polymerase and l igase. A particular example i s the endonucleasespeci fic for the repair of thymine dimers . Strand breaks are repaired by l igases or, when complex, by excis ion and subsequentretrans lation/l igation. Cel l cycle check points (see below) a lso offer the body the chance to veri fy the accuracy of the DNA sequence prior tocommitment to cel l repl ication and divis ion.

Thymidine Dimer Repair and Xeroderma Pigmentosum: Al though a relatively uncommon disorder Xeroderma pigmentosa i s a di rect example ofhow an autosomal recess ive mutation in the enzyme(s ) required for the repair of thymidine dimers can lead to a disease. The loss of thisimportant repair mechanism resul ts in diseases such as basal cell carcinoma, squamous cell carcinoma, and malignant melanoma—al l as a resul tof unrepaired, ul traviolet l ight damage. The only treatment for affected individuals i s reducing the exposure to sunl ight and supportivecare, a l though most patients die by the age of 20 years .

REGULATION OF CELL GROWTH AND DIFFERENTIATIONCell cycle i s the ordered pathway undertaken by a l l mammal ian cel l s , which defines not only times of both cel l divis ion and repl ication, buta lso those times of cel l growth and functional activi ties . The cel l cycle i s divided into G1 (gap 1), S (synthesis), G2 (gap 2/interphase), and M(mitosis) phases . Cel l s can a lso enter a G0 (“resting”) phase. The progress ion through the cel l cycle depends on a variety of proteins , whichinsure that a cel l i s ready for the next cel l cycle phase (“checkpoints”). These proteins include two types , the cyclins and the cyclin-dependentkinase (CDKs), which join in di fferent heterodimer combinations that regulate various parts of the cel l cycle. The cycl in component of thisprotein complex regulates the function of the phosphorylation activi ty of the bound CDK. Di fferent concentrations of cycl ins during the cel lcycle, as determined by regulation of thei r protein synthes is in response to various molecular s igna ls , lead to activation or inhibi tion of a CDK.An overview of the cel l cycle and the role of cycl ins and CDKs are shown in Table 9-2 and Figure 9-7.

TABLE 9-2. Overview of the Cel l Cycle

Figure 9-7. Overview of Cell Cycle Control. Review of various s ignals that regulate progress ion through the cel l cycle. Checkpoints include G1–S andG2–M trans i tion points where DNA damage i s detected and repaired, S-phase where completion of repl ication i s assessed, and M-phasewhere the integri ty of the spindle apparatus i s veri fied. Cycl ins A, D, E, CDK2, and CDK4 and the protein regulators p16, p 27, and p53, as wel l asDNA i rradiation damage and extracel lular s igna ls play roles as i l lus trated. Transforming growth factor-β a lso increases p27 inhibi tory activi ty(not shown). CDK, cycl in-dependent kinase; DNA, deoxyribonucleic acid. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus tratedBiochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Microtubules in the Cell Cycle and Cancer Treatment Strategies: The growth of microtubules during G2 and their subsequent breakdown during Mphases of the cel l cycle have led to varying s trategies in the treatment of cancers . One such cancer agent i s vincristine, which binds to tubul inprotein and blocks microtubule polymerization, s topping mitos is at metaphase. Vincris tine i s used in non-Hodgkin’s lymphoma, Wilm’s tumor,and acute lymphoblastic leukemia treatments among others . Taxol i s another important cancer treatment medication obta ined from the Paci ficyew tree and i s used for patients suffering from cancers of the breast, lung, ovaries , head and neck, and for Kapos i ’s sarcoma. Taxol worksby s tabi l i zing microtubules , thereby inhibi ting the progress ion of cancer cel l s undergoing s i s ter chromatid separation. Vincris tine and taxola lso affect a patient’s hea l thy cel l s , but to a lesser extent than the much faster repl icating and dividing cancer cel l s .

Various inhibi tors can s top the cel l cycle by inactivating the cycl in–CDK heterodimer complex. When DNA damage i s present, the tumorsuppressor protein p53 senses the problem and activates the inhibi tor p21, which blocks the cycl in D–CDK4 complex and, subsequently, the G1

to S trans i tion. The inhibi tors p16 and p27, the latter activated by the growth inhibi tor transforming growth factor-β (not shown in figure), canalso inhibi t the cel l cycle by inhibi ting the cycl in D–CDK4 or cycl in E–CDK2 complex, respectively. The overa l l control of the cel l cycle by cycl insand CDKs i s shown in Figure 9-7.

REVIEW QUESTIONS1. What are the bas ic s tructura l components of the nucleus and their roles?2. What are the key events involved in DNA repl ication and the role of relevant proteins and enzymes?3. What are the key events involved in transcription and the role of relevant proteins and enzymes?4. What are the key events involved in protein synthes is including the roles of mRNA, tRNA, and accessory proteins?

5. What i s the role of chaperones?6. What i s the s igni ficance of protein trafficking?7. What are the key aspects in the control of gene express ion?8. What are the various types of mutations and how do repair mechanisms relate to these mutations?9. What are the various phases of the cel l cycle and their functions?

SECTION IIINTEGRATED USMLE-STYLE QUESTIONS AND ANSWERS

QUESTIONSII-1. A previous ly normal 2-month-old female presents with ji ttery spel l s severa l hours after meals . She has low blood glucose. Phys ica l

exam reveals a l iver edge 4 cm below the right costa l margin. Percuss ion of the right chest and abdomen confi rms hepatomegaly. Theinfant increases her blood glucose after breastfeeding but i t i s not mainta ined at normal levels upon fasting. Which of the fol lowing i sthe most l ikely diagnos is of this patient?A. Fructosemia with inabi l i ty to l iberate sucrose from glucoseB. Galactosemia with inabi l i ty to convert lactose to glucoseC. Glycogen s torage diseaseD. Growth hormone deficiency with inabi l i ty to mainta in glucoseE. Intestina l malabsorption of lactose

II-2. A wel l , 2-year-old gi rl contracted a vi ra l i l lness at day care with vomiting, diarrhea, and progress ive lethargy. She presents to the cl inicwith disorientation, a barely rousable sensorium, cracked l ips , sunken eyes , lack of tears , flaccid skin with “tenting” weak pulse withlow blood pressure, and increased deep tendon reflexes . Laboratory tests show low blood glucose, normal electrolytes , elevated l iverenzymes , and (on chest X-ray) a di lated heart. Urina lys is reveals no infection and no ketones . The chi ld i s hospi ta l i zed and s tabi l i zedwith 10% glucose infus ion. Admiss ion laboratories show elevated medium-chain fatty acylcarni tines in blood and 6–8 carbondicarboxyl ic acids in the urine. Which of the fol lowing abnormal i ties i s the most l ikely diagnos is in this chi ld?A. Carni tine deficiencyB. Defect of medium-chain coenzyme A dehydrogenaseC. Defect of medium-chain fatty acyl synthetaseD. Mitochondria l defect in fatty acid transportE. Mitochondria l defect in the electron transport cha in

II-3. Which of the fol lowing i s the correct order of the fol lowing s teps in protein synthes is?1. A peptide bond i s formed.2. The smal l ribosomal subunit i s loaded with ini tiation factors , messenger ribonucleic acid (mRNA), and ini tiation aminoacyl–transfer

RNA (tRNA).3. The intact ribosome s l ides forward three bases to read a new codon.4. The primed smal l ribosomal subunit binds with the large ribosomal subunit.5. Elongation factors del iver aminoacyl–tRNA to bind to the A s i te.

A. 1, 2, 5, 4, 3B. 2, 3, 4, 5, 1C. 2, 4, 5, 1, 3D. 3, 2, 4, 5, 1E. 4, 5, 1, 2, 3

II-4. A middle-Eastern fami ly presents for eva luation because their infant son died in the nursery with severe hemolys is and jaundice. Thecouple had two prior female infants who are a l ive and wel l , and the wi fe relates that she lost a brother in infancy with severehemolys is induced after a vi ra l infection. The phys ician suspects glucose-6-phosphate dehydrogenase deficiency, implying defectivesynthes is of which of the fol lowing compounds?A. Deoxyribose and nicotinamide adenine dinucleotide phosphate (NADP)B. Glucose and lactateC. Lactose and NADPHD. Ribose and NADPHE. Sucrose and nicotinamide adenine dinucleotide

II-5. Which of the fol lowing i s an important intermediate in the biosynthes is of unesteri fied fatty acids?A. Carni tineB. CholesterolC. Fatty acyl -coenzyme A (CoA)D. GlucoseE. Malonyl -CoA

II-6. Severa l disorders , such as one form of α1-anti tryps in deficiency, can resul t from mistargeting of proteins into the wrong cel lularcompartments . New proteins destined for secretion are synthes ized in which of the fol lowing?A. Free polysomesB. Golgi apparatusC. NucleusD. Rough endoplasmic reticulumE. Smooth endoplasmic reticulum

II-7. Chi ldren with urea cycle disorders present with elevated serum ammonia and consequent neurologic symptoms including a l teredrespiration, lethargy, and coma. Severa l amino acids are intermediates of the urea cycle, having s ide ammonia groups that join withfree carbon dioxide (CO2) and ammonia to produce net excretion of ammonia as urea (NH2CONH2). Which of the fol lowing amino acidshas an ammonia group in i ts s ide cha in and i s thus l ikely to be an intermediate of the urea cycle?A. ArginineB. AspartateC. GlutamateD. MethionineE. Phenyla lanine

II-8. Cholera toxin causes mass ive and often fata l diarrhea by caus ing the continual synthes is of cycl ic adenos ine monophosphate (cAMP).Which of the fol lowing mechanisms would account for this effect?A. Activation of Gi protein

B. Irrevers ibly activation of adenylyl cyclaseC. Locking of Gs protein into an inactive form

D. Prevention of guanos ine triphosphate (GTP) from interacting with Gq protein

E. Rapid hydrolys is of G protein GTP to guanos ine diphosphate (GDP)II-9. Which of the fol lowing events occur during the formation of phosphoenolpyruvate from pyruvate during gluconeogenes is?

A. Acetyl -CoA i s uti l i zedB. Adenos ine triphosphate (ATP) i s generatedC. CO2 i s required

D. GTP i s generatedE. Inorganic phosphate i s consumed

II-10. Regulation of which of the fol lowing enzymes i s most important in control l ing l ipogenes is?A. Acetyl -CoA carboxylaseB. Acyl -CoA synthetaseC. Carni tine–acylcarni tine trans locaseD. Carni tine–palmitoyl transferaseE. Fatty acid synthase

II-11. The sequence of the template deoxyribonucleic acid (DNA) s trand i s 5′-GATATCCATTAGTGAC-3′. What i s the sequence of the RNAproduced?A. 5′-CAGUGAUUACCUAUAG-3′B. 5′-CTATAGGTAATCACTG-3′C. 5′-CUAUAGGUAAUCACUG-3′D. 5′-GTCACTAATGGATATC-3′E. 5′-GUCACUAAUGGAUAUC-3′

II-12. Which of the fol lowing i s an enzyme that i s activated by hydrolys is of a proenzyme form?A. HeparinB. KeratinC. LactaseD. Peps inE. Phenyla lanine hydroxylase

II-13. Which of the fol lowing i s an accurate description of s igna l transduction in response to a peptide hormone?A. Ca lcium activation of ca lmodul in-dependent kinase.

B. Diacylglycerol (DAG) caus ing an increase of intracel lular Ca 2+.C. Inos i tol tri sphosphate (IP3) binding to and activating protein kinase B.

D. Phosphol ipase A2 cata lyzing the cleavage of membrane phosphol ipids to release DAG.

E. Release of potass ium ions from the endoplasmic reticulum.II-14. Which of the fol lowing i s an energy-requiring s tep of glycolys is?

A. HexokinaseB. Phosphoenolpyruvate carboxykinaseC. Phosphoglycerate kinaseD. Pyruvate carboxylaseE. Pyruvate kinase (PK)

II-15. Which of the fol lowing processes generates the most ATP?A. Ci tric acid cycleB. Fatty acid oxidationC. Glycogenolys isD. Glycolys isE. Pentose phosphate pathway

II-16. Which of the fol lowing s tatements accurately describes features of ribosomes?A. An integra l part of transcriptionB. Bound together so tightly they cannot dissociate under phys iologic conditionsC. Composed of RNA, DNA, and proteinD. Composed of three subunits of unequal s i zeE. Found both free in the cytoplasm and bound to membranes

II-17. Chi ldren with cystinos is have growth delay, photosens i tivi ty with crysta ls in the lens of thei r eyes , and progress ive renal fa i lure becauseof accumulation of cystine in cel lular lysosomes. The defect involves a speci fic lysosomal membrane receptor that faci l i tates cystineegress , and an effective therapy has been found us ing ora l cysteamine, a compound s imi lar in s tructure to cystine. This therapy reflectsthe genera l principle that competi tive inhibi tors typica l ly resemble the s tructure of which of the fol lowing?A. An a l losteric regulator of enzyme/receptor activi tyB. Enzyme or receptor proteinC. Enzyme reaction productsD. Substrates or l igands that bind the enzyme/receptorE. The cofactor

II-18. Which of the fol lowing kinases plays a role in s igna l transduction for growth hormone, leptin, and prolactin?A. Adenylate kinaseB. Janus kinaseC. Mitogen-activated protein (MAP) kinaseD. Protein kinase AE. Pyruvate kinase (PK)

II-19. A 2-month-old boy i s brought to the emergency department in a coma after s leeping through the night and fa i l ing to awaken in the

morning. He i s given intravenous glucose and awakens . Serum levels of pyruvate, lactate, and a lanine are elevated, whereas asparticacid levels are reduced. A muscle biopsy shows no abnormal i ties , and vi tamin supplementation i s ineffective. Which of the fol lowingis the most l ikely diagnos is in this patient?A. Isoci trate dehydrogenase deficiencyB. Phosphofructokinase (PFK) deficiencyC. Pyruvate carboxylase deficiencyD. Pyruvate dehydrogenase (PDH) complex deficiencyE. Pyruvate kinase deficiency

II-20. Which of the fol lowing best expla ins why s tatin therapy i s effective for individuals with hypercholesterolemia?A. Bind to low-dens i ty l ipoprotein (LDL) receptor, displacing cholesterol and inhibi ting cholesterol synthes is .B. Inhibi t 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, a key regulator of cholesterol synthes is .C. Inhibi t HMG-CoA synthase, key s tep for synthes is of mevalonate that inhibi ts fatty acid synthes is .D. Stimulate synthes is of trans-unsaturated fatty acids .E. Stimulate thiolase, thus making more malonyl -CoA for inhibi tion of the tricarboxyl ic acid cycle.

II-21. GTP i s required by which of the fol lowing s teps in protein synthes is?A. Aminoacyl–tRNA synthetase activation of amino acidsB. Attachment of mRNA to ribosomesC. Attachment of ribosomes to endoplasmic reticulumD. Attachment of s igna l recognition protein to ribosomesE. Trans location of tRNA–nascent protein complex from A to P s i tes

II-22. Inheri ted deficiency of the enzyme methylmalonyl -CoA (MMA-CoA) mutase causes serum and urine accumulation of methylmalonic acid.Recognition that pernicious anemia (due to deficiency of vi tamin B12) can involve accumulation of methylmalonic acid led to success fultreatment of some patients with MMA-CoA mutase deficiency us ing excess B12. Studies of puri fied MMA-CoA mutase enzyme fromnormal individuals then showed enhanced mutase activi ty when B12 was added to the reaction mixture. These facts are best reconci ledby which of the fol lowing roles for Vi tamin B12?

A. Cofactor for the MMA- CoA mutase enzymeB. Competi tive inhibi tor of MMA-CoA mutase enzymeC. Covalently attached group for the enzyme methylmalonic acid-CoA mutaseD. Feedback inhibi tor of MMA-CoA mutase enzymeE. Precursor for methylmalonic acid synthes is

II-23. Which of the fol lowing s tatements describes where integra l proteins are located?A. Associated with DNA in the nucleusB. In the lumen of the endoplasmic reticulumC. In the mitochondria l matrixD. Mostly in the bloodE. Predominantly within cel l membranes

II-24. A 7-year-old boy arrives at the emergency department as leep in his father’s arms. The boy’s mother expla ins that the boy spent the nightthrowing up and experiencing severe diarrhea. She i s concerned about the vomiting and his inabi l i ty to s tay awake. His tory indicatesthe boy was heal thy yesterday, but became i l l a t dinnertime after spending time playing in the basement of thei r apartment complexthat afternoon. Further inquiry reveals that an exterminator had been hi red to take care of a rat problem in the apartment. He had useda poison (Rotenone) that blocks complex I in the respiratory cha in of oxidative phosphorylation. The boy i s pa le and not cyanotic. Ananalys is of this patient’s metabol i sm would l ikely indicate impaired function of which of the fol lowing enzymes?A. Glucose-6-phosphate dehydrogenaseB. PFKC. Pyruvate carboxylaseD. PDH complexE. Succinate dehydrogenase

II-25. A prime diagnostic indicator for the fi rs t presentation of diabetes mel l i tus type 1 i s the presence of ketonuria and severe acidos is ; theacidos is produces exaggerated attempts at respi ratory compensation known as Kussmaul breathing. Which of the fol lowing are“ketone bodies” that would be elevated in serum and urine from a chi ld with diabetic ketoacidos is?A. Acetone and ethanolB. Fumarate and succinateC. Oxaloacetate and pyruvateD. Pyruvate and lactateE. β-Hydroxybutyrate and acetoacetate

II-26. Aminoacyl–tRNA synthetases must be capable of recognizing which of the fol lowing?A. A speci fic amino acid and the 40S ribosomal subunitB. A speci fic amino acid and the 60S ribosomal subunitC. A speci fic ribosomal RNA (rRNA) and a speci fic amino acidD. A speci fic tRNA and a speci fic amino acidE. A speci fic tRNA and the 40S ribosomal subunit

II-27. Your patient presents with a deficiency of the enzyme that cata lyzes synthes is of N-acetylglutamic acid. Which of the fol lowing would bea consequence of this deficiency in the patient?A. Decreased digestion of dietary proteinB. Decreased excretion of uric acidC. Increased amino acids in the bloodD. Increased fatty acids in the bloodE. Increased glucose in the blood

II-28. A patient presents with anemia and hyperbi l i rubinemia, reflecting excess ive red blood cel l hemolys is . Her symptoms include fatigue,pa l lor, and jaundice. Further eva luation shows a defect in spectrin of her red blood cel l membranes . Which of the fol lowing i s thel ikely diagnos is?

A. Essentia l fatty acid (EFA) deficiencyB. Pyruvate kinase deficiencyC. Spherocytos isD. Tarui ’s diseaseE. Zel lweger syndrome

II-29. Which of the fol lowing describes the malate shuttle system?A. Carries NADH from the cytoplasm directly into the mitochondria l matrix.B. Generates three molecules of ATP in mitochondria per each NADH from glycolys is .C. Moves NADH from the mitochondria to the cytoplasm.D. Rel ies on malate dehydrogenase in the inner mitochondria l membrane.E. Requires reduction of pyruvate to lactate for oxidizing NADH.

II-30. Which of the fol lowing i s the fina l primary product of the fatty acid synthase reaction in adipose ti ssue?A. Acetyl -CoAB. Malonyl -CoAC. Pa lmitic acidD. Pa lmitoyl -CoAE. Propionic acid

II-31. A mutation that resul ts in a va l ine replacement for glutamic acid at pos i tion 6 of the β-chain of hemoglobin S hinders normalhemoglobin function and resul ts in s ickle cel l anemia when the patient i s homozygous for this mutation. This i s an example of whichof the fol lowing types of mutation?A. DeletionB. Frameshi ftC. InsertionD. MissenseE. Nonsense

II-32. In l iver, which of the fol lowing inhibi tory effects i s the key regulatory event that ensures newly synthes ized pa lmitoyl -CoA i s notimmediately oxidized?A. Acyl -CoA synthetase by malonyl -CoA.B. Carni tine–acylcarni tine trans locase (CAT) by pa lmitoyl -CoA.C. Carni tine–palmitoyl transferase-I (CPT-I) by malonyl -CoA.D. CPT-I by pa lmitoylcarni tine.E. Carni tine–palmitoyl transferase-II (CPT-II) by acetyl -CoA.

II-33. Some patients with fami l ia l hypercholesterolemia produce a truncated form of the LDL receptor, termed the “Lebanese” a l lele, whichlacks three of the five domains of the protein and causes i t to be reta ined in the endoplasmic reticulum. Analys is of the mutant geneindicated that the sequence of the protein was normal up to the point where i t terminated. The genetic change that produced themutant LDL receptor in these cases can be class i fied as which type of mutation?A. DeletionB. InsertionC. MissenseD. NonsenseE. Si lent

ANSWERSII-1. The answer is C. Important carbohydrates include the disaccharides maltose (glucose–glucose), sucrose (glucose–fructose), and lactose

(ga lactose–glucose), and the glucose polymers s tarch (cerea ls , potatoes , and vegetables ) and glycogen (animal ti s sues). Humans mustconvert dietary carbohydrates to s imple sugars (mainly glucose) for fuel , employing intestina l enzymes and transport systems forenzymatic digestion and absorption. Simple sugars (ga lactose and fructose) are converted to glucose by l iver enzymes , and the glucoseis revers ibly s tored as glycogen. Enzymatic deficiencies in intestina l digestion (e.g., lactase deficiency in those with lactoseintolerance), in sugar to glucose convers ion (e.g., ga lactose to glucose convers ion in ga lactosemia), or in glycogenes is/glycogenolys is(e.g., in those glycogen s torage diseases) resul t in glucose deficiencies (low blood glucose or hypoglycemia) with potentia laccumulation and toxici ty to hepatic ti s sues . The infant had been normal during breastfeeding, excluding low glucose due to growthhormone deficiency, and could readi ly digest breast mi lk lactose with absorption and convers ion to glucose. Low glucose during fastingand l iver enlargement impl ies a l tered regulation of glycogen synthes is/ release due to one of the enzyme deficiencies within thecategory of glycogen s torage disease. The hepatomegaly resul ts from the accumulation of excess ive amounts of glycogen (Table 6-1;Figure 6-9).

II-2. The answer is B. Fatty acid oxidation i s a major source of energy after glycogen i s depleted during fasting. Fatty acids are fi rs t coupledwith CoA, transferred for mitochondria l import as acylcarni tines , and degraded in s teps that remove two carbons . The fatty acyl -CoAdehydrogenases , enoyl hydratases , hydroxyacyl -CoA dehydrogenases , and thiolases that carry out each oxidation s tep are present inthree groups with speci fici ties for very long-/long-, medium-, and short-cha in fatty acyl es ters . As would be expected, deficiencies oflong-chain oxidizing enzymes have more severe consequences than those for short cha ins because they impair many more cycles oftwo-carbon removal . Long-chain deficiencies may be letha l in the newborn period, whereas medium- or short-cha in deficiencies maybe undetected unti l a chi ld goes without food for a prolonged time and must resort to extens ive fatty acid oxidation for energy.Medium-chain CoA dehydrogenase deficiency can be fata l i f not recognized, and sometimes presents as sudden unexpla ined deathsyndrome (usual ly at older ages than sudden infant death syndrome, that i s , mostly from respiratory problems). The defici t of acetyl -CoA from fatty acid oxidation impacts gluconeogenes is with hypoglycemia, and the energy defici t leads to heart, l i ver, and muscledisease that may be letha l . Unl ike most causes of hypoglycemia, the impaired fatty acid oxidation does not produce ketones(nonketotic hypoglycemia). Carni tine i s tied up as medium-chain acylcarni tines and i s secondari ly deficient in fatty acid oxidationdisorders . Rare primary carni tine deficiencies [as in answer option (e)] impair oxidation of a l l fatty acids because they cannot beimported into mitochondria .

II-3. The answer is C. Despi te some di fferences , protein synthes is in prokaryotes and eukaryotes i s qui te s imi lar. The smal l ribosomal subuniti s 30S in prokaryotes and 40S in eukaryotes . The large ribosomal subunit i s 50S in prokaryotes and 60S in eukaryotes . The intactribosome is consequently larger in eukaryotes (80S) and smal ler in prokaryotes (70S). At the s tart of trans lation, ini tiation factors ,

mRNA, and ini tiation aminoacyl–tRNA bind to the dissociated smal l ribosomal subunit. The ini tiation tRNA in prokaryotes i s N-formylmethionine in prokaryotes and s imply methionine in eukaryotes . Only after the smal l ribosomal subunit i s primed with mRNA andini tiation aminoacyl–tRNA does the large ribosomal subunit bind to i t. Once this happens , elongation factors bring the fi rs t aminoacyl–tRNA of the nascent protein to the A s i te. Then peptidyl transferase forges a peptide bond between the ini tiation amino acid and thefi rs t amino acid of the forming peptide. Now uncharged ini tiation tRNA leaves the P s i te and the peptidyl–tRNA from the A s i te moves tothe now vacant P s i te with the two amino acids attached. The ribosome advances three bases to read the next codon and the processrepeats . When the s top s ignal i s reached after the complete polypeptide has been synthes ized, releas ing factors bind to the s tops ignal , caus ing peptidyl transferase to hydro-lyze the bond that joins the polypeptide at the A s i te to the tRNA. Factors prevent thereassociation of ribosomal subunits in the absence of new ini tiation complex (Figure 9-5).

II-4. The answer is D. Glucose-6-phosphate dehydrogenase (G6PD) i s the fi rs t enzyme of the pentose phosphate pathway, a s ide pathway forglucose metabol i sm whose primary purpose i s to produce ribose and NADPH. Its deficiency i s the most common enzymopathy, a ffecting400 mi l l ion people worldwide. It contrasts with glycolys is in i ts use of NADP rather than NAD for oxidation, i ts production of CO2, i tsproduction of pentoses (ribose, ribulose, and xylulose), and i ts production of the high-energy compound (5-phosphoribosyl -1-pyrophosphate) rather than ATP. Production of NADPH by the pentose phosphate pathway i s crucia l for reduction of glutathione, whichin turn removes hydrogen peroxide via glutathione peroxidase. Erythrocytes are particularly susceptible to hydrogen peroxideaccumulation, which oxidizes red blood cel l membranes and produces hemolys is . Stresses such as newborn adjustment, infection, orcerta in drugs can increase red blood cel l hemolys is in G6PD-deficient individuals , leading to severe anemia, jaundice, plugging ofrenal tubules with released hemoglobin, renal fa i lure, heart fa i lure, and death. Because the locus encoding G6PD is on the Xchromosome, the deficiency exhibi ts X-l inked recess ive inheri tance with severe affl i ction in males and transmiss ion throughasymptomatic female carriers . Ribose-5-phosphate produced by the pentose phosphate pathway i s an important precursor forribonucleotide synthes is , but a l ternative routes from fructose-6-phosphate a l low ribose synthes is in ti s sues without the completecohort of pentose phosphate enzymes or with G6PD deficiency. The complete pentose phosphate pathway i s active in l iver, adiposetissue, adrenal cortex, thyroid, erythrocytes , testi s , and lactating mammary gland. Skeleta l muscle has only low levels of some of theenzymes of the pathway but i s s ti l l able to synthes ize ribose through fructose-6-phosphate (Figure 6-7).

II-5. The answer is E. Acetyl -CoA i s carboxylated to form malonyl -CoA through the addition of CO2 by acetyl -CoA carboxylase. The acetyl - andmalonyl -CoA groups are added to sul fydryl groups of fatty acid synthase multienzyme complex (one on each subunit) throughtransacylation reactions . Condensation forms acetoacetyl -S-enzyme on one subunit and a free sul fhydryl group of the other subunit—asequence of enzyme reactions then converts the acetoacetyl -S-enzyme to acyl (acetyl ) enzyme. A second round of two-carbon additionbegins , as another malonyl -CoA res idue displaces the acyl -S-enzyme to the other sul fhydryl group, and then condenses to extend theacyl group by two carbons . Fatty acid synthes is then proceeds by success ive addition of malonyl -CoA res idues with condensation,caus ing the acyl cha in to grow by two carbons with each cycle (Figure 7-1).

II-6. The answer is D. Protein synthes is occurs in the cytoplasm, on groups of free ribosomes ca l led polysomes, and on ribosomes associatedwith membranes , termed the rough endoplasmic reticulum. However, proteins destined for secretion are only synthes ized onribosomes of the endoplasmic reticulum and are synthes ized in such a manner that they end up ins ide the lumen of the endoplasmicreticulum. From there, the secretory proteins are packaged in ves icles . The Golgi apparatus i s involved in the O-glycosylation andpackaging of macromolecules into membranes for secretion.

II-7. The answer is A. Arginine i s an amino acid used in proteins that i s a l so part of the urea cycle. Ci trul l ine and orni thine are amino acidsnot used in proteins but important as urea cycle intermediates . Aspartate i s condensed with ci trul l ine to form argininosuccinate in theurea cycle, and acetylglutamate i s a cofactor in the joining of CO2 wi th ammonia to form carbamoyl phosphate at the beginning of theurea cycle (Figure 5-9A; a lso see Table 1-1).

II-8. The answer is B. Cholera toxin i s an 87-kDa protein produced by Vibrio cholerae, a Gram negative bacterium. The toxin enters intestina lmucosa l cel l s by binding to GM1 gangl ios ide. It interacts with Gs protein, which s timulates adenyl cyclase. By ADP-ribosylation of Gs, thetoxin blocks i ts capaci ty to hydrolyze bound GTP to GDP. Thus , the G protein i s locked in an active form, and adenyl cyclase s taysi rrevers ibly activated. Under normal conditions , inactivated G protein conta ins GDP, which i s produced by a phosphatase cata lyzing thehydrolys is of GTP to GDP. When GDP is so bound to the G protein, the adenyl cyclase i s inactive. Upon hormone binding to the receptor,GTP i s exchanged for GDP and the G protein i s in an active s tate, a l lowing adenyl cyclase to produce cAMP. Because cholera toxinprevents the hydrolys is of GTP to GDP, the adenyl cyclase remains in an i rrevers ibly active s tate, continuous ly producing cAMP in theintestina l mucosa l cel l s . This leads to a mass ive loss of body fluid into the intestine within a few hours (Figures 8-8, 8-9A; Table 8-2).

II-9. The answer is C. In the formation of phosphoenolpyruvate during gluconeogenes is , oxa loacetate i s an intermediate. In the fi rs t s tep,cata lyzed by pyruvate carboxylase, pyruvate i s carboxylated with the uti l i zation of one high-energy ATP phosphate bond:

Pyruvate + ATP + CO2 → Oxaloacetate + ADP + Pi

The pyruvate in gluconeogenes is i s derived mostly from lactate and to a large extent from a lanine, as wel l as to a lesser extent fromsome other amino acids .

In the second s tep, cata lyzed by phosphoenolpyruvate carboxykinase, a high-energy phosphate bond of GTP drives the decarboxylationof oxa loacetate:

Oxaloacetate + GTP → Phosphoenolpyruvate + GDP + CO2

In contrast to gluconeogenes is , the formation of pyruvate from phosphoenolpyruvate during glycolys is requires only PK, and ATP i sproduced (Figure 6-6).

II-10. The answer is A. Acetyl -CoA carboxylase cata lyzes the fi rs t s tep of l ipogenes is in which acetyl -CoA i s l inked to malonyl -CoA. This enzymeis activated by ci trate via polymerization and inactivated by pa lmitoyl -CoA that causes depolymerization to the monomeric form. Themonomeric form can undergo phosphorylation by epinephrine or glucagon to put i t into an inactive conformation that cannot readi lypolymerize. Insul in activation of a phosphatase reverses this cova lent modi fication. Acetyl -CoA does not readi ly cross themitochondria l membrane. Instead, ci trate trans locates to the cytosol where i t i s cleaved to acetyl -CoA and oxa loace-tate by ATP-ci tratelyase. Ci trate increases in the fed s tate and indicates an abundant supply of acetyl -CoA for l ipogenes is . Al though fatty acid synthase i sin the pathway, i ts regulation i s far less important. Acyl -CoA synthetase i s not in the l ipogenes is pathway per se because i t activatesfatty acids to the fatty acyl -CoA form regardless of thei r source. Carni tine–acylcarni tine trans locase and carni tine–palmitoyl transferaseare both involved in the process of fatty acid degradation (Figure 7-1).

II-11. The answer is E. The template s trand refers to the DNA strand that i s transcribed into mRNA. As for DNA, mRNA is synthes ized in the 5′ to3′ di rection. The template s trand i s a lways read in the 3′ to 5′ di rection. The oppos i te DNA strand i s known as the coding s trand andhas the same sequence as the mRNA transcript, except that U replaces T in mRNA. Choices b and d are DNAs, as they conta in T insteadof U.

II-12. The answer is D. Peps in i s secreted in a proenzyme form (peps inogen) in the s tomach. Unl ike the majori ty of proenzymes , i t i s notactivated by protease hydrolys is but instead by spontaneous acid hydrolys is . Hydrochloric acid secreted by the s tomach l ining createsthe acid envi ronment. Al l the enzymes secreted by the pancreas exis t a lso in a proenzyme form including tryps inogen,chymotryps inogen, procarboxypeptidase, and proelastase. Lactase and phenyla lanine hydroxylase are active in thei r native forms.Heparin and keratin are not enzymes.

II-13. The answer is A. Hormone s ignal transduction ul timately leads to the activation of a variety of kinases . Some of these depend on ca lciumfor thei r activi ty. One of these involves ca lcium fi rs t attaching to a ca lcium-binding protein, ca lmodul in. Hence, this kinase i s referred toas ca lmodul in-dependent kinase. Protein kinase C depends on interaction with both ca lcium and DAG for i ts ful l activi ty. The DAG isgenerated by the cleavage of membrane phosphol ipids cata lyzed by the enzyme phosphol ipase C. The other product of this cleavage i sIP3 that causes the release of ca lcium from the endoplasmic reticulum where i t i s s tored (Figure 8-9B).

II-14. The answer is A. Hexokinase cata lyzes the convers ion of glucose to glucose-6-phosphate in the energy-requiring fi rs t s tep of glycolys is .ATP i s a lso required in the convers ion of fructose-6-phosphate to fructose 1,6-bisphosphate by PFK. ATP i s generated in the convers ionof 1,3-bisphosphoglycerate to 3-phosphoglycerate by phosphoglycerate kinase and in the convers ion of phosphoenolpyruvate topyruvate by PK. Both phosphoenolpyruvate carboxykinase and pyruvate carboxylase are energy-requiring reactions except that theseoccur in the gluconeogenes is pathway (Figure 6-2).

II-15. The answer is B. The pentose phosphate pathway does not generate any ATP but instead forms NADPH and ribose phosphate. Glycolys isproduces a net two ATP molecules per glucose. The ci tric acid cycle produces a net 12 ATP per turn of the cycle. Fatty acid oxidation ofpa lmitate resul ts in a tota l of 129 ATP. Electron transport in the respiratory cha in resul ts in five ATP for each of the fi rs t seven acetyl -CoAproduced by the oxidation of pa lmitate for a tota l of 35 ATP. Each of the eight acetyl -CoA molecules produced from palmitate resul ts in12 ATP from the ci tric acid cycle for 96 tota l ATP. This gives a tota l of 131 ATP per pa lmitate oxidized, minus two ATP for the ini tia lactivation of pa lmitate for a grand tota l of 129 ATP per pa lmitate.

II-16. The answer is E. The two subunits of ribosomes are composed of proteins and rRNA. Ribosomes are found in the cytoplasm,mitochondria , and bound to the endoplasmic reticulum. Transcription refers to the synthes is of RNA complementary to a DNA templateand has nothing immediately to do with ribosomes.

II-17. The answer is D. Ligand–receptor and substrate–enzyme reactions are both saturable processes with s imi lar dependence of reaction rateon l igand/substrate and receptor/substrate concentrations . Competi tive inhibi tors function by binding to the substrate or l igand-binding portion of the active s i te and thereby block access to the substrate (Figure 5-3). Thus , the s tructures of competi tive inhibi torstend to resemble the s tructures of the substrate and are often ca l led substrate or l igand analogs . The effects of competi tive inhibi torscan be overcome by ra is ing the concentration of the substrate. The amount the substrate must be increased i s dependent on theconcentration of the inhibi tor, the affini ty of the inhibi tor for the enzyme, and the affini ty of the substrate for the enzyme. Formembrane receptors such as that in the lysosome that i s defective in cystinos is , the reaction may be one of membrane transport suchthat internal substrate/l igand i s in equi l ibrium with external substrate/l igand. Thus , lysosome–internal cystine i s substrate andlysosomal–external cystine a product, in a sense, such that lysosomal–external cysteamine wi l l effectively decrease external cystineconcentration and lead to egress of lysosomal cystine through i ts defective transporter.

II-18. The answer is B. Janus kinase i s a soluble receptor-associated tyros ine kinase that helps ful ly activate the receptor after binding of theappropriate hormone (e.g., growth hormone, prolactin, and leptin). Adenylate kinase cata lyzes the revers ible interconvers ion of 2 ADP↔ ATP + AMP. MAP kinase i s associated with transduction of s igna ls from growth factors (e.g., epidermal growth factor, fibroblastgrowth factor, insul in-l ike growth factor, and other cytokines and growth factors ) but not from growth hormone, which i s more s imi lar tothe metabol ic hormones than the growth factors . Protein kinase A i s involved in transducing s ignals from hormones that act via Gs

protein (e.g., catecholamines , glucagon, fol l i cle-s timulating hormone, parathyroid hormone, among others ). PK i s the las t enzyme of theglycolytic pathway (Figure 8-9C; Table 8-1).

II-19. The answer is C. Deficiencies of PK and PFK are ruled out by the elevated serum lactate levels , as these are glycolytic enzymes. The comais associated with a fasting hypoglycemia, which i s indicative of pyruvate carboxylase deficiency. The elevated lactate and a lanineoccurs because the pyruvate required for pyruvate carboxylase in gluconeogenes is i s derived mostly from lactate and to a large extenta lso from a lanine, as wel l as to a lesser extent some other amino acids . The reduction of aspartic acid levels occurs because there i sreduced formation of oxa loacetate, the product of the pyruvate carboxylase reaction and oxa loacetate provides the carbon backbone forsynthes is of aspartate (Figure 6-6).

II-20. The answer is B. Cholesterol i s formed in five s teps . The fi rs t s tep, biosynthes is of mevalonate, i s cata lyzed by three enzymes—acetyl -CoAthiolase, HMG-CoA synthase, and HMG-CoA reductase. Thiolase cata lyzes the condensation of two molecules of acetyl -CoA to formacetoacetyl -CoA. HMG-CoA synthase cata lyzes the addition of a thi rd molecule of acetyl -CoA to form HMG-CoA. This compound i sreduced to mevalonate by HMG-CoA reductase. This enzyme is the principa l regulatory s tep in the pathway. In the second s tep ofcholesterol synthes is , mevalonate i s phosphorylated and decarboxylated to produce i sopentyl diphosphate. Six of these i soprenoidunits are condensed to form squalene in the thi rd s tep. Lanosterol i s formed in the fourth s tep and i s subsequently converted tocholesterol (Figure 7-9).

II-21. The answer is E. Two molecules of GTP are used in the formation of each peptide bond on the ribosome. In the elongation cycle, bindingof aminoacyl–tRNA del ivered by EF-2 to the A s i te requires hydrolys is of one GTP. Peptide bond formation then occurs . Trans location ofthe nascent peptide cha in on tRNA to the P s i te requires hydrolys is of a second GTP. The activation of amino acids with aminoacyl–tRNAsynthetase requires hydrolys is of ATP to AMP plus PPi.

II-22. The answer is A. Smal l molecules may be integra l (cova lently attached) parts of enzymes (prosthetic groups) or cofactors that participatein enzyme–substrate interaction or convers ion. Prosthetic groups cannot be dissociated from the enzyme by di lution and thus wi l l notbe obvious components of the enzyme reaction when reconsti tuted in the test tube. Cofactors , such as vi tamin B12 for MMA-CoA mutase,associate revers ibly with enzymes or substrates and can be added in vi tro to obta in enhancement of the cata lyzed reaction(s ).Competi tive or feedback inhibi tors interact at substrate or a l losteric binding s i tes of the enzyme, reducing effective substrateconcentration and reaction rate or converting the enzyme to a less active conformation. Vi tamin B12 (cyanocobalamin) i s a cofactor forMMA-CoA mutase, accelerating the convers ion of methylmalonic acid to succinyl -CoA through activi ty of i ts cobal t group. Certa in defectsin MMA-CoA mutase can be amel iorated by intramuscular B12 injections so that effective B12 concentration and mutase activi ty areincreased.

II-23. The answer is E. Membrane proteins are class i fied as extrinsic—predominantly on the inner or the outer surface of the membrane bi layer—and intrinsic—predominantly within the membrane. Intrins ic proteins are usual ly composed of uncharged, hydrophobic amino acidsso they can enter and s tay in the hydrophobic envi ronment of the l ipid bi layer. Intrins ic proteins serve severa l functions includingchannels , “carrier proteins”—transporting molecules through the membrane as a “carrier protein” or as a s igna l ing protein—changing aportion of thei r internal cytoplasmic s tructure in response to a s igna l at an exposed external part of thei r s tructure to activateadditional , closely associated molecules leading to a resul ting function (e.g., turning on a gene) (Figure 8-4).

II-24. The answer is D. This patient exhibi ts severa l s igns of acute complex I poisoning. Because complex I of oxidative phosphorylation i s atleast partia l ly inhibi ted by the chi ld consuming this poison, the abi l i ty of mitochondria l NADH to be oxidized i s impaired.Consequently, NAD wi l l become l imiting for the PDH complex (Chapter 6). Al though glycolys is a lso requires regenerating NAD, enzymesin this pathway can operate normal ly because NADH can be oxidized by the convers ion of pyruvate to lactate. Glucose-6-phosphatedehydrogenase in the pentose phosphate pathway wi l l be unaffected because i t uses NADP not NAD (Figure 6-7A). Likely pyruvatecarboxylase wi l l have increased activi ty because pyruvate not used by the PDH complex could be processed to oxa loacetate. Fina l ly,succinate dehydrogenase wi l l be unaffected because i t produces FADH2 rather than NADH. Hence, i t feeds electrons into complex IIins tead of complex I of the respiratory cha in.

II-25. The answer is E. The ketone bodies , β-hydroxybutyrate and acetoacetate, are synthes ized in l iver mitochondria from acetyl -CoA. The l iverproduces ketone bodies under conditions of fas ting associated with high rates of fatty acid oxidation. The inabi l i ty to get glucose intoextrahepatic cel l s because of insul in deficiency in diabetes a lso increases fatty acid oxidation and ketogenes is . The acid groups of β-hydroxybutyrate and acetoacetate cause acidos is and an anion gap (sum of serum sodium and potass ium minus the sum of chlorideand bicarbonate) that i s greater than normal (over 8–15). In the case of diabetes , the “hidden anions” that add to bicarbonate inbalancing the cations can be recognized as ketones through urine ketostix testing. In metabol ic disorders such as methylmalonicaciduria or fatty acid oxidation defects , there are scanty abnormal or no ketones (i f fat cannot be oxidized) so the hidden anions mustbe identi fied by plasma acylcarni tine or urine organic acid profi les . Acetone i s a ketone body produced in diabetes that produces anacid breath during ketoacidos is (Figure 7-8A).

II-26. The answer is D. Aminoacyl–tRNA synthetases are respons ible for charging a tRNA with the appropriate amino acid for trans lation.Charging a tRNA is a two-step reaction. In the fi rs t s tep, the enzyme forms an aminoacyl–AMP enzyme complex in a reaction thatrequires one ATP. In the second s tep, the activated amino acid i s attached to the appropriate tRNA and the enzyme and AMP arereleased.

II-27. The answer is C. N-acetylglutamic acid i s a s timulant of the urea cycle. A patient with a deficiency of the enzyme that cata lyzes i tsformation would have a decreased activi ty of the urea cycle. Because the ni trogen for the cycle largely comes from amino acids , theblood concentration of amino acids would increase. A decreased abi l i ty to process these amino acids in fasting/starvation for glucoseproduction could potentia l ly lead to decreased blood glucose. Fatty acid usage would be unaffected. Al though the urea cycle processesthe ni trogen from amino acids derived from dietary proteins , thei r digestion would be unaffected. Instead, blood amino acids woulda lso ri se after a meal . Al though secretion of urea would be diminished, uric acid i s derived from the degradation of purine nucleotidesand that would be unaffected.

II-28. The answer is C. Diseases of red blood cel l s often resul t from changes in l ipid compos i tion and/or defects in proteins associated withthe red blood cel l membrane. Spherocytos is i s one such disease that resul ts from defects in spectrin, ankyrin, or other red blood cel lmembrane proteins important in the s tabi l i zation of the normal biconcave shape of red blood cel l s . This unique shape i s required forred blood cel l s to travel eas i ly and undamaged through blood vessels . As the name impl ies , red blood cel l s appear as spheres andpatients often suffer from low red blood cel l numbers (anemia) due to the resul ting destruction of the affected cel l s . EFA deficiency, PKdeficiency, and Tarui ’s disease (PFK deficiency) can a l l lead to anemia for various reasons . EFA deficiency causes instabi l i ty of the redblood cel l membrane and hence hemolys is . Both PK and PFK deficiencies resul t in insufficient energy production to mainta in red bloodcel l membrane integri ty. Al though Zel lweger syndrome can lead to jaundice, this i s related to diminished l iver function and not to redblood cel l i s sues .

II-29. The answer is B. The role of the malate shuttle i s , under aerobic conditions , to oxidize cytosol ic NADH produced during glycolys is toregenerate NAD to keep the glycolytic pathway operating. Because there i s no transporter to carry NADH into the mitochondria , theelectrons retrieved during the cytosol ic oxidation of NADH must be carried across the mitochondria l inner membrane to the matrix asmalate. In the matrix, malate dehydrogenase in the ci tric acid cycle (Figure 6-4) uses the malate to produce NADH that i s subsequentlyprocessed via oxidative phosphorylation (Figure 6-5) to produce three molecules of ATP. Under anaerobic conditions , NAD isregenerated for glycolys is by the reduction of pyruvate to lactate.

II-30. The answer is C. The primary fatty acid that i s s tored for energy purposes i s the 16-carbon pa lmitic acid. The product of fatty acid synthaseis a lways the free (unesteri fied) fatty acid. In a subsequent s tep cata lyzed by acyl -CoA synthetase, the fatty acid i s activated to i ts fattyacyl -CoA form. Acetyl -CoA i s a precursor for the synthes is of fatty acids , and malonyl -CoA i s an intermediate in the process . Propionicacid i s a product of odd-chain fatty acid degradation (Figure 7-2).

II-31. The answer is D. Missense mutations are those in which a s ingle base change (point mutation) resul ts in a codon that encodes for adi fferent amino acid res idue. The effects of these types of mutations can range from very minor or even undetectable to major,depending on the importance of the a l tered res idue to protein folding and function. Nonsense mutations are a lso point mutations inwhich the affected codon i s a l tered to a s top (nonsense) codon, resul ting in a truncated protein. Frameshi ft mutations are due to oneor two base pa i r insertions or deletions such that the reading frame is a l tered. These mutations genera l ly lead to truncated proteinsas wel l because, in most protein coding regions , the unused reading frames conta in numerous s top codons .

II-32. The answer is C. Malonyl -CoA i s a unique intermediate in the pathway for fatty acid synthes is and wi l l only be high in concentration inthe cel l when fatty acid synthes is i s active. When the cel l i s synthes izing fatty acids , i t would be energetica l ly i l logica l to immediatelyoxidize the newly formed fatty acid. The s implest means of s lowing fatty acid oxidation i s to prevent the formation of the fattyacylcarni tine derivative that must be formed for transport of the fatty acid into the mitochondria l matrix for oxidation. Inhibi tion of acyl -CoA synthetase would not work because the fatty acyl -CoA form must be produced for esteri fi cation of glycerol to form triacylglycerol fors torage. Any control by pa lmitoyl -CoA would be ineffective because this i s the substrate for fatty acid oxidation. Inhibi tion of CAT wouldbe i l logica l because the fatty acylcarni tine derivatives would accumulate and be useless for any other function. Inhibi tion of carni tine–palmitoyl transferase-I by pa lmitoylcarni tine would be feedback inhibi tion by i ts product and would not be effective. Inhibi tion ofcarni tine–palmitoyl transferase-II by acetyl -CoA would cause accumulation of pa lmitoylcarni tine in the mitochondria l matrix, leading toa depletion of carni tine (Figure 7-3).

II-33. The answer is D. Production of a truncated protein indicates that a mutation has occurred, but this phenomenon may have arisen from aframeshi ft mutation (insertion or deletion) or by a nonsense mutation. The most l ikely poss ibi l i ty i s a nonsense mutation becausesequence analys is of the truncated protein showed that i t had normal (wi ld-type) sequence. Insertion and deletion events oftenproduce a s tretch of garbled or abnormal protein sequence at the C-terminal end of the truncated protein ari s ing from out-of-frametrans lation of the mRNA downstream of the mutation unti l a s top codon i s encountered.

SECTION IIIAPPLIED BIOCHEMISTRY

CHAPTER 10METABOLISM AND VITAMINS/MINERALS

Co-authors/Editors: Maria L. Valencik and Cynthia C. MastickUnivers i ty of Nevada School of Medicine, Department of Biochemistry, Reno, NV

Metabol ic Roles of Major Biochemica l MoleculesIntegration and Regulation of Metabol i smHormonal Control of Metabol i smVitamins and Minera lsReview Questions

OVERVIEWThe integration of metabol i sm is a s tory of supply and demand. Food i s ingested to supply energy but must be converted to the carbohydrate,l ipid, and amino acid forms the body can use, primari ly glucose and fatty acids . Individual cel l s then convert the fuels to usable energy,adenos ine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH). The body demands energy to function but individual organsand ti ssues require particular sources of energy under varying conditions .

To convert consumed food into the needed energy, the body uses a variety of organs , each with unique metabol ic profi les , to integrate andregulate the use and s torage of energy. Speci fic regulatory points of biochemica l pathways provide immediate control of the usage,convers ion, or s torage of food energy. Various hormones can a lso regulate these biochemica l pathways to provide longer term control of foodconvers ion and energy usage. Essentia l to both of these processes i s the maintenance of glucose homeostas is . Fina l ly, vi tamins and minera lsserve important functions as cofactors in many of these metabol ic reactions . Their deficiency or excess can lead to numerous disease s tates .

METABOLIC ROLES OF MAJOR BIOCHEMICAL MOLECULESThe fi rs t cons ideration i s the major sources of energy that can be used by the body, the nutrients required for thei r metabol i sm, and thebiochemica l pathways that integrate them.

Amino acids (Chapter 1, Figure 10-1) provide severa l major biochemica l functions , including serving as (1) the bui lding blocks of proteins ; (2)the precursors of hormones , neurotransmitters , and other important s igna l ing molecules (such as ni trous oxide); and (3) contributors to thepurine and pyrimi-dine components of nucleic acids , co-enzymes [NADH and flavin adenine dinucleotide (FADH2)], and other fundamenta lbiologica l molecules . Additional ly, excess amino acids can enter the ci tric acid cycle and can be used to generate or s tore biologica l energy(Chapter 5). Furthermore, the metabol i sm of some amino acids can be funneled into glucose synthes is (gluconeogenes is ) during fooddeprivation.

Figure 10-1. Summary of Amino Acid Metabolism. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

Carbohydrates (Chapter 2, Figure 10-2) perform a fundamenta l role as the primary energy-production source for the human body. Glycolys isand the subsequent metabol ic pathways form the primary energy molecules ATP, NADH, and FADH2 via the oxidation of glucose and othercarbohydrates (Chapter 6). Storage of carbohydrates as glycogen offers a readi ly ava i lable source of energy when dietary carbohydrate intake i slow (Chapter 2). Carbohydrates are a lso important in the synthes is of nicotinamide adenine dinucleotide phosphate (NADPH) (Chapter 6) andnucleic acids (Chapter 4).

Figure 10-2. Transport and Fate of Major Carbohydrates and Amino Acids. [Reproduced with permiss ion from Murray RA, et a l .: Harper’s I l lus tratedBiochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Lipids (Chapter 3, Figure 10-3) are nonpolar biomolecules . In most ti s sues , they serve a primary s tructura l role as the components ofbiologica l membranes , creating a l ipid bi layer via thei r hydrophobic and hydrophi l i c enti ties (Chapters 7 and 8). Their roles in membranes aswel l as in pathologica l processes such as atheroscleros is (Chapter 16) have ra ised the awareness of saturated, mono-unsaturated, and poly-unsaturated forms with regard to thei r role in diet. However, in adipose ti ssue, triglycerides are the major s torage form of biologica l energyand their oxidation yields more energy per carbon than carbohydrates (Chapter 7). Lipolys is of triglycerides mobi l i zes fatty acids that generateenergy through β-oxidation and produces the substrates necessary for ketone body (acetoacetate and β-hydroxybutyrate) synthes is , anessentia l fuel source during prolonged s tarvation. Oxidation of both fatty acids and ketone bodies spares glucose by preventing i ts oxidation.The consumption of dietary cholesterol and fats has a large impact on l ipid metabol i sm through the generation of plasma l ipoproteins[chylomicrons and low-dens i ty l ipoprotein (LDL) via very-low-dens i ty l ipoprotein (VLDL)]. The resul tant elevation of harmful l ipids/l ipoproteins(dys l ipidemia) has negative metabol ic consequences that di rectly impact heal th and disease throughout a l l socioeconomic classes of modernsociety.

Figure 10-3. Transport and Fate of Major Lipid Substrates and Metabolites. FFA, free fatty acids ; LPL, l ipoprotein l ipase; MG, monoacylglycerol ; TG,triacylglycerol ; and VLDL, very-low-dens i ty l ipoprotein. [Reproduced with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry,28th edi tion, McGraw-Hi l l , 2009.]

Vitamins, both l ipid and nonl ipid derived, serve important roles as cofactors in metabol ic pathways and reactions (see end of this chapter).Severa l diseases , including scurvy, rickets , and Wernicke–Korsakoff syndrome, resul t di rectly from deficiencies of vi tamins or, as in perniciousanemia, from the body’s inabi l i ty to properly absorb them. Minerals, including sodium, potass ium, chloride, ca lcium, phosphate, i ron, andothers , play major roles in the regulation of metabol ic enzymes involved in digestion, in the use and/or s torage of food metabol i tes , and inthe el imination of waste products .

Even more important i s the integration of metabol i sm of these molecules in the human body and how regulation can be mainta ined byinterrelationships between their anabol ic and catabol ic metabol i sm. In this regard, the body’s abi l i ty to sense energy levels , respond tohormone s ignal ing, and upregulate and downregulate particular metabol ic pathways i s paramount for the body to mainta in the proper andcontrol led level of metabol ic function and for the myriad of s tructura l and functional processes to occur, which a l low l i fe.

INTEGRATION AND REGULATION OF METABOLISM

ATP, associated with magnes ium (Mg2+) for s tabi l i ty, i s the primary form of biologica l energy uti l i zed by the human body (Figure 10-4).

Figure 10-4. Adenosine Triphosphate Structure with Its Magnesium Cofactor. [Reproduced with permiss ion from Murray RA, et a l .: Harper’s I l lus tratedBiochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

As such, the catabol ic oxidation of carbohydrates (glycolys is , ci tric acid cycle, and oxidative phosphorylation), fatty acids/ l ipids/ketonebodies (fatty acid degradation), and amino acids a l l lead eventual ly to the production of ATP. In contrast, anabol ic metabol ic processes(gluconeogenes is , glycogen synthes is , l ipid synthes is , triglyceride synthes is , and amino acid synthes is ) consume ATP, NADH, and/or NADPH tostore energy (glucose), to s tore energy, or to bui ld essentia l biomolecules . Coupled to a l l of these processes i s the need to el iminate wasteproducts , including CO2 (exhalation, acid–base ba lance), reactive and/or free-radica l species (antioxidants ), and urea (urea cycle). Theseconcepts are summarized in Figure 10-5.

Figure 10-5. Interrelationship Between Proteins, Carbohydrates, and Fats. ATP, adenos ine triphosphate; CoA, coenzyme A. [Adapted with permiss ionfrom Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

These metabol ic pathways are intimately l inked at severa l points in biochemica l pathways , but are a lso separated into dis tinctcompartments and/or organel les (e.g., cytoplasm versus mitochondria versus nucleus , etc.) to a l low the necessary regulation and control .Additional ly, each organ has unique metabol ic needs and functions as summarized in Figure 10-6. These functions and needs must becoordinated in a variety of organs to mainta in a constant supply of energy whi le preserving some energy for the future. The body accompl ishesthis goal by us ing the nervous system and hormonal s igna ls to di fferentia l ly s timulate and inhibi t biochemica l pathways within variousorgans in response to supply and demand. The main s ignals used to regulate metabol i sm are insul in, glucagon, catecholamines ,glucocorticoids , and growth hormone (in chi ldren).

Figure 10-6. Integration of Metabolism Among Major Organs. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

The remainder of this chapter wi l l focus on the metabol i sm in three major ti s sues , the l iver, adipose ti ssue, and skeleta l muscle (Figure 10-6). The l iver actively provides the quick fuel (glucose) your body needs , whereas adipose ti ssue provides long-term energy s torage. Fina l ly,skeleta l muscle and the rest of your body constantly demand this energy. For example, the bra in consumes approximately 90 g of glucose in aday, 20% of the average diet.

The supply and demand of energy must be continuous ly provided via dietary intake or breakdown of s tores to ba lance with the energyrequirements of respi ration, transport, moti l i ty, and synthes is of cel l s and ti ssues (Figure 10-7). Overa l l , the average adult uses approximately24 kca l of energy per ki logram of body mass to insure proper heal th and to mainta in proper weight.

Figure 10-7. Factors Affecting Blood Glucose. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

Severa l key biomolecules (glucose-6-phosphate or G6-P, pyruvate, and acetyl coenzyme A or acetyl -CoA) l ink the biochemica l pathways forcarbohydrates , l ipids , and amino acids/ proteins and the pathways they funnel into are tightly regulated and ti ssue speci fic (Figure 10-8). G6-P, pyruvate, and acetyl -CoA l ink the anabol ic and catabol ic pathways of carbohydrate metabol i sm to mainta in a constant supply of energy tomainta in homeostas is under constantly changing conditions . The particular pathways and regulation a lso depend on the speci fic functionsand needs of each ti ssue type.

Figure 10-8. Summary of Important Control Points of Metabolism. The three important intermediaries , glucose-6-phosphate, pyruvate, and acetyl -CoA are indicated. Metabol ic pathways of importance are indicated in red. See text for ful l discuss ion. ATP, adenos ine triphosphate; CoA,coenzyme A; NADPH, nicotinamide adenine dinucleotide phosphate. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, JaypeeBrothers Medica l Publ i shers (P) Ltd., 2009.]

GLUCOSE-6-PHOSPHATE

Metabol ic regulation at this fi rs t branch point, G6-P, i s clearly i l lus trated in the l iver (Figure 10-8). After the ingestion of carbohydrates , glucosetaken up by the l iver i s converted to G6-P by glucokinase. This phosphorylation uses one ATP molecule and traps the glucose within l iver cel l s(hepatocytes ). Subsequently, G6-P i s metabol i zed via one of the fol lowing three pathways : (a ) glycogenesis—the s torage of carbohydrates asglycogen, (b) glycolysis—the production of ATP, or (c) the pentose phosphate pathway—the production of NADPH and/ or five-carbon (pentose)sugars (Chapter 6, Figure 10-8). The pathway chosen depends upon the activation s tate of key enzymes (glycogen synthase andphosphofructokinase-1), substrate ava i labi l i ty (G6-P, ATP, and NADP+), and a l losteric effectors [ATP, adenos ine monophosphate (AMP), fructose2,6-bisphosphate (F2,6BP), hydrogen ions (H+), and ci trate]. The key enzymes in glycogenes is and glycolys is are predominantly regulated byhormone-stimulated, cova lent modi fication (phosphorylation), whereas the a l losteric effectors fine-tune the flow of carbons through thesepathways . In contrast, the pentose phosphate pathway i s primari ly regulated by the ava i labi l i ty of G6-P and NADP+ (Chapter 6).

In the wel l -fed s tate, when ATP and ci trate concentrations are high, phosphofructokinase-1 i s a l losterica l ly inhibi ted, s lowing down thecommitted s tep of glycolys is (the production of fructose 1,6-bisphosphate) leading to increased concentrations of G6-P. The increasedconcentration of G6-P can s timulate carbohydrate s torage in two ways . Fi rs t, G6-P i s a pos i tive a l losteric effector of glycogen synthase, leadingto the formation of glycogen. Second, G6-P indirectly inhibi ts glycogen phosphorylase thereby inhibi ting glycogenolysis (glycogen degradation).Al ternatively, when the ratio of NADP+ to NADPH is high, G6-P can be shuttled into the pentose phosphate pathway to generate NADPH(reductive energy). This reducing power i s used to synthes ize a variety of biomolecules such as , fatty acids , cholesterol , nucleotides and othercofactors as needed. Under conditions where the ratio of NADP+ to NADPH is low, the pentose pathway wi l l not operate regardless of theconcentration of G6-P.

The production of glycogen in the l iver i s further control led by the hormones , insul in and glucagon (see below), and the resul tingphosphorylation or dephosphorylation of glycogen synthase. Degradation of glycogen i s decreased concomitantly by counterregulatorydephosphorylation or phosphorylation of glycogen phosphorylase. Eating breakfast, a fter an overnight fast, s timulates glycogen synthes is(and inhibi ts glycogen breakdown) in preparation for the next period of fas ting. This replenishment i s control led by the favorable high ratio ofinsul in to glucagon, leading to activation of glycogen synthase activi ty and decreased glycogen phosphorylase activi ty. Under these conditions ,the demand for de novo synthes is of l ipid wi l l ri se after the glycogen i s replaced, us ing carbons from excess dietary carbohydrate (tosynthes ize fatty acids ). Additional ly, i f cholesterol biosynthes is i s active, excess acetyl Co A from fatty acid catabol i sm can be synthes ized intocholesterol . Once l ipid biosynthes is commences , the uti l i zation of NADPH increases the NADP+/NADPH ratio favoring flux through the pentosephosphate pathway.

After a meal , the key regulator that restarts glycolys is in l iver i s F2,6BP. F2,6BP concentration i s control led by a bifunctional enzyme thatincludes both kinase and phosphatase active s i tes . Under conditions of a high ratio of insul in to glucagon, the bi functional enzyme(phosphofructokinase-2/fructose 2,6-bisphosphatase) i s dephosphorylated, leading to s timulation of phosphofructokinase-2. The resul ting F2,6BPformed a l losterica l ly activates phosphofructokinase-1 and hence increases glycolys is whi le s imultaneous ly inhibi ting fructose 1,6bisphosphatase, therefore shutting down gluconeogenes is . Fol lowing food deprivation, these events are reversed with a high ratio ofglucagon to insul in, favoring phosphorylation of the bi functional enzyme s timulating the fructose 2,6-bisphosphatase activi ty and leading todecreased phosphofructokinase-1 activi ty.

PYRUVATE

The second major branch point in the integration of metabol i sm is at pyruvate (Chapter 6, Figure 10-8). Pyruvate can be converted into fourdi fferent substrates : lactate, a lanine, oxa lo-acetate, and acetyl -CoA, depending upon the energy needs of a cel l . Therefore, i t i s an importantintegration point where carbons are shuttled between energy s torage, energy generation, and/or biosynthetic reactions . In the l iver, pyruvatecan undergo oxidative decarboxylation to enter the ci tric acid cycle and ul timately generate ATP when energy levels are low. Speci fica l ly, lowenergy levels inhibi t the activi ty of an important regulatory enzyme, pyruvate dehydrogenase kinase. This inhibi tion prevents phosphorylation of

the pyruvate dehydrogenase complex to an inactive s tate. Furthermore, this kinase i s inhibi ted by NAD+, pyruvate, and sul fhydryl form of CoA(non-acetylated), substrates of pyruvate dehydrogenase. Therefore, when substrates are plenti ful , pyruvate i s oxidatively decarboxylated toacetyl -CoA. In the l iver, pyruvate i s a lso the point where lactate and a lanine (see below) can be actively funneled into ei ther the ci tric acidcycle or gluconeogenes is via pyruvate carboxylation to oxa loacetic acid (Chapter 6) when l iver glycogen or blood glucose levels are low. Duringstarvation, gluconeogenes is can produce up to 160 g of glucose in a day, ha l f of this from amino acids . Ha l f of this glucose wi l l be used by thebra in. As blood glucose levels s tabi l i ze and gluconeogenes is i s no longer required, oxa loacetate can re-enter the glycolytic pathway atphosphoenolpyruvate or shuttle back into the mitochondria , as malate, to be used in the ci tric acid cycle. If energy i s abundant, high NADHand acetyl -CoA concentrations activate pyruvate dehydrogenase kinase and a lso serve as a l losteric inhibi tors of enzymatic activi ties withinthe PDH complex. This effectively turns off the pyruvate dehydrogenase complex by phosphorylation and a l losteric control and shuts down theci tric acid cycle. High ATP and acetyl -CoA concentrations a lso s timulate pyruvate carboxylase, the fi rs t s tep of gluconeogenes is (hormoneregulation of gluconeogenes is i s even more important; see below).

Skeleta l muscle i l lus trates another important way that pyruvate can be metabol i zed (Figure 10-9). If oxygen levels are low and anaerobicrespiration becomes important (such as during a quick sprint), pyruvate can be converted to lactate by lactate dehydrogenase wi th an oxidation ofone NADH to NAD+, the latter being essentia l for susta ining glycolys is . In this scenario, ATP i s solely derived from anaerobic glycolys is . Lactatecan subsequently be converted back to glucose for energy production via the Cori cycle (in the l iver); when lactate concentrations get too high,feedback inhibi tion blocks further convers ion of pyruvate to lactate. High lactate concentrations a lso create the sensation of “burning” inmuscles , which serves as a s igna l to the body to l imit further use of these muscles . Furthermore, pyruvate can a lso be converted in muscleti ssue to the amino acid alanine via the alanine transaminase reaction. In a manner analogous to the Cori cycle, the alanine cycle then convertsthis a lanine back to pyruvate in the l iver where i t i s used to produce new glucose via gluconeogenes is as a source of energy for anaerobicglycolys is in muscle. Once oxygen levels are restored in skeleta l muscle, production of ATP via ci tric acid cycle/oxidative phosphorylationresumes.

Figure 10-9. The Lactic Acid (Cori) and Glucose–Alanine Cycles. Carbons from glucose metabol i sm in muscle are recycled to the l iver ei ther as lactateor a lanine for reconvers ion to glucose. Hence, when these cycles operate glucose carbons are spared. [Adapted with permiss ion from MurrayRA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

ACETYL-COA

Acetyl-CoA i s the thi rd branch point of primary metabol ic control , and coordinates carbohydrate, ketone, and fat/l ipid pathways (Chapter 6,Figures 10-8 and 10-10). Acetyl -CoA i s a substrate for the ci tric acid cycle and can be oxidized to generate energy. However, when energy levelsare high (high NADH/ NAD+ ratio), NADH inhibi ts the ci tric acid cycle at the i soci trate dehydrogenase and α-ketoglutarate dehydrogenase s teps .Accumulation of FADH2 a l so occurs , leading to an increase in succinyl -CoA that inhibi ts the cycle as wel l . Hormones a lso play a key, longerterm role in the regulation of fatty acid synthes is and degradation (see below). Acetyl -CoA i s a lso required for production of theneurotransmitter acetylchol ine (see below and Chapter 19).

Figure 10-10. Overview of Acetyl-CoA Metabolism. ATP, adenos ine triphosphate; CoA, coenzyme A; TCA, tricarboxyl ic acid. [Adapted with permiss ionfrom Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

In the fed s tate, excess acetyl -CoA can be di rected toward synthes is of cholesterol and/or fats/triacylglycerols in the l iver. During s tarvation,fatty acid oxidation suppl ies energy for hepatocytes to drive gluconeogenes is . Furthermore, any excess acetyl -CoA generated wi l l be used forthe synthes is of ketone bodies . The ketones cannot be oxidized by the l iver and are exported and used as an a l ternate fuel for the bra in,heart, and muscles . In fact, during fasting/starvation, the bra in wi l l be heavi ly rel iant on ketone bodies , us ing them for up to 70% of i ts energyrequirements , especia l ly in prolonged s tarvation. Hormones a lso regulate ketone body synthes is (see below).

In both nutri tional ci rcumstances , acetyl -CoA may activate pyruvate carboxylase, a l though for di fferent purposes . In the fed s tate, pyruvatecarboxylase converts pyruvate to oxa loace-tate, which condenses with acetyl -CoA producing ci trate, the fi rs t product of the ci tric acid cycle. Theci trate i s transported to the cytoplasm for fatty acid synthes is . High ci trate activates acetyl-CoA carboxylase to promote the formation of fattyacids (Chapter 7). Ci trate, when too high, inhibi ts phosphofructokinase-1, thus blocking glycolys is to prevent unnecessary metabol i sm of moreglucose to pyruvate. The G6-P that backs up can be cycled through the pentose pathway to provide NADPH for fatty acid synthes is , as describedabove, or may be di rected toward glycogen synthes is . In the s tarvation s tate, high acetyl -CoA from oxidation of fatty acids s timulates pyruvatecarboxylase to promote gluconeogenes is .

Low concentrations of citrate and the other intermediates of the ci tric acid cycle as wel l as low ATP/NADH/FADH2 promote continuation ofthe ci tric acid cycle and oxidative phosphorylation. The ci tric acid cycle intermediates can a lso be used for the production of amino acids or asan energy source (Chapter 5). Low ci trate/high pa lmitoyl -CoA (from l ipolys is ) concentrations prevent fatty acid synthes is . The resul tantdecrease of malonyl-CoA, an a l losteric inhibi tor of carni tine pa lmitoyl transferase 1 (CPT1), favors formation of pa lmitoyl carni tine by CPT1, withsubsequent transport across the mitochondria l membrane and oxidation in the mitochondria .

HORMONAL CONTROL OF METABOLISMThe coordination of metabol ic pathways to achieve this essentia l ba lance primari ly depends on hormone; nerve and s ignal ing pathways ,including insul in, glucagon, catecholamines (Chapter 19), glucocorticoids (s lower, s tress -related changes); and cytokines . Errant control leadsto disease s tates i f glucose levels are high (diabetes mel l i tus or DM) or low (hypoglycemia) and, i f too low, even death due to coma.

INSULIN

Insul in (Figure 10-11) i s the anabol ic hormone of the wel l -fed s tate and an important s igna l to s timulate s torage of excess nutrients asglycogen and triglycerides (fat in adipose ti ssue).

Figure 10-11. Preproinsulin Processing. Preproinsul in (top) i s composed of a leader sequence (blue), A (green) and B (yel low) insul in cha ins , and C(red) peptide. Removal of the leader sequence produces proinsul in (bottom left). Cleavage of C-peptide from proinsul in leads to theproduction of active insul in. Because C-peptide i s produced in equal amounts to insul in, i t has become an important measure of insul inproduction. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

The action of insul in (Figure 10-12) i s experienced by three main targets , the l iver, adipose ti ssue, and s triated muscle. The synthes is andrelease of insul in i s s timulated by glucose and potentiated by amino acids . In the l iver, insul in s timulates glycogenes is (glycogen synthes is ),fatty acid synthes is , glycolys is , and the pentose phosphate pathway. In the adipose ti ssue, i t s timulates glucose and fatty acid uptake andtriglyceride synthes is (energy s torage). Simi larly, in skeleta l muscle, i t s timulates glucose uptake, glycogenes is , and protein synthes is . It i snoteworthy that insul in does not influence glucose metabol i sm in ei ther the bra in or red blood cel l s .

Figure 10-12. Metabolic Systems Affected by Insulin. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

The release of insul in from the pancreatic β-cel l s (Figure 10-13) i s the resul t of increased blood glucose concentrations . Glucose enters theβ-cel l s via the glucose transporter 2 (GLUT2) (pass ive transport). The GLUT2 has a weak affini ty for glucose so that i t favors glucose uptake onlyafter a meal , when blood glucose levels are high, rather than in the fasted s tate. Fol lowing glucose oxidation, the increased ATPconcentration s timulates K+ channels and depolarizes the cel l membrane. This depolarization opens vol tage-gated Ca 2+ channels . Others ignals related to production of inos i tol tri sphosphate, a second messenger, s timulate Ca 2+ release from the endoplasmic reticulum,resul ting in high intercel lular Ca 2+ concentration and triggering the release of insul in.

Figure 10-13. Regulation of Secretion of Insulin via Glucose. Pancreatic β-cel l s are induced to secrete insul in by (1) the uptake of glucose and i tsoxidative metabol i sm in mitochondria , which produces increased concentration of ATP. (2) Increased ATP causes closure of the ATP-sens i tive K+

channels , leading to depolarization. (3) The depolarization of the cel l causes in influx of Ca 2+ from vol tage-gated Ca 2+ channels . (4) Theincreased Ca 2+ a long with IP3 and other s igna l ing induces further release of Ca 2+ from the endoplasmic reticulum (ER), which promptsexocytos is of insul in, produced in the ER, and subsequently processed and released from secretory granules of the Golgi apparatus . ATP,adenos ine triphosphate; GLUT2, glucose transporter 2; IP3, inos i tol tri sphosphate. [Adapted with permiss ion from Kibble JD and Halsey CR: TheBig Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Insul in affects the metabol i sm of cel l s that have insul in receptors : l i ver cel l s (hepatocytes ), fat cel l s (adipocytes ), and muscle cel l s (Table10-1). The bra in and red blood cel l s are not affected by insul in. Insul in works via a tyros ine kinase receptor, which phosphorylates targetproteins that lead to a number of metabol ic effects . One effect i s the rapid trans location of a glucose transporter 4, GLUT4, from ves icles to thecel l surface of skeleta l and cardiac muscle and fat cel l s , increas ing glucose transport into these cel l s . Insul in a lso regulates metabol ic

enzymes such as glycogen synthase and phosphorylase through activation of type I phosphatase and dephosphorylation.

TABLE 10-1. Insul in Effects on Metabol i sm

GLUCAGON

Glucagon (Figure 10-14) i s the hormone of fas ting produced by pancreatic α-cel l s , adjacent to the insul in-producing α-cel l s . Glucagon s ignalsvia G-protein coupled receptors and the secondary messenger molecule cycl ic AMP. In contrast to many mammals , glucagon acts a lmostexclus ively on the l iver in humans (Table 10-2). Primari ly, i t s timulates glycogenolys is , gluconeogenes is , and fatty acid oxidation. Glucagonlevels increase two-to threefold in response to hypoglycemia, and the l iver begins production of glucose from glycogen. During times of highblood glucose, glucagon i s reduced to ha l f of i ts normal level . Glucagon a lso s timulates the release of insul in, thereby a l lowing insul in-sens i tive cel l s to take up the released glucose. The del icate ba lance of glucagon and insul in levels i s how the body mainta ins glucosehomeostas is under varying conditions .

Figure 10-14. Preproglucagon. Preproglucagon, produced by pancreatic α-cel l s , i s processed to active glucagon (orange). [Adapted withpermiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

TABLE 10-2. Glucagon Effects on Metabol i sm

CATECHOLAMINES

Catecholamines , including norepinephrine and epinephrine, the latter being primari ly the hormone respons ible for the “fight or fl ight”response to external s tresses , can provide a lmost immediate (within seconds) regulation of metabol i sm (Figure 10-15). Speci fica l ly, theystimulate glycogenolys is and glycolys is for the production of ATP in the muscle. At the same time, they inhibi t glycolys is in the l iver andstimulate glycogenolys is to provide glucose for the blood. More recently, synaptica l ly released catecholamines have emerged as the mainphys iologica l pathway for the activation of l ipolys is under conditions of fas ting (a condition of chronic s tress ). The effects of epinephrine onmetabol i sm are summarized in Table 10-3.

Figure 10-15. Integrated Control of Blood Glucose Concentration. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica lPhys iology, 1st edi tion, McGraw-Hi l l , 2009.]

TABLE 10-3. Epinephrine Effects on Metabol i sm

GLUCOCORTICOIDS

Cortisol , a glucocorticoid, i s a chronic s tress hormone that a lso regulates metabol i sm but in the time frame of hours to days . With prolongedstress , the hypothalamus increases secretion of corticotrophin-releas ing factor, which subsequently leads to production and secretion ofadrenocorticotropic hormone from the anterior pi tui tary gland and then corti sol from the adrenal glands . Corti sol has much of the sameinfluence on metabol i sm as epinephrine but functions via activation of transcription and trans lation of genes rather than modulation ofenzyme activi ty. Under conditions where insul in decl ines and/ or corti sol levels ri se, corti sol s timulates transcription of l ipases involved inl ipogenes is (glucose sparing), enzymes involved in gluconeogenes is and glycogenes is in the l iver, and in the breakdown of muscle protein.The net effect i s restored blood glucose and larger glycogen s tores in the l iver. However, this increase i s at the expense of muscle and boneand ul timately impairs immunologica l function.

DIABETES MELLITUS (DM)

DM i s a condition characterized by ei ther the tota l lack of insul in (Type 1) or res is tance of periphera l ti s sues to the effects of insul in (Type 2).Both diseases lack the s ignal ing effect of insul in in the presence of normal or high glucagon and other metabol ic s igna ls (Figure 10-16). Thedisease of DM is due to the imbalance in carbohydrate metabol i sm and i ts effects on other metabol ic pathways .

Figure 10-16. Metabolic Events Occurring in Diabetes Mellitus. Overview of effects incurred by the deficiency of insul in and excess glucagon,including those on glucose, proteins/amino acids , and l ipids . Al l effects lead to dehydration and the condition of diabetic ketoacidos is , whichcan be fata l . [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

In type 1 DM, autoimmune destruction of the pancreatic β-cel l s leads to a complete loss of insul in production. Al though the l iver can makeglucose, glycogen synthes is i s impeded. In the absence of insul in, gluconeogenes is i s unrestra ined, elevating blood glucose (Figure 10-17).However, muscle and fat cel l s cannot take up ava i lable blood glucose via the GLUT4. Thus , the body i s unable to clear the elevated bloodglucose, and the periphera l ti s sues (muscle and fat) are s tarved for glucose even when present at very high levels in the blood. Furthermore,in the absence of insul in, glucagon secretion i s uncoupled from the blood glucose levels (insul in i s an important phys iologica l regulator ofglucagon secretion). Unopposed glucagon, together with the other counter regulatory hormones (catecholamines , corti sol , and growthhormone), inhibi ts glycogen synthes is and s timulates gluconeogenes is , glycogenolys is , and l ipolys is . Increased l ipolys is leads to elevation offree fatty acids in the blood s tream. These fatty acid molecules are partly taken up by l iver and incorporated into lipoproteins to increase VLDLand LDL levels , a ri sk factor for heart disease. Ketone bodies are a lso produced because of the excess of l ipolys is , which cannot be inhibi ted inthe absence of insul in. This can resul t in the dangerous condition ketoacidos is , i f the ketone body level becomes too elevated. The onlyava i lable treatment i s the injection of exogenous insul in into the body. However, even with optimal control , the damaging effects of elevatedglucose and l ipids eventual ly lead to medica l compl ications .

Figure 10-17. Type 1 Diabetes Mellitus. The effect of this disease on organs and major metabol ic pathways i s i l lus trated. The absence of insul inin type 1 diabetes inhibi ts (red bars ) the uptake/convers ion of glucose by muscle and adipose ti ssue, leading to an increase in glucose (redarrow) synonymous with the disease. Uptake of fatty acids from triacylglycerols by adipose ti ssue i s a lso inhibi ted (red bars ), leading to anincrease in i ts levels (red arrow). Resul ting changes in metabol i sm lead to increased fatty acids and ketone bodies (red arrows), the latter ofwhich contributes to diabetic ketoacidos is seen in type 1 diabetic patients . See the text for further discuss ion. VLDL, very-low-dens i tyl ipoprotein. [Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Diabetes and the Polyol Pathway. DM i s a disease with the ha l lmark of elevated blood glucose. Al though the mechanism is not completelyagreed, the high glucose levels are speci fica l ly detrimenta l to the kidneys, retina, and nerves because of thei r abi l i ty to transport glucosewithout the a id of insul in. In these ti ssues , excess glucose enters the sorbitol–aldolase reductase pathway (a l so known as the polyol pathway)where i t i s reduced to sorbi tol and then fructose, oxidizing NADPH to NADP+ and reducing NAD+ to NADH, during the enzymatic reactions .

Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.

When glucose concentrations are normal , this pathway i s minimal ly active because of low affini ty of glucose for the enzyme aldolasereductase. However, when glucose levels are high, this reaction i s more prominent. The resul ting decrease in NADPH and increase in NADHaffects other enzymatic reactions that use these molecules as cofactors . NADPH is required for production of reduced glutathione and ni tricoxide required for detoxi fication of reactive oxygen species. Lowered NAD+ a l so leads to additional reactive oxygen species , and production ofinositol (s igna l ing, including insul in receptor) i s a l so decreased. The effects of lowered NADPH and NAD+ resul t in continual damage tothose ti ssues where the polyol pathway i s most prominent, caus ing kidney, eye, and nerve problems seen in many diabetic patients .

Type 2 DM is characterized by the production of insul in but res is tance of i ts effects on target ti s sues . As a resul t of this res is tance, thehuman body acts as i f there i s a relative deficiency of insul in, even when present at high levels (Figure 10-18). The disease shares many tra i tswith type 1 DM. As in type 1, gluconeogenes is i s unrestra ined, and muscle and fat cel l s do not take up glucose via the GLUT4. As a resul t, highlevels of blood glucose are present. However, the l iver s ti l l can make glycogen, and l ipolys is i s kept in check because of decreased butpresent insul in. However, plasma l ipoproteins are typica l ly elevated, often as a consequence of obes i ty and poor nutri tion. Ketoacidos is i snot a common sequela to type 2 DM. However, i t can occur in type 2 DM patients under conditions of addi tional metabol ic s tress and afterpancreatic fa i lure leads to decreased production and secretion of insul in. Some older people with type 2 DM may experience a di fferentserious condition ca l led Hyperosmolar hyperglycemic nonketotic syndrome (HHNS), a condition in which the body tries to get rid of excess sugar bypass ing i t into the urine. HHNS i s usual ly brought on by an i l lness , infection, or other factors .

Figure 10-18. Type 2 Diabetes Mellitus. The effect of this disease on organs and major metabol ic pathways i s i l lus trated. Insul in res is tance intype 2 diabetes inhibi ts (red bars ) the uptake/convers ion of glucose by muscle and adipose ti ssue, leading to an increase in glucose (redarrow) synonymous with the disease. Uptake of fatty acids from triacylglycerols by adipose ti ssue i s a lso inhibi ted (red bars ), leading toincrease in i ts levels (red arrow). Unl ike type 1 diabetes , fatty acids and ketone bodies are not increased (not shown) and diabeticketoacidos is i s , therefore, rare. See the text for further discuss ion. VLDL, very-low-dens i ty l ipoprotein. [Adapted with permiss ion from KatzungBG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

DM and Advanced Glycation End Products: Increased levels of sorbi tol and fructose in diabetic patients lead to nonspeci fic attachment ontoproteins of carbohydrate molecules via carbohydrate–nitrogen l inks (see the figure below), for example, lys ine and arginine amino acids ,proportional to the level of glucose in the body. These erroneous ly modi fied proteins are referred to as advanced glycated (also known asglycosylation) endproducts (AGEs). The best known AGE i s hemoglobin A1C in ci rculating red blood cel l s , which forms the bas is for testing ofdiabetic control . AGE proteins and their breakdown products cause oxidant damage to the kidney and a lso increase the production ofcytokines (e.g., tumor necros is factor-β), which damages the glomerulus . In addition, they increase the permeability of blood vessels, increaseoxidized LDL levels and increase cytokine-related oxidative stress.

Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.

AGE proteins a lso bind to a receptor for advanced glycation endproducts (RAGE). Activation of RAGE leads to nuclear factor kappa B induction ofsevera l inflammatory gene products resul ting in a chronic inflammatory envi ronment. This long-term inflammation promotes such varieddisease s tates as atheroscleros is with accompanying heart attacks and s trokes . Damage to the nerves and retina i s a l so prominent,

leading to diabetic neuropathy and retinopathy, the latter of which often leads to avoidable bl indness .

VITAMINS AND MINERALS

VITAMINS

Vitamins and minera ls serve as important cofactors in severa l , essentia l enzymatic reactions , including protein, carbohydrate, and fatmetabol i sm as wel l as the formation of ti s sues . Vi tamins are usual ly divided into fat soluble (vi tamins A, D, E, and K) and water soluble(vi tamins B and C).

Al though the human body or res ident, benevolent bacteria can produce some vi tamins , most, a long with the required minera ls , areobta ined from the diet. Some vi tamins can a lso be s tored in the body for di fferent times . Deficiencies of particular vi tamins in the diet,therefore, often do not mani fest themselves for a lengthy period of time. Vi tamins A, D, and B12, for example, are s tored in large enoughquanti ties so that absence in the diet wi l l not be noticed for months or years . Deficiencies in vi tamins and minera ls may lead to ti ssuedamage and/or a number of diseases (e.g., beriberi , scurvy, night bl indness , pel lagra , and rickets ), many of them being fata l . At the other endof the spectrum, excess amounts of l ipid-soluble vi tamins can be toxic. The contribution of vi tamins during feta l development i s especia l lyimportant; excess maternal vi tamin A during pregnancy can lead to s igni ficant bi rth defects . Vi tamins required by humans are summarized inTable 10-4.

TABLE 10-4. Summary of Vi tamins

MINERALS

Minera ls , speci fica l ly those essentia l atoms in the human diet, are s imple chemica l elements required for the exis tence and subs is tence ofl i fe. The bas ic chemica l elements include carbon, hydrogen, ni trogen, oxygen, phosphate, and sul fur. Additional minera ls that play a smal lerbut s ti l l important role are sodium, potass ium, ca lcium, magnes ium, iodine, and zinc. Other minera ls are required by the human body,a l though the exact number of essentia l minera ls i s s ti l l controvers ia l . A summary of minera ls important to biochemica l functions of thehuman body i s included in Table 10-5.

TABLE 10-5. Summary of Important Minera ls

REVIEW QUESTIONS1. What are the genera l metabol ic roles of the major biochemica l molecules—carbohydrates , l ipids , and amino acids?2. What are the primary metabol ic contributions of smal l intestine, l iver, adipose ti ssue, pancreas , and muscle?3. Why are glucose-6-phosphate, pyruvate, and acetyl -CoA cons idered primary intermediates in metabol i sm of the major biochemica l

molecules?4. How does the concentration of glucose-6-phosphate determine the fate of glucose after entry into the l iver cel l or muscle cel l?5. What i s the role of fructose 2,6-bisphosphate in the control of l iver carbohydrate metabol i sm?6. What factors in the fed and s tarved s tates determine the fate of pyruvate in terms of i ts oxidation, use for glucose synthes is , or role in

fatty acid formation? How do these factors ba lance each other?7. What factors in the fed and s tarved s tates determine the fate of acetyl CoA in l iver in terms of i ts oxidation, use for fatty acid synthes is , or

convers ion to ketone bodies? How do these factors ba lance each other?8. How does the ratio of insul in to glucagon determine the ba lance of carbohydrate metabol i sm in the body in terms of effects on metabol ic

pathways in speci fic ti s sues?9. How does the ratio of insul in to glucagon determine the ba lance of fat metabol i sm in the body in terms of effects on metabol ic pathways

in speci fic ti s sues?10. What are the key effects of epinephrine on metabol i sm and how do these relate to the body’s needs in the fight or fl ight response?11. What are the roles of glucocorticoids in s tress or s tarvation?12. What are the key metabol ic dis turbances associated with type 1 and type 2 diabetes?13. What are the fat-soluble vi tamins , thei r functions , and diseases of deficiency?14. What are the B vi tamins , thei r functions , and diseases of deficiency?15. What are the functions of vi tamin C and diseases of deficiency?16. What are the functions of the fol lowing minera ls and the consequences of thei r deficiency and/or excess—sodium, potass ium, ca lcium,

magnes ium, phosphorus , i ron, and iodine?

CHAPTER 11THE DIGESTIVE SYSTEM

Editor: Kshama JaiswalDepartment of Surgery, Denver Heal th

Medica l Center, Denver, Colorado

Summary of the Digestive SystemMouthStomachLiverGal l BladderPancreasSmal l Intestine (Duodenum, Jejunum, and I leum)Large Intestine/AnusReview Questions

OVERVIEWThe digestive or gastrointestina l system is roughly defined as the anatomica l component from the mouth to the anus , including organsrespons ible for trans i t, mechanica l breakdown, digestion and absorption of foodstuffs , as wel l as the efficient el imination of sol id waste.Included are the mouth and denti tia , pharynx and esophagus , s tomach, smal l intestine, l iver, ga l l bladder, pancreas , large intestine, rectum,and anus . As with the complex integration and control of metabol i sm, the digestive system is , i tsel f, under the influence of neurologica l andhormonal regulation that both activates and inhibi ts many of i ts complex actions . Most of these complex actions are, themselves , s implebiochemica l processes of proteins , carbohydrates , l ipids , and nucleos ides/ nucleotides to include enzyme reactions with associatedactivating and inhibi tory molecules , membrane-spanning protein channels , and pumps a l l leading to the production and s torage of energyand essentia l bui lding blocks for current or future use.

SUMMARY OF THE DIGESTIVE SYSTEMThe digestive system is the col lection of organs respons ible for the digestion of ingested foods and l iquids (Figure 11-1). This system isclass ica l ly cons idered to s tart at the mouth and continue via the esophagus , s tomach, smal l and large intestines , and end at the rectum/anus .Bes ides thei r mechanica l and enzymatic breakdown of foodstuffs , cons iderable contributions toward digestions are suppl ied from the l iverand pancreas .

Figure 11-1. Overview of the Digestive System. Components of digestion and transport of food from the mouth to the rectum/anus are shown,including a summary of thei r contributions and the average amount of time for food to reach their location after ingestion. [Reproduced withpermiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

MOUTHTeeth provide the ini tia l mechanica l breakdown of food via chewing or mastication and, therefore, the formation and heal th of teeth i simportant. Proteins serve as an important s tructura l component of teeth enamel . Like bone, teeth require the col lagen-l ike proteinsamelogenin (over 90% composed of prol ine, glutamine, and his tidine amino acids ), ameloblastin, enamelin, and tuftelin (a phosphorylated

glycoprotein), which organize, ini tiate, and di rect ca lcium phosphate crysta l formation and help anchor teeth to the gums.The poss ible breakdown of enamel i s equal ly important and i s a lso dependent on biochemica l processes . Denta l plaque formation and

tooth decay both rely on enzymatic processes for formation as wel l as prevention. Plaque i s caused mainly by the normal ora l bacteriaStreptococcus mutans, Lactobacillus acidophilus, Fusobacterium nucleatum, Actinomyces viscosus, and Nocardia spp. When these organisms form a layeron teeth, those closest to the teeth exis t in an oxygen-deficient envi ronment and convert to anaerobic respi ration for energy production. Thisenzymatic process turns carbohydrates into lactic acid from pyruvate (Chapter 6) and resul ts in a pH of below 5.5, leading to tooth decay, thedeminera l i zation process that causes cavi ties . Glucose, fructose, and especia l ly sucrose (common table sugar) are the main carbohydratesources .

The mouth i s a lso the location for the s tart of the processes of carbohydrate, l ipid, and protein digestion via important enzymes conta inedin sa l iva . Sa l iva production, a long with feel ings of hunger and satiety, i s control led by a variety of neuro-biochemica l processes , which s tartwith the ini tia l thoughts or cephalic phase of eating. The phys ica l presence and act of chewing and tasting food, known as the orosensory orgustatory phase, el i ci ts further s igna ls that enhance sa l iva formation and express ion. The various factors affecting hunger and satiety arediscussed in Chapter 19. Sa l iva a lso traps molecules produced by normal ora l bacteria that adds to the taste sensation of otherwise odorlessand tasteless food compounds . The hormone gustin, produced in sa l iva and an activator of a ca lmodul in-dependent cycl ic adenos inemonophosphate (cAMP) phosphodiesterase (Chapter 8), i s a l so thought to play an important part in taste bud formation. Digestive enzymesconta ined in sa l iva and their roles in digestion are l i s ted in Table 11-1.

TABLE 11-1. Compos i tion of Sa l iva

“Meth Mouth”: Patients who abuse methamphetamines are prone to marked denta l decay, known col loquia l ly as “Meth Mouth.”Methamphetamine acts on the αadrenergic receptors of the vasculature of the sa l ivary glands , caus ing vasoconstriction and reducing sa l ivaryflow, depriving the ora l envi ronment of sa l iva ’s buffering activi ty to counteract acidi ty and prevent deminera l i zation of enamel .Methamphetamine-induced vomiting a lso exposes teeth to acids . In addition, methamphetamine overstimulates the sympathetic nervoussystem, eventual ly depleting norepinephrine and dopamine and a l tering concentrations of other centra l nervous system (CNS)neurotransmitters such as serotonin, acetylchol ine, and glutamate. This reduction increases the demand for adenos ine triphosphate (ATP);methamphetamine users may compensate by consuming more carbohydrates in the form of sugars and s tarches . Observers speci fica l lyreport a high intake of carbonated soft drinks among meth users . At the same time, users typica l ly abandon ora l hygiene. In short,methamphetamine use encourages an envi ronment that maximizes caries ri sk—decreased sa l iva , frequent exposure to sugar, and lack ofplaque control .

STOMACHStomach i s the location of continued mechanica l breakdown of food via the actions of i ts smooth muscle layers but, perhaps moreimportantly, the s i te of the activation and activi ty of a number of regulated enzymes and exposure to acid (pH 1–2) that ini tiates metabol i sm.The other main function of the s tomach i s the regulated and coordinated secretion of hydrogen atoms and various digestive enzymes underthe control of the autonomic nervous system and severa l hormones . These molecules are produced from a variety of cel l types found invarious parts of the s tomach as shown in Figure 11-2 and summarized below. Hormones that affect the s tomach are described in Table 11-2.

Figure 11-2. Hormone Production from the Stomach. Speci fic portions of the s tomach are respons ible for production of hormones as i l lus tratedabove (see text for further description). [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1stedi tion, McGraw-Hi l l , 2009.]

TABLE 11-2. Hormones Affecting the Stomach

1. Mucus (Neck) Cells. Found in a l l parts of the s tomach, these cel l s produce a viscous mixture of protective enzymes and mucins, largeglycoproteins (Chapter 1), via s timulation of a myristylated alanine-rich C kinase. Mucins have low glycosylation on the amino and carboxyterminal ends but very high levels of glycosylation (via serine, threonine, and asparagine amino acids ) in the centra l part of i ts amino acidsequence. These mucin glycoproteins are a lso interl inked by cysteine–cysteine disul fide bonds (Chapter 1) that form large aggregate gelsfi l led with water and protected from enzymes by their dense carbohydrate coating. The secreted mucus provides a protective barrier aga instthe digestive enzymes and highly acidic conditions found in the s tomach.

2. Parietal (Oxyntic) Cells. Found in a l l parts of the s tomach, parieta l cel l s are s timulated by the hormones histamine via the H2 receptor (Gs—

adenyl cyclase/cAMP) and gastrin via a CCK2 receptor [Gq—phosphol ipase C/inos i tol triphosphate (IP3)/Ca 2+] as wel l as by the vagus

(parasympathetic) nerve via acetylchol ine and the M3 receptor (Gq—phosphol ipase C/IP3/Ca 2+). Stimulation of adenyl cyclase i s known to

speci fica l ly activate the H+/K+ ATPase active transport channel and gastric acid production. These cel l s produce three main components thatare as fol lows:

a. Gastric acid [mainly hydrogen (H+) and chloride (Cl −) ions] via a unique H+/K+ ATPase active transport channel that pumps hydrogen ions intothe s tomach aga inst a very high concentration gradient (approx. 3 mi l l ion to 1). Gastric acid functions in protein denaturization,peps inogen activation (see below), and inhibi tion of bacteria l growth.

b. Bicarbonate ion (HCO3−). Excreted into the blood as part of the overa l l H+ pumping mechanism.

c. Intrinsic factor. Required for intestina l absorption of vi tamin B12 (see Chapter 10).

Achlorydia: The destruction or damage of parietal cells leads to a marked reduction in the production of gastric acid, essentia l for the ini tia lbreakdown of food and activation of s tomach enzymes (e.g., peps in). Al though severa l disease s tates and/or surgica l interventions canaffect parieta l cel l s , immune destruction of the cel l s leads to the condition of achlorydia/hypochloridia in which decreased amounts ofhydrochloric acid are produced. The resul ting higher s tomach pH leads to symptoms of gastroesophageal reflux, pa in and ful lness frominadequate digestion, and increased growth of bacteria , normal ly l imited by low pH, which can lead to diarrhea and decreased absorptionof essentia l ions (e.g., magnes ium and zinc) and vi tamins (e.g., C, K, B-complex), which themselves lead to other disease s tates . Treatmentis via supplementation with Betaine HCl, a form of hydrochloric acid, which survives into the s tomach, and any required vi tamins andminera ls .

3. Chief (Zymogenic) Cells. Produce pepsinogen, the proenzyme form of the important enzyme pepsin, which cleaves peptide bonds , preferably athydrophobic and aromatic [phenyla lanine (Phe), tryptophan, and tyros ine] amino acids . Stimulation of chief cel l secretions i s by the vagusnerve, acidic conditions per gastric acid, or by the hormones gastrin or secretin (produced in the duodenum). In infancy, Chief cel l s a l so

produce the enzyme rennin, which a ids mi lk absorption by breaking the Phe–methionine peptide bond in mi lk protein kappa-casein.Secretion of rennin i s s timulated by ingestion of mi lk by the human infant but the gene product i s turned off past this s tage.

4. Enterochromaffin-like Cells (ELCs). Found in the gastric glands . These cel l s secreted his tamine, which activates parieta l cel l s and gastric acidproduction. ELCs are, themselves , activated by the hormone gastrin, pi tui tary adenyl cyclase-activating peptide, and vagus nerve. ELCs areinhibi ted by somatostatin.

5. G Cells. Found in the antrum. Secrete the hormone gastrin (see Table 11-2), which both increases the secretion of and works a long withhis tamine to s timulate parieta l cel l s to produce hydrochloric acid and Chief cel l s to produce peps in. Secretion of gastrin i s increased byparasympathetic vagus nerve activi ty via release of gastrin-releasing peptide or by the presence of amino acids in the s tomach.

6. Prostaglandin E2. Binding to i ts receptor s timulates smooth muscle contraction of the gastrointestina l tract and decreases parieta l cel lsecretion of gastric acid whi le increas ing mucus production. The action i s via the Gi protein receptor, which inhibi ts the production of cAMP

by adenyl cyclase and, therefore, parieta l cel l H+/K+ ATPase pump activi ty.

Misoprostol and Gastric Ulcers. Misoprostol (see the figure), a synthetica l ly produced prostaglandin E1, i s sometimes used to prevent gastriculcers because of i ts abi l i ty to inhibi t parieta l cel l production of gastric acid. Misoprostol i s normal ly used only for treatment of orprophylaxis aga inst nonsteroida l anti -inflammatory drug-induced peptic ulcers because other medication classes (H2-receptor blocker andprotein pump inhibi tors , PPIs ) are more effective for long-term care of acid reflux and s imi lar disorders .

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

The actions of each of the above cel l types and, therefore, the envi ronment of the s tomach are control led by vagus nerve s ignals as wel l asthe hormones l i s ted below. Of note i s the fact that severa l hormones that affect the s tomach are produced and a lso act on organs outs ide thestomach (e.g., smal l intestine) to decrease s tomach moti l i ty and/or digestion (Figure 11-3). In this manner, the body not only “turns on” thestomach when food i s present but a lso turns i t “off” when food has traveled further a long the digestive tract.

Figure 11-3. Hormone Production by the Digestive System. Si tes of production of the five major gastrointestina l hormones a long the length of thegastrointestina l tract. The width of the bars indicates the relative abundance at each location. CCK, cholecystokinin; GIP, gastric inhibi torypeptide. [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Acid Reflux (Gastroesophageal Reflux Disease or GERD), Barrett’s esophagus, and the Vagus Nerve. GERD a ffects a large percentage of thepopulation ei ther acutely or chronica l ly. Untreated GERD can sometimes lead to precancerous changes in the lower part of the esophagusca l led Barrett’s esophagus, which can progress to potentia l ly fata l adenocarcinoma of the esophagus . The surgica l severing of the vagus nerve(vagal nervectomy or vagotomy) was once used for treatment due to this nerve’s prominent role in parieta l and G cel l (producers of gastrin),secretory activi ty. The procedure, a l though usual ly effective for GERD and peptic ulcer disease, a lso carried a number of unwanted s ideeffects because of the innervation of other organs by the vagus nerve unless careful and selective surgery was performed. Many treatmentmethods have s ince been developed, including the H2 blocker class of medications , which inhibi t the promotion of acid secretion by

his tamine and PPIs, which block the unique H+/K+ ATPase pump found in the s tomach. As a resul t, a vagotomy is now rarely performed forGERD treatment.

LIVERThe l iver plays multiple, cri ti ca l roles (see Table 11-3) in such diverse functions as a major part in carbohydrate, protein, and l ipidmetabol i sm/regulation; synthes is and secretion of severa l blood clotting (coagulation) factors ; synthes is of bi le; the degradation ofhemoglobin/bi l i rubin from decompos ing red blood cel l s (Figure 11-4); synthes is and clearance of cholesterol ; s torage of glycogen, vi tamins , A,D, B12, i ron, and copper; breakdown and el imination of a variety of toxic substances (including a lcohol ); and the synthes is of a variety ofmolecules from a lbumin to insul in-l ike growth factor 1 to angiotens in (see Chapters 12 and 16). These functions are carried out byhepatocytes , the functional cel l s of the l iver.

TABLE 11-3. Summary of Liver Functions

Figure 11-4. Degradation of Hemoglobin to Bilirubin. Overview of breakdown in the reticuloendothel ia l sys tem of heme molecules that arereleased from decompos ing red blood cel l s . Steps producing bi l iverdin and bi l i rubin are shown. The gradual breakdown of heme resul ts insevera l intermediate products that create the colors seen in an evolving and heal ing bruise. NADPH, nicotinamide adenine dinucleotidephosphate. [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Bilirubin Measurement and Disease: Laboratory measurement of bilirubin i s a lways reported as the conjugated (direct), unconjugated (indirect), andtotal bilirubin. A dye used to detect bi l i rubin quickly produces a red-violet azobi l i rubin compound with conjugated bi l i rubins ; these are,

therefore, referred to as “di rect” bi l i rubin. The addition of ethanol to the sample makes a l l bi l i rubins (conjugated and unconjugated) reactquickly with the dye and yields “tota l bi l i rubins .” Unconjugated bi l i rubin levels are ca lculated by subtracting di rect bi l i rubin from tota lbi l i rubin and are, therefore, ca l led “indirect” bi l i rubin. Urobilinogen i s measured by the addition of a reagent conta ining p-dimethylaminobenzaldehyde, which turns a dark pink/red proportional to the urobi l inogen in the test sample. Urobi l in does not react withthis compound and, therefore, usual ly only urobi l inogen i s measured. Stercobl in, found exclus ively in feces , i s usual ly not measured.

Diseases of the l iver, ga l lbladder, and red blood cel l s a l l a ffect the amount of unconjugated/conjugated bi l i rubin as wel l as theamounts of resul tant urobi l inogen and s tercobi l in. Simple observations and laboratory measurement of these molecules can greatly ass is tin determining these disease s tates . For example, ga l l s tones , chronic l iver disease with ci rrhos is , or other diseases that block or decreasethe secretion of conjugated bi l i rubin into the bi le wi l l lead to an increased leakage of bi l i rubin from l iver cel l s and a resul ting ri se in urinebi l i rubin. This ri se can both be measured and seen in urine by the resul ting dark amber color. The lack of bi l i rubin secretion into theintestine wi l l a l so decrease the amount of s tercobi l in creating white or pa le feces . Diseases that cause increased breakdown of red bloodcel l s (e.g., hemolytic anemias) wi l l increase the amount of unconju-gated bi l i rubin in blood tests ; however, as the unconjugated form is notsoluble in water, urine bi l i rubin wi l l not increase and urine color wi l l remain the same. However, the excess heme from the destroyed redblood cel l s wi l l increase the amount of urobi l inogen/urobi l in that can be measured.

In newborns , the ini tia l lack of l iver enzymes respons ible for conjugation and intestina l bacteria , which convert bi l i rubin to urobi l inogen,may lead to the diseases of hyperbilirubinemia and potentia l ly letha l kernicterus. These same conditions a lso lead to pa ler feces innewborns and elevated levels of bi l i rubin create a yel low skin color (jaundice).

LIPID METABOLISM IN THE LIVER

The l iver plays a centra l role in l ipid metabol i sm. It i s the only organ capable of the disposa l of s igni ficant quanti ties of cholesterol, ei ther viaexcretion into the bi le or by metabol i sm to bi le acids , both of which are lost to some degree in the feces . Dietary cholesterol , which i spackaged into chylomicrons in the intestine for transport in the blood, ul timately ends up in the l iver where i t combines with the pool ofcholesterol synthes ized from acetyl coenzyme A derived from β-oxidation of saturated fatty acids and i s ei ther excreted via the bi le as bi lesa l ts or dis tributed to other ti s sues via low-density lipoprotein (LDL). In the fed s tate, the l iver converts excess dietary glucose to fatty acids thatare esteri fied to glycerol (Chapter 7). The resul ting triglycerides are packaged, with cholesterol , into very-low-density lipoprotein (VLDL), which i ssecreted into the blood to del iver fatty acids primari ly to adipose cel l s and muscle. Fina l ly, bi le sa l ts made in the l iver are needed for theabsorption of dietary l ipids (e.g., triglycerides and cholesterol ) and fat-soluble vi tamins .

The l iver i s the source of the l ipoprotein VLDL and most apol ipoproteins (apo) and i s actively involved in the endogenoustransport/metabol ic pathway of cholesterol and associated l ipoproteins (high-dens i ty l ipoprotein, LDL, and VLDL; see Chapter 3). Its majorfunction i s to transport the endogenous ly synthes ized triacylglycerol from the l iver to extrahepatic ti s sues . VLDL secreted into the ci rculationalso conta ins apo B100 (a lso synthes ized in l iver) that i s required for the proper assembly and export of VLDL particles . The second majorfunction of VLDL i s to carry l iver-generated cholesterol es ters to periphera l cel l s a fter i ts convers ion to LDL (see Chapter 16). The further s tepsin cholesterol transport and del ivery to periphera l ti s sues are described in Chapter 16.

GALL BLADDERThe ga l l bladder serves to s tore and concentrate bile produced in the l iver. Bi le i s released into the duodenum a t the ampulla of Vater by smoothmuscle contraction of the muscularis externa layer and relaxation of the Sphincter of Oddi. Release i s in response to secretion of cholecystokinin(CCK), the name of a group of related peptide hormones secreted from I-cel l s in the duodenum with s imi lar s tructure to gastrin (see above).CCK secretion i s increased by entrance of fat- or protein-conta ining food into the duodenum but decreased by the actions of somatostatin (seeabove). Bi le i s composed of water, a variety of ions [Na +, K+, Ca 2+, Cl −, and HCO3

− (bicarbonate)], l ipids (fatty acids , phosphol ipids , andcholesterol ), proteins , and, most importantly for digestion, more than 30 g/l of anion forms of bi le acids (see Chapter 3). As noted in Chapter 3,glycine- and taurine-conjugated bi le acids are the major forms. These bi le acids have detergent properties enabl ing them to surround andbreak up triglycerides and phosphol ipids fat particles in food (Figure 11-5), a l lowing l ipid-degrading enzymes (e.g., pancreatic l ipase) to act.Bi le acids a lso promote improved absorption of fats , including the fat-soluble vi tamins A, D, E, and K (Chapters 3 and 10).

Figure 11-5. A–B. Role of Bile Salts in Triglycerides Metabolism. A. Bi le sa l ts form micel les with fatty acids released from dietary triglycerides bypancreatic l ipase. The hydrophobic, charged s ide cha in and hydroxyl (OH) groups of bi le sa l t monomers (see Chapter 3) are wel l sui ted formicel le formation. [Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.] B.These micel les promote increased degradation of the triacylglycerol , by the l ipase/col ipase complex, to two fatty acids and monoacylglycerolfol lowed by transport of these products to the mucosa, as part of new micel les , where they are absorbed for reassembly of triacylglycerols .

Bes ides express ion of bi le from the ga l l bladder, CCK serves an important role in s timulating the pancreas to release digestive enzymes ,including trypsin, chymotrypsin, pancreatic lipase, and pancreatic amylase, to a id in fat and protein digestion. CCK a lso s lows down stomachemptying to a l low proper digestion of these food particles and decreases the production of gastric acid (see above). Fina l ly, CCK serves as aneuropep-tide regulating hunger/satiety working via CCK receptors found in the CNS. As discussed above, the CCK receptor a lso binds gastrinand functions via a Gq protein-coupled s ignal ing process , which activates phosphol ipase C with subsequent production of IP3 and Ca 2+. In the

CNS, CCK works via regulation of the activi ty of dopamine and poss ibly GABA (Chapter 19). CCK activi ty i s a l so involved in the activi ty of opioidsin the CNS.

Cholesterol Gallstones: The formation of cholesterol ga l l s tones , one type of cholel i thias is , occurs most often in women older than 40 yearswho have had severa l pregnancies . Obes i ty increases the ri sk. Ga l l s tones are a lso increased in women of Caucas ian and Hispanic origin.These ri sk factors are sometimes described by the five “Fs”—female, forty, ferti le, fat, and fa i r—but the mechanism appears to be far morecompl icated than these s imple factors . High cholesterol and relatively low bi le sa l t levels are known ri sk factors as wel l as bi le s tas is inthe ga l lbladder due to infrequent, weak, and incomplete contractions and emptying of i ts contents . Particular proteins found in bi le havealso been noted to enhance or inhibi t the precipi tation of cholesterol into s tones . Interestingly, levels of the dietary supplementmelatonin may a lso play a role in ga l l s tone formation as , among many suggested functions , i t a l so promotes lower levels of cholesteroland reduces oxidation levels in the ga l l bladder.

PANCREASThe pancreas plays a predominant role in the digestion of fats and proteins because of the pancreatic juice and various digestive enzymesthat i t produces and secretes into the duodenum (exocrine functions), sharing the ampul la of Vater with the common bi le duct. In addition, thepancreas produces severa l hormones (endocrine functions). The exocrine functions occur in the pancreatic acini, smal l clusters of cel l s and ducts .One function of these acini i s the production of bicarbonate ions (HCO3

−), under control of the hormone secretin, for a lka l inization of the acidcontents leaving the s tomach (Figure 11-6).

Figure 11-6. Mechanism for Production of Bicarbonate Ions (HCO3−) by the Pancreas. Ach, acetylchol ine. [Reproduced with permiss ion from Kibble JD

and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

A second function, mainly under the control of CCK, i s the synthes is and secretion of enzyme proteases , including the proenzymestryps inogen and chymotryps inogen as wel l as pancreatic l ipase, pancreatic amylase, phosphol ipase A2, lysophosphol ipase, and cholesterolesterase (Table 11-4).

TABLE 11-4. Exocrine Pancreatic Enzymes and Their Functions

As an endocrine organ, the pancreas produces and secretes severa l hormones from five di fferent cel l types forming the Is lets ofLangerhans , summarized in Table 11-5.

TABLE 11-5. Endocrine Pancreatic Hormones , Cel l source, and Functions

The endocrine pancreas i s regulated not only by other hormones but a lso by the autonomic nervous system (Chapter 19). Sympathetic(adrenergic) innervation speci fica l ly increases α-cel l secretions whi le decreas ing that from β-cel l s . Parasympathetic (muscarinic) innervationincreases secretions from both α- and β-cel l s .

SMALL INTESTINE (DUODENUM, JEJUNUM, AND ILEUM)The small intestine i s the approximately 16–20 ft. long section of the digestive tract in which a majori ty of the digestion of food and absorptionof nutrients occurs . The effective functional length of the smal l intestine i s increased by a factor of about 500 by folds/ invaginations (plicaecirculares and rugae) of the intestina l wal l and a lso the projections (vi l l i ) of the enterocyte cel l borders which l ine i t. The three di fferent partsof the smal l intestine—the duodenum, the jejunum, and the ileum—perform di fferent functions , depending on the presence of digestiveenzymes and absorptive capabi l i ties of thei r particular cel l types (Figure 11-7).

Figure 11-7. Summary of Absorption by Small Intestine. Absorption of various nutrients i s noted for the duodenum, jejunum, and i leum.[Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Short Gut Syndrome: Short gut (bowel) syndrome resul ts from ei ther congenita l or surgica l ly caused shortening of the smal l intestine withresul ting loss of the essentia l digestive and absorptive functions . Surgery for Crohn’s disease, cancers , trauma, and bariatric (obes i ty)treatments are the primary causes . Symptoms which include diarrhea, pa in, and secondary disorders from l imited vi tamin and nutrientabsorption usual ly do not present unless more than two-thi rds of the smal l intestine (i .e., less than 7 ft. remaining) i s a ffected. Somepatients can improve via enlargement and increased functioning of the remaining intestina l length and/or s lowing of movement of foodthrough the intestine to optimize digestion and absorption. Treatment i s symptomatic and by supplementation of required nutrients ,a l though surgica l attempts at intestina l lengthening or transplant are being attempted.

Infectious/Inflammatory Diseases of the Small Intestine: The constant exposure of the digestive tract and, in particular, the smal l intestine toingested microorganisms leads to multiple occurrences of gastroenteritis or inflammation of the s tomach and/or smal l intestine. Al though amajori ty of causes are vi ra l (e.g., rotavi rus ), various bacteria (e.g., Escherichia coli, Shigella, Salmonella, Campylobacter, Vibrio cholerae, andClostridium), protozoan (e.g., Giardia), and paras i tes (e.g., Ascaris lumbricoides, flat-worms, and tapeworms) can a lso infect the smal lintestine. Al l infections lead to acute changes in the abi l i ty of the intestina l l ining to digest and absorb nutrients and water, leading toacute diarrhea and nutrient deficiencies .

Digestion and absorption of each of the three parts of the smal l intestine are summarized in Table 11-6.

TABLE 11-6. Overview of Functions of the Smal l Intestine

Pernicious Anemia: Pernicious anemia i s a decrease in red blood cel l s count caused by a decrease of absorbed vitamin B12 due to the lack ofintrinsic factor. Intrins ic factor i s normal ly produced by the parieta l cel l s of the s tomach and i s essentia l in a l lowing the absorption of thisessentia l vi tamin in the i leum. The cause of pernicious anemia i s normal ly by atrophic gastritis and resul ting immune attack aga inst intrins icfactor and these cel l s . Gastric bypass surgery can a lso cause an arti fi cia l ly produced form of nonfunctional parieta l cel l s . Symptoms includeloss or a l teration of nerve sensation (paresthesia) usual ly in the fingers and toes , inflammation of the tongue (glossitis), and weakness(secondary to the anemia) among others . Pernicious anemia i s one of the megaloblastic anemias, one cause of which i s folate deficiency,leading to defects in red blood cel l s DNA synthes is and abnormal ly enlarged red blood cel l s . Treatment i s by ora l and subl ingualsupplementations , both of which a l low absorption of vi tamin B12 via locations other than the i leum, or by injections or other absorptivemethods .

LARGE INTESTINE/ANUSThe fina l part of the gastrointestina l tract i s the approximately 5 ft. long, large intestine and anus/rectum, which mainly serves as a locationfor continued, pass ive water reabsorption, vi tamin absorption, and transport of indigestible food for el imination as feces (Figure 11-8). Manyof these functions are dependent on “gut flora ,” a variety of from 300 to 1000 di fferent, symbiotic bacteria l species and four or more fungalspecies that l ive in the intestine and a l low a large number of biochemica l reactions that are essentia l to l i fe. The Bacteroides genus ofbacteria appears to be most abundant and play an especia l ly important role.

Figure 11-8. Summary of Functions of the Large Intestine. Absorption of water and ions by the ascending and proximal transverse colon and s torageand transport of waste materia ls by the dis ta l transverse, descending, and s igmoid colon and the rectum is i l lus trated. [Reproduced withpermiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

One role of gut flora i s the fina l digestion of dietary fiber, s tarches , and carbohydrates that the human body cannot metabol i ze, includinglactose in lactose-intolerant individuals . These polysaccharide chains are converted to short-chain fatty acids (e.g., acetic acid, propionic acid,butyric acid, lactic acid, and i sova leric acid) by bacteria l fermentation and pass ively absorbed into the blood s tream. Indigestible proteins (e.g.,col lagen and elastin) are a lso broken down by bacteria l fermentation pathways . Bicarbonate ions secreted by the large intestine help toreduce acidi ty produced by the fermentation reactions . In addition, symbiotic, intestina l bacteria a lso increase the absorption of remainingl ipids and minera ls such as ca lcium, magnes ium, and i ron. These bacteria l processes not only a l low uti l i zation of these energy sources buta lso increase water absorption and reduce levels of dangerous intestina l bacteria whi le increas ing growth of beneficia l bacteria .

Gut flora a lso augment levels of vitamin K and biotin (vitamin B7), which are absorbed by the large intestine, as a normal by-product of thei rmetabol i sm. Gut flora are a lso respons ible for production of some modified (secondary) bile salts. In addition, these bacteria are important inthe development and growth of essentia l , intestina l lymph tissue; the development of antibodies to harmful pathogens ; metabol i sm andel imination of ingested carcinogens; the continued repl ication and growth of the cel l s l ining the intestine; and protective changes in theexpress ion of cel l -surface molecules on these cel l s .

Antibiotic Use and Clostridium difficile: Gut flora provides a number of essentia l functions , which includes keeping the growth of unwantedbacteria and yeast reduced. This abi l i ty to prevent harmful species from overproducing in the human intestine i s sometimes ca l led the“barrier effect.” Antibiotics used in the treatment of diseases can sometimes adversely affect this ba lance by inadvertently reducing thenumber of helpful gut flora . The increas ing use of broad-spectrum antibiotics that el iminate multiple bacteria l species , especia l ly thefami ly of fluoroquinolones, has augmented this problem.

One common bacterium that grows in the large intestine in the wake of overaggress ive antibiotic use i s C. difficile. Overgrowth by thisorganism and i ts release of harmful toxins leads to chronic diarrhea, bloating, and abdominal pa in. These toxins are bel ieved to inactivatea fami ly of G-proteins/GTPase receptors by a l tering essentia l recognition and binding sugar res idues on their surface. Continued growth ofC. difficile can lead to the serious condition of pseudomembranous colitis, a severe infection of the colon. This disease resul ts from markedinflammation of the intestina l l ining and a resul ting membrane-l ike s tructure composed of fibrin, lymphocytes and monocytes , and deadand dying l ining cel l s . Patients who have been in the hospi ta l or nurs ing homes are increas ingly susceptible to this problem due toincreased numbers of background Clostridium in thei r intestines . Treatment of the condition i s ei ther by cessation of antibiotic use or withthe ora l use of the antibiotics metronidazole or vancomycin.

REVIEW QUESTIONS1. What are the genera l roles of the mouth, s tomach, l iver, ga l lbladder, pancreas , and smal l intestine in digestion?2. What are the major digestive enzymes of sa l iva and the speci fic function of each?3. What i s the role of mucus?4. What are the functions of the parieta l (oxyntic) and Chief cel l s in the s tomach?5. What are the functions of the major digestive hormones and where i s each produced?6. What are the primary metabol ic roles of l iver?7. What factors related to clotting and clot dissolution are secreted by the l iver and what are thei r functions?8. What transport/carrier proteins are secreted by l iver?9. What roles does the l iver play in l ipid transport and metabol i sm, especia l ly with regard to cholesterol and associated l ipoproteins?

10. What are the functions of each of the exocrine pancreatic hormones?11. What are the functions of each of the endocrine pancreatic hormones and in which cel l type i s each produced?12. What are the main functional molecules/glands of each section of the smal l intestine?13. What i s intrins ic factor and the consequence of i ts deficiency?14. What are the main functions of the large intestine?

CHAPTER 12MUSCLES AND MOTILITY

Co-author/Editor: Darren CampbellDivis ion of Sports Medicine,

U.S. Ai r Force Academy

The Bas ic Components of MuscleExci tation–Contraction Coupl ingSkeleta l MuscleCardiac MuscleSmooth MuscleEnergy Production and Use in MusclesMicrotubule-Based Moti l i tyIntermediate Fi lamentsNonmuscle Cel l sReview Questions

OVERVIEWMovement and i ts regulation, on a microscopic and/or macroscopic sca le, are essentia l for human l i fe. Muscle, composed of actin, myos in,and a variety of s tructura l and regulatory proteins , i s one of the main cel l /ti ssue types involved in this movement. Skeleta l , cardiac, andsmooth muscles offer a coordinated and regulated means to move the human body from place to place; interact with i ts surroundings ; keepnutrients flowing to and waste products flowing from various cel l s ; or move nutrients , blood, lymph, and other molecules . Specia l i zed proteinsand the means to provide energy for these processes have evolved for particular ti s sues and functions .Microtubules with tubul in, dynein, and kines in molecules and associated s tructures such as ci l ia , flagel la , centrioles , basa l bodies ,centromeres , and mitotic spindles provide another important mechanism for a variety of cel l movements and internal cel l functions that affecta l l types of cel l s and a l low the divis ion of cel l s . Intermediate fi laments (IFs ) a lso serve severa l essentia l roles of cel l moti l i ty. Nonmusclecel l s uti l i ze a l l of these mechanisms—actin/myos in, microtubule/dynein/kines in, and IFs—to achieve a wide array of functions throughoutthe human body.

THE BASIC COMPONENTS OF MUSCLEMuscle i s an organ that specia l i zes in transforming chemica l energy into mechanica l work or movement. The muscular system comprises a l lthe individual anatomic muscles . Muscle ti s sue i s derived from the mesodermal layer of embryologic germ cel l s and i s divided into threemain types : skeleta l muscle, cardiac muscle, and smooth muscle (Figure 12-1). The s tructure of these di fferent types of muscles i s s imi lar butthe archi tecture and regulation are often very di fferent, making their functions unique. Nonmuscle cel l s are a fourth cel l type that uti l i zesmany of the same proteins and processes for moti l i ty.

Figure 12-1. A–C. The Three Types of Muscle. Light micrographs of each type, accompanied by labeled drawings . A. Skeleta l muscle i s composed oflarge, elongated, multinucleated fibers that show strong, quick, voluntary contractions . B. Cardiac muscle i s composed of i rregular branchedcel l s bound together longi tudina l ly by interca lated disks and shows s trong, involuntary contractions . C. Smooth muscle i s composed ofgrouped, spindle-shaped cel l s with weak, involuntary contractions . The dens i ty of intercel lular packing seen reflects the smal l amount ofextracel lular connective ti ssue present. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12thedition, McGraw-Hi l l , 2010.]

There are two major proteins involved in contraction in a l l types of muscle: actin and myosin. These proteins convert chemica l energy intomechanica l work through an interaction with adenosine triphosphate (ATP). The triggering and control of this process are di fferent for eachmuscle type. Skeleta l , cardiac, and smooth muscles have a repeating uni t ca l led the sarcomere, conta ining the muscle fibers composed of actinand myos in and accessory proteins (see below). Surrounding these muscle fibers i s the equiva lent of a plasma membrane ca l led thesarcolemma. Within the sarcolemma is the sarcoplasm conta ining mitochondria and a sarcoplasmic reticulum (SR), the equiva lent of a smoothendoplasmic reticulum (SER). Like the SER in other cel l types , the SR conta ins ca lcium ions (Ca 2+) essentia l for the ini tiation of muscleactivation. More speci fica l ly, specia l i zed invaginations of the SR, ca l led transverse tubules (T-tubules), interdigi tate between the muscle fibers

to provide efficient del ivery of ca lcium.

ACTIN

The term actin describes both the globular, s ingle amino acid (monomer) cha in (G-actin) and the thin filament (F-actin) s tructure formed from twopara l lel s trands of multiple G-actin molecules , which wind to form a double hel ix s tructure (Figure 12-2A). Three major classes of actin exis t,including α-actin (found in skeleta l , cardiac, and smooth muscles ) and β- and γ-actins (found in nonmuscle cel l s ). These F-actin fi lamentsprovide s tructure as part of the cel l ’s cytoskeleton. The cytoskeleton i s a lso di rectly l inked to the plasma membrane via integra l membraneproteins and intracel lular junctions (Chapter 8) and, as such, i s important in many s ignal ing functions .

Actin thin fi laments interact with myos in thick fi laments (Figure 12-2C and see below) and are integra l in a multi tude of cel l moti l i tyfunctions , including muscle contraction, the movement of intracel lular ves icles , cel l divis ion and cytokines is , immune response, phagocytos is ,wound heal ing, and others . Actin i s a l so required in the nucleus for RNA polymerase I, I I , and II I complex formation and function, export ofRNA and proteins from the nucleus , and some aspects of chromatin remodel ing.

Figure 12-2. A–C. Molecules Composing Thin and Thick Filaments. The contracti le proteins are the thin and thick myofi laments within myofibri l s . A.Each thin fi lament i s composed of F-actin, tropomyos in, and troponin complexes . B. Each thick fi lament cons is ts of many myos in heavy cha inmolecules bundled together a long their rod-l ike ta i l s , wi th thei r heads exposed and di rected toward neighboring thin fi laments . C. Bes idesinteracting with the neighboring thin fi laments , thick myofi lament bundles are held in place by less wel l -characterized, myos in-bindingproteins within the M-l ine. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-

Hi l l , 2010.]

TROPOMYOSIN–TROPONINS

Tropomyosin i s a regulatory protein that binds to F-actin thin fi laments and revers ibly blocks myos in head binding s i tes (Figure 12-2A–C). Innoncontracting muscle, troponin T and troponin I molecules (part of a troponin complex, Figure 12-2A) hold tropomyos in over the myos in-bindings i te. Upon exposure to Ca 2+ released from the SR, troponin C, the thi rd protein of the troponin complex, creates a conformational change of thetropomyos in–troponin complex expos ing the s i te. When ca lcium concentrations fa l l , the change reverses and the block i s aga in present.Smooth muscle and nonmuscle cel l s do not conta in troponin and rely on other regulatory processes to control actin–myos in contraction/forcegeneration. Troponin C conta ins a speci fic amino acid sequence named an EF hand. In this tertiary s tructure moti f (Chapter 1), an α-hel ix(termed E for i ts sequence in a series of α-hel ices ) i s joined to an F α-hel ix by a loop of approximately 12 amino acids . The hel ix–loop–hel ixforms a s tructure reminiscent of an extended forefinger (E hel ix) and thumb (F hel ix) with the curve between representing the loop. Among the12 amino acids of the loop are pos i tions , 1, 3, 5, 7, 9, and 12, which bind the Ca 2+. Amino acid 12 i s a lways glutamate or aspartate whose R-group provides two partia l ly negatively charged oxygen molecules (Chapter 1) for binding the pos i tively charged ca lcium. The smal l s i ze of ahighly conserved glycine amino acid at res idue 6 provides space for this s tructure to form. The other amino acids are mainly hydrophobic thatstabi l i ze the E and F hel ices and, therefore, the hel ix– loop–hel ix s tructure.

MYOSIN

The human genome holds more than 40 di fferent types of myosin genes . These di fferent genes produce variations of myos in shapes , which inturn affect the speed at which the fi laments move. To date, there are 12 classes of myos in defined in the human genome. Al l myos inmolecules have three domains , including a head, neck, and tail portion of the amino acid sequence (Figure 12-3A). The myos in head binds actinthin fi laments and uses hydrolys is of ATP to adenos ine diphosphate (ADP) to generate force. The ta i l domain varies depending on the type ofmyos in but, in genera l , can bind to transport ves icles or combine with other myos in molecules . Some myos in ta i l domains can a lso regulatethe proteins ’ activi ty by folding over and effectively blocking the myos in head domain. The neck domain serves as a l ink or lever arm for thetransfer of this force between the head and ta i l . The varying types of myos in proteins produce di fferent speeds of movement, depending onthe length of the neck region. Four myosin light chains: two essential light chains, and two regulatory light chains are a lso bound to the neck regions(Figure 12-3A). The function of these l ight cha ins i s discussed below.

Figure 12-3. A–C. Myosin I and II. A. Basic Myosin Molecule. The heavy and l ight cha ins (essentia l–red, regulatory–dark blue) form the bas ics tructure of a l l myos in molecules , including the long ta i l composed of two coi led α-hel ices and the globular head region. B. Myosin I. Ves iclesfor transport are attached by the myos in I ta i l section. The s ingle head of myos in I moves down the actin fi lament in a s imi lar manner to othermyos in heads . Myos in I molecules can a lso l ink between F-actin fi laments , resul ting in l imited moti l i ty s imi lar to myos in II molecules .[Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.] C. Myosin II. The ta i l sectionof the two heavy cha ins of myos in II forms a coi led-coi l arrangement. This s tructure can interact with severa l other myos in II ta i l s to form thickfi laments . The two head regions of each myos in molecule conta in an actin-binding s i te (red ci rcle) and an ATP-binding s i te (blue l ine).Coordinated movement of the myos in heads produces force and, therefore, movement when interacting with actin fi laments . See text forfurther deta i l s . [Adapted with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Myosin I i s the s implest form cons is ting of a bas ic myos in head, neck, and a ta i l of varying lengths (Figure 12-3A–B). The bas ic function ofmyos in I i s most often described as ves icle transport, a l though other functions are emerging. Myosin II i s composed of two identica l aminoacid cha ins , termed the “heavy cha ins” (Figure 12-3A). The ta i l section of each chain coi l s around the other, making a dimer myos in fi lamentwith two heads . Multiple myos in II dimers bind at the ta i l domain, forming a s tructure ca l led a thick filament (Figures 12-2B–C and 12-3C).Additional myos in subtypes have s tructures s imi lar to ei ther myos in I or myos in II and are respons ible for diverse types of cel l moti l i ty. Thesefunctions include intracel lular transport (both to and from the nucleus to the cel l periphery), maintenance and movement of ves icles andorganel les in subregions of the cytoplasm, and, poss ibly, the perception of l ight in the retina and sound waves in the inner ear.

MYOSIN LIGHT CHAINS

Each myos in head conta ins two essential myosin light chains and two regulatory myosin light chains bound to the neck region (Figure 12-3A).Di fferent forms of myos in l ight cha in are found on di fferent myos in types and influence their activi ty and regulation. Myos in l ight cha ins servean important regulatory role for myos in activi ty in smooth and nonmuscle cel l contraction where the tropomyos in–troponin mechanism isabsent. In skeleta l muscle, the myos in l ight cha ins influence the speed of contraction but are not essentia l for myos in activi ty. In cardiacmuscle, myos in l ight cha ins affect myos in head ATPase and are bel ieved to have some role in the development of heart fa i lure. Activi ty ofmyos in in smooth/nonmuscle cel l s rel ies on phosphorylation of serine 19 on the regulatory cha ins by myosin light chain kinase (MLCK). MLCKactivi ty i s regulated by Ca 2+ that binds to the regulatory protein ca lmodul in, a l so by an EF hand-binding moti f (see above). Ca lmodul insubsequently activates MLCK. When ca lcium concentrations fa l l , myosin light chain phosphatase (MLCP) removes the phosphate group and

inactivates the regulatory l ight cha ins and, therefore, myos in. Ca lcium a lso inhibi ts MLCP by the actions of another serine/ threonine enzymecal led Rho kinase; other s igna l ing pathways and enzymes have a lso been impl icated in myos in l ight cha in phosphorylation anddephosphorylation. One enzyme, sphingos ine-1-phosphatase, may play an important role in modulating contraction and relaxation of bloodvessels and, therefore, maintenance of blood flow in response to changes in blood pressure.

ACTIN-BINDING PROTEINS

Actin-binding proteins (ABPs) are a varied group of proteins that regulate actin fi lament formation and length as wel l as actin–myos ininteractions . Al though more prominent in non-muscle cel l and smooth muscle contraction, some, including tropomyos in and the troponins(see above), are found in skeleta l and cardiac muscles . One such ABP, α-actinin binds actin thin fi laments to skeleta l muscle Z-l ines andsmooth muscle dense bodies (Figures 12-4 and 12-8A). α-Actinin i s a l so bel ieved to bind to actin fi lament bundles and separates each thinfi lament by approximately 35 nm. This separation a l lows myos in thick fi laments the proper spacing for optimal contraction. Additional ABPscross -l ink thin fi laments (filamin), sever thin fi laments (gelsolin, tropomodulin, and villin) and cap them to restrict thei r length (capZ), regulateactin–tropomyos in activi ty in smooth muscle cel l s where troponins are absent (caldesmon), regulate myos in ATPase activi ty (calponin), andconnect microfi laments to the cel l membrane (dystrophin, vinculin, and integrins). Many other ABPs a lso exis t and perform other functions in avariety of cel l types .

Figure 12-4. Excitation–Contraction Coupling. Actin and myos in form sarcomere s tructures in skeleta l muscle as shown. The influx of ca lcium ions(Ca 2+) a l lows myos in heads from thick fi laments to bind to the actin fi laments . Ca 2+ a l so activates myos in head cross -bridge cycl ing, leading toforce generation and, ul timately, shortening of the sarcomere. Shortening of multiple sarcomeres leads to muscle contraction. The process i ss imi lar in cardiac and smooth muscle contraction, a l though s tructura l organization and regulation di ffer. ADP, adenos ine diphosphate; ATP,

adenos ine triphosphate. [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

EXCITATION–CONTRACTION COUPLINGMuscle achieves i ts major function of generating movement by contraction through a series of binding events and enzymatic reactions . Thisprocess i s ca l led excitation–contraction coupling (Figure 12-4). Certa in parts of this mechanism are conserved in a l l types of actin–myos incontraction/moti l i ty, whereas a l ternative forms of ini tiation and/or regulation are found in particular muscle types . Many of the componentsare s imi lar in nonmuscle cel l s , but many di fferences exis t because of the variations in organization and additional proteins and theirfunctions . Nonmuscle cel l contraction wi l l be discussed separately below.

Two bas ic processes must happen in a l l muscle types for contraction to occur: (1) binding of myos in heads to the actin thin fi lament and (2)s timulation of myos in to generate force. Both of these processes rely, at least in part, on Ca 2+. In a l l muscle types , a s igna l from a nerve orpacemaker cel l s causes an ini tia l increase in Ca 2+ concentrations . This relatively smal l influx of Ca 2+ leads to a larger release from the SR.This concept of a smal l ca lcium s ignal leading to higher ca lcium concentrations and contraction i s termed calcium-induced calcium release (CICR).The release from the SR varies depending on the type of muscle and i s discussed below. T-tubules a l low del ivery of this ca lcium to a l l areasof a muscle uni t to permit synchronous contraction. The released ca lcium interacts with an ABP—troponin/tropomyos in in skeleta l and cardiacmuscle and ca ldesmon in smooth muscle—to a l low myos in head binding to actin thin fi laments .

Myos in force generation i s the second bas ic function required for contraction. In a l l muscle types , ATP binds to the myos in molecule head.An ATPase on the myos in head cleaves ATP into ADP and a phosphate molecule (PO4

3–), producing a charged form of the myos in protein thatbinds to the now open F-actin fi laments at an angle. Release of the phosphate molecule from the myos in head causes i t to swivel to a moreacute angle, resul ting in a ratcheting movement between i t and actin. This ratcheting movement s l ides the thick and thin fi laments past eachother, converting the chemica l energy of the ATP into the mechanica l energy moving the fi laments . Release of the ADP molecule and binding ofa new ATP molecule breaks the bond between myos in head and F-actin thin fi lament. As this process repeats many times , the overa l l lengthof the sarcomere shortens . Ca lcium a lso plays a prominent role in smooth muscle myos in activi ty via activation of regulatory myos in l ightchains (see above).

The cycle continues as long as the concentration of ca lcium in the sarcoplasm of the cel l remains elevated. At the end of this process , theca lcium entry into the cel l s s lows and begins to be col lected aga in by the SR by a sarco/endoplasmic reticulum Ca2+-ATPase pump, a lso poweredby ATP. The pump can produce an approximately 10,000-fold higher concentration ins ide the SR versus the sarcoplasm. An additional proteincalsequestrin a l so binds Ca 2+ ins ide the SR to reduce the effective, free Ca 2+ concentration that the pump must work aga inst. Ca lsequestrin canbind over 40 Ca 2+ per protein molecule not by an EF hand s tructure but, ins tead, by us ing charged amino acids res idues and changes ofsecondary s tructure to α-hel ices . Once the ca lcium concentration lowers , ca lcium is released from troponin C, the previous conformationalchange i s reversed and troponin T and I aga in block the binding of myos in to the actin binding s i te. As the cycle ends , a new molecule of ATPthen binds to the myos in head, which displaces the ADP and the ini tia l sarcomere length i s restored.

SKELETAL MUSCLE

STRUCTURE AND GENERAL OVERVIEW

Skeleta l muscle comprises the bulk of the muscle mass found in our bodies (Figure 12-5). The average adult male i s made up of approximately42% skeleta l muscle and an average adult female cons is ts of 36% skeleta l muscle. Each muscle has an origin and insertion on the skeleton,with the individual muscle fibers running para l lel or obl ique to the long axis of the muscle. Skeleta l muscles are capable of producing aforceful contraction and moving the elements of the muscle over a relatively large dis tance. With the shortening of length during thecontraction, this muscle wi l l often change in diameter, l ike the swel l produced when contracting the biceps muscle. In skeleta l muscle, the F-actin thin fi laments are bound end to end at a s tructure ca l led the Z-l ine (Figure 12-4). The repeated Z-l ines and a l ternating thin and thickfi laments give the muscle the s triated appearance seen on l ight microscopy.

Figure 12-5. Skeletal Muscle. Skeleta l muscle i s highly organized. At the molecular level , repeti tive sarcomeres (see text above) form a myofibri l .Multiple myofibri l s form muscle cel l s , columns, and, ul timately, muscle. [Reproduced with permiss ion from Kibble JD and Halsey CR: The BigPicture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Centronuclear/Myotubular Myopathies: Centronuclear/ Myotubular myopathies (CNMs) are a rare series of inheri ted disease s tates in whichskeleta l muscle nuclei are located in the center of the muscle cel l s ins tead of at the periphera l edges . The exact causes have not beenfound but errors in the development of embryologica l muscle cel l s appear to be common. A suspect enzyme ca l led myotubularin, which hasdephosphorylating activi ty, may be part of the X-l inked form, properly termed myotubular myopathy. X-l inked CNM presents at bi rth withsevere loss of muscle tone (hypotonia), a ffects muscles required for breathing, and often resul ts in death before a cause can bedetermined. Patients a lso often have an associated narrow and long head with pa late, finger, and chest s tructura l abnormal i ties .Autosomal dominant and recess ive forms a lso exis t, properly termed centronuclear myopathies . The autosomal dominant vers ion may bebecause of mutations affecting the protein dynamin, a guanos ine triphosphate-dependent transport protein. Symptoms of CNM may neverpresent (e.g., autosomal recess ive form) or may include variable ranges of increas ing muscular weakness , which often presents in theteens or 20s . There i s no cure for CNM diseases and treatment i s only supportive.

Growth, regeneration, and activi ty of skeleta l muscle i s under the control of severa l s igna l ing pathways , which activate speci fic genetranscription/trans lation, leading to the production of muscle proteins as wel l as regulatory proteins , including those involved in energyproduction for muscle contraction. Some are dependent on motor neuron activi ty and/or contraction [e.g., more glucose transporter type 4(GLUT4) receptors are brought to the surface in highly contracting muscles to increase glucose uptake] and, therefore, are di rectly influencedby speci fic muscle activi ty.

Skeleta l muscle i s under voluntary control and the contraction i s driven by motor neurons in the spina l cord and the neurotransmitteracetylcholine (Chapter 19). Skeleta l muscle neurons are di rectly associated with ryanodine receptors in the SR, which receive the nerve s ignaland depolarize L-type vol tage-dependent ca lcium channels in the T-tubules . This depolarization causes a change in the foot s tructures thatbridge the T-tubules and SR, a l lowing release of ca lcium into the sarcoplasm. The free ca lcium binds with the regulatory molecule calmodulinand activates troponin C, unmasking the myos in-binding s i te by displacing troponin-I and tropomyos in.

Acetylcholine and Skeletal Muscle Disease: Skeleta l muscle rel ies on the neurotransmitter acetylcholine to ini tiate muscle contraction.Dis ruption of acetylchol ine’s activi ty leads to disease s tates including myasthenia gravis and Lambert–Eaton syndrome. The major form ofmyasthenia gravis (MG) i s caused by antibodies aga inst the body’s own receptor for acetylchol ine at the postsynaptic junction (nicotinic–acetylcholine receptor). The antibodies block or destroy the receptor negating the action of acetylchol ine. The body’s natura l cholinesteraseactivi ty degrades the neurotransmitter before activation of muscle contraction can be achieved. The cumulative effect of both leads toweakening of muscles after extended periods of activi ty (fatigability). Muscles often affected include eyes and eyel ids , facia l/neck musclesrespons ible for express ion, speech and swal lowing, muscles involved in breathing, and arm/leg muscles . Examination can often be normalunless measurement to fatigue of muscles i s included. Blood tests for the antibodies are not a lways conclus ive, leading to otherneurologica l or muscle biopsy tests . Al though rarely performed, intravenous adminis tration of the chol inesterase inhibi tors edrophonium orneostigmine can rel ieve symptoms and ass is t in diagnos is . Patients diagnosed with MG are treated with s imi lar chol inesterase inhibi tors(neostigmine or pyridostigmine) and/or immune suppressants (s teroids and other medications). With proper treatment, l i fe expectancy i snormal . Lambert– Easton syndrome a ffects patients in a s imi lar manner to MG but i s caused by antibodies aga inst a presynaptic ca lciumchannel that s tops the release of acetylchol ine. Like MG, Lambert–Eaton syndrome is rare and often di ffi cul t to diagnose.

SKELETAL MUSCLE TYPES

Skeleta l muscle can be divided into di fferent types , depending on the myos in fibers present. There are two genera l categories : s low twitch(type I) and fast twitch (type II) wi th type II divided into three additional subtypes (type IIa , I Ib, and IIx). The types of muscle fibers have manydi fferences including contraction time, abi l i ty to be fatigued, and the maximum duration of use.

Slow-twitch (type I) fibers fi re more s lowly than the fast-twitch fibers . They conta in the heme-conta ining, oxygen-binding protein myoglobin,which serves as an important oxygen s torage protein capable of releas ing oxygen during periods of hypoxia . Myoglobin i s a l so used cl inica l lyas a marker for damaged muscle and can be elevated during periods of muscle cel l damage such as rhabdomyolysis (see s idebar). Type I fibersare sometimes referred to as “red” because of the presence of heme/Fe2+ in the myoglobin as wel l as increased numbers of mitochondria andsmal l blood vessels/capi l laries . These fibers , therefore, are able to primari ly use oxygen for more efficient oxidative phosphorylation (Chapter 6)to produce energy, mainly from fatty acids mobi l i zed from triglycerides. As a resul t, these fibers can participate in s low force generation andcontraction for long periods of time without fatigue, especia l ly when compared with fast-twitch fibers . This muscle fiber type helps enduranceathletes , who often have a greater percentage of s low-twitch muscle fibers than nonendurance athletes , compete over long dis tances for anextended period of time. Type I myos in i s a l so found in muscles involved in posture.

Rhabdomyolysis: Rhabdomyolysis or “rhabdo” i s the medica l term for the abnormal breakdown of skeleta l muscle caused by trauma (e.g., majorcrush injuries ), disease s tates , and/or medication s ide effects . Rhabdo i s marked by the appearance of abnormal ly high levels of musclecomponents in the blood s tream. Myoglobin i s a speci fic laboratory marker used by cl inicians who suspect rhabdomyolys is in thei rpatients . The sudden release of proteins and various ions can cause kidney fa i lure, neurologica l problems, and i rregular heart rhythms,which can be fata l . Inflammation caused by the damage to muscle can a lso lead to serious conditions such as compartment syndromewhere portions of the body can lose their blood supply because of swel l ing. The sudden swel l ing can a lso lead to shock. Treatment i s byintravenous fluids and supportive care often including dia lys is to preserve kidney function and to correct ion imbalances . Compartmentsyndrome may require surgica l opening of muscle connective ti ssue (fasciotomy) to rel ieve the pressure from swel l ing. Prompt andprofess ional care of anyone in danger of or showing s igns of rhabdomyolys is usual ly resul ts in a good outcome for the patient.

Type IIa fast-twitch fibers are a lso known as intermediate fast-twitch fibers and conta in high amounts of myoglobin, mitochondria , andcapi l laries . To some extent, they bridge the gap between the s low-twitch and fast-twitch fibers in that they can use both aerobic andanaerobic metabol i sm to create energy us ing creatine phosphate and glycogen. These fibers do not generate the same force as type IIb fibersnor are they as fatigue res is tant as the s low-twitch fibers . Type IIb fast-twitch fibers are the class ic fas t-twitch muscle fibers . These fibers arecapable of producing quick and powerful muscle contractions necessary for the sudden explos ive need for s trength of a power l i fter or theburst of speed needed from a sprinter. This muscle fiber has the highest rate of fi ring of a l l the muscle fiber types . Type IIb fibers do notconta in myoglobin and have fewer capi l laries than type I. Therefore, they primari ly use anaerobic metabol i sm a lso us ing creatine phosphateand glycogen. As a resul t, they are sometimes ca l led white fibers and are a lso more sens i tive to fatigue. Less i s known about type IIx fibers,which may have some of the oxidative metabol i sm of s low-twitch fibers a long with the biophys ica l properties of fas t-twitch fibers .

In individual muscles , there i s usual ly a combination of di fferent types of fibers (Figure 12-6). Each individual motor uni t, however, conta insa s ingle α-motor neuron and the muscle fibers i t innervates wi l l conta in the same type of muscle fibers . These fibers a l l have the same

contracti le characteris tics and fi re as an a l l or none phenomenon. The dis tribution of these di fferent types of motor uni ts within a s ingleskeleta l muscle i s determined genetica l ly and by the speci fic function of the muscle. It i s this mix that determines the function andfatigabi l i ty of the individual muscle. A muscle with more fast-twitch motor uni ts wi l l have quicker and more powerful contractions but be moresusceptible to fatigue, whereas a muscle with fatigue-res is tant s low-twitch motor uni ts wi l l generate less powerful contractions but be ableto susta in the contractions for longer periods of time. There i s some evidence that muscles are able to adapt and switch muscle fiber typeswith speci fic tra ining.

Figure 12-6. Skeletal Muscle Fiber Types. Cross section of skeleta l muscle s ta ined his tochemica l ly to detect the dens i ty of myofibri l lar myos inATPase can be used to demonstrate the dis tribution of s low (S)-type I fibers , intermediate (I)-type IIa fibers , and fast (F)-type IIb fibers .[Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Muscle Mass Loss from Sarcopenia to Astronauts: Severa l causative factors can lead to the loss of skeleta l muscle mass . One common cause i ssarcopenia, the loss of skeleta l muscle ti s sue due to aging, estimated at 0.5%–1% per year from age 25 years on. Al though decreased exerciseand use may be part of the reason, biochemica l changes a lso resul t in decreased muscle fiber growth, including smal ler muscle fiberci rcumference and replacement of muscle by fat or fibrous ti ssue. Part of the mechanism may be the increas ing fa i lure during aging ofmuscle satel l i te cel l s that help in muscle growth and repair. Skeleta l muscle a lso appears to exhibi t decreased response to growthhormone and testosterone as part of i ts s low decl ine.

Loss of muscle mass i s a l so seen in astronauts exposed to a period of microgravi ty. During space fl ight, the weightless envi ronmentmeans that muscles normal ly used on an a lmost constant bas is for posture and movement wi l l no longer be bearing weight, resul ting inloss of muscle ti s sue ca l led atrophy. Loss of bone accompanies muscle loss and adds to a genera l s tate of deconditioning when theastronauts return to the earth’s gravi ty. Often the most s igni ficant bone loss i s at s i tes of muscle attachment. Attempts to counteract thisproblem include regular exercise on specia l ly constructed exercise equipment and even a space sui t with bungee cords adapted to providecounterforce for major muscle groups . The problem of muscle mass loss for both ground-based patients and space travelers i s oftenstudied us ing lengthy (days to weeks) research s tudies wherein individuals are restricted to horizonta l bed rest. Animal models in whichmuscles are made nonweight bearing have been developed to a lso s tudy this problem.

CARDIAC MUSCLECardiac cel l s , ca l led cardiomyocytes, share some of the same s tructures as skeleta l muscle, including having severa l nuclei in one cel l and as imi lar organization of thin and thick fi laments , giving the resul ting muscle a s triated appearance (Figure 12-7). Like some types of skeleta lmuscle, they have a high content of myoglobin and a large number of mitochondria to drive oxidative phosphorylation. However, cardiac cel l smay be branched instead of the normal ly s tra ight and uni form structure found in skeleta l muscle.

Figure 12-7. Cardiac Muscle Structure. Diagram of cardiac muscle cel l s indicates characteris tic features of this muscle type. The fibers cons is t ofseparate cel l s with interdigi tating processes wherein they are held together. These regions of contact are ca l led the interca lated disks (IDs ),which cross an enti re fiber between two cel l s . The transverse regions of the s tep-l ike ID have abundant desmosomes and other adherentjunctions , which hold the cel l s fi rmly together. Longi tudina l regions of these disks conta in abundant gap junctions , which form “electrica lsynapses” a l lowing contraction s ignals to pass from cel l to cel l as a s ingle wave. Cardiac muscle cel l s have centra l nuclei and myofibri l s thatare less dense and organized than those of skeleta l muscle. Also the cel l s are often branched, a l lowing the muscle fibers to interweave in amore compl icated arrangement within fascicles that produces an efficient contraction mechanism for emptying the heart. [Reproduced withpermiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Cardiac muscle normal ly derives energy aerobica l ly from triglycerides/fat (65%), glucose (30%), and proteins/ketone bodies (5%) or a l ternativelyfrom lactic acid transported from skeleta l muscle. In fact, cardiac cel l s are a lmost tota l ly rel iant on aerobic respi ration (only about 10% of theenergy needs can ever be derived from anaerobic respi ration) and oxygen del ivery i s , therefore, key to cardiac cel l function and surviva l .Decreased blood supply and oxygen as wel l as removal of waste and carbon dioxide (CO2) via the coronary arteries lead to heart-related chestpain (angina) and heart attacks .

Cardiac muscle produces a s trong and forceful contraction but i s not under voluntary control . Ins tead of nerve input, specia l internalpacemaker cel l s contract on a regular bas is and then propagate this contracti le impulse to the remainder of the heart. Contractions are furthercontrol led by specia l cardiac muscle fibers that are innervated by the autonomic nervous system. T-tubules are fewer but larger and run onlya long the Z-l ines or discs (Figure 12-7). Cardiac cel l s are a lso joined to each other by interca lated discs (IDs ), which serve both s tructura l andimpulse transmiss ion roles (Figure 12-7). The IDs serve as cytoskeleta l anchors between individual cel l s to a l low the coordination of thecontraction into a multicel l compos i te. IDs a lso conta in gap junctions (Figure 12-7; Chapter 8), which a l low the rapid propagation of flow ofions (Chapter 19) from one cel l to the next. Both functions are essentia l for the rapid and synchronized contraction required by the heart.

CICR i s the predominate mechanism in cardiac muscle. Vol tage triggering of a dihydropyridine receptor induces ca lcium to flow into thesarcoplasm. Once ins ide the sarcolemma, the ini tia l influx of ca lcium binds to a ryanodine receptor on the SR and releases much larger s toresof Ca 2+ in a manner s imi lar to inos i tol triphosphate. Ca 2+ flow through L-type calcium channels to activate the cardiomyocytes . These L (“long”)channels s tay open for an extended period of time to susta in the influx of ca lcium. This ri se in ca lcium concentration causes troponin–tropomyos in conformation changes as previous ly described, actin–myos in binding and contraction. The release of tempora l and spatia lca lcium wave and the integrated actions resul ting from the IDs lead to the organized contraction of various parts of the heart muscle toproduce the force to propel blood throughout the body. See Chapter 16 for additional deta i l s .

SMOOTH MUSCLESmooth muscle not only shares severa l qual i ties with skeleta l and/or cardiac muscle but a lso di ffers in others . Smooth muscle activi ty rel ieson the interaction of actin thin fi laments and myos in thick fi laments . Al though smooth muscle conta ins actin and myos in fi laments and ABPs ;in contrast to skeleta l and cardiac muscle, there i s much less s tructura l organization. Smooth muscle, therefore, has a smooth appearance(i .e., not s triated) under a l ight microscope. However, closer examination does show loca l zones of contracti le elements that somewhat mimicthe sarcomere s tructure. This s tructure a l lows smooth muscle cel l s to perform speci fic and di rected functions , both tempora l ly and spatia l ly.

Unl ike skeleta l and cardiac muscle cel l s , smooth muscle cel l s have only one nucleus and lack troponins to regulate actin– myos in headbinding (Figure 12-8A–B). Smooth muscle cel l s a lso do not conta in T-tubules , and the SR i s organized in only a loose network around the cel l .Ins tead, ca lmodul in, ca ldesmon, and ca lponin regulate contraction via ca lcium (see Figure 12-9 and associated text below). Like cardiacmuscle, the cytoskeleta l elements of individual smooth muscle cel l s are anchored together as dense bodies (Figure 12-8A), which mimic the Z-l ine of skeleta l muscle. Dense bodies , composed of the intermediate fi lament (IF) protein desmin, anchor the actin thin fi laments . Other IFs(e.g., vimentin) and s tructures known as adherens junctions are a lso prominent in the organization of smooth muscle. Thesecytoskeleta l/membrane connections a l low transduction of force generation between individual smooth muscle cel l s . Smooth muscle cel l sa lso conta in gap junctions , which a l low the quick spread of Ca 2+ fluxes between cel l s . Together, adherens and gap junctions a l lowcoordinated, multicel lular contractions s imi lar to cardiac muscle.

Figure 12-8. A–B. Smooth Muscle Contraction. Most molecules that a l low contraction are s imi lar in the three types of muscle, but the fi laments ofsmooth muscle are arranged di fferently and appear less organized. A. The diagram shows thin fi laments attach to dense bodies located inthe cel l membrane and deep in the cytoplasm. Dense bodies conta in α-actinin for thin fi lament attachment. Dense bodies at the membraneare a lso attachment s i tes for intermediate fi laments and for adhes ive junctions between cel l s . This arrangement of both the cytoskeleton andcontracti le apparatus a l lows the multicel lular ti s sue to contract as a uni t, providing better efficiency and force. B. Contraction decreases thelength of the cel l , deforming the nucleus and promoting contraction of the whole muscle. The micrograph shows a region of contracted ti ssuein the wal l of a urinary bladder. The long nuclei of individual fibers assume a cork-screw shape when the fibers contract, reflecting thereduced cel l length at this time. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion,McGraw-Hi l l , 2010.]

Like cardiac muscle, smooth muscle contractions can be ini tiated by pacemaker cel l s , which contract on a regular bas is and are innervatedby the autonomic nervous system. However, nerve impulses , hormone s ignals , and even s tretching can a lso ini tiate contraction. Smoothmuscle cel l s do not produce the forceful contraction of skeleta l muscle or cardiac muscle but they have the abi l i ty to undergo ei ther a s lowcontraction for long periods of time with l i ttle energy use (tonic contraction) or a rapid contraction and relaxation (phasic contraction). Toniccontractions are seen in blood vessel wal l s . Phas ic contractions are important for portions of the gastrointestina l tract involved in peris ta ls i s(e.g., esophagus or smal l intestine).

Paramount to any contraction by smooth muscle i s an influx of Ca 2+. Smooth muscle SR conta ins speci fic and specia l i zed areas where ionchannels and s ignal ing pathway receptors are found together. Examples of these receptors include those for G proteins , neurohormones ,activation of individual kinases , L-type ca lcium channels (see above), and ca lcium-sens i tive and insens i tive potass ium channels . Areas of theSR are a lso near the external cel l membrane a l lowing extracel lular s igna ls (e.g., hormones) and ca lcium influxes to a lso activate contraction.Signals for contraction (s igna l from an adjacent smooth muscle cel l via a gap junction, nerve, hormone, and s tretching) resul t in release ofca lcium from the SR (via the ryanodine receptor, see above) as wel l as poss ible influx of external ca lcium through the L-type channels . Theresul ting increase in Ca 2+ concentration ini tiates actin–myos in interactions and force generation.

Regulation of contraction by ca lcium di ffers in many ways from skeleta l and cardiac muscle. Smooth muscle cel l s do not conta in troponin,and tropomyos in only serves an accessory role in actin–myos in force generation. Instead, the proteins ca ldesmon and ca lponin inhibi t myos inATPase enzymatic function and, poss ibly, actin–myos in binding through mechanisms that are s ti l l being s tudied. A genera l overview is that aninflux of ca lcium activates the protein ca lmodul in, which leads to ca ldesmon and ca lponin phosphorylation and loss of inhibi tory effects .Ca lmodul in activation a lso activates MLCK, which leads to the phosphorylation of the regulatory myos in l ight cha in (Figure 12-9). MLCP removesthe phosphate group when ca lcium concentrations fa l l , inactivating myos in and s topping contraction.

Figure 12-9. Regulation of Smooth Muscle Myosin Force Generation by Calcium Ions. The influx of ca lcium ions (Ca 2+) leads to activation of ca lmodul inand then myos in l ight cha in kinase (MLCK). MLCK phosphorylates the regulatory l ight cha ins on the myos in molecule and activates i ts ATPaseand force-generating capabi l i ties . Myos in l ight cha in phosphatase (MLCP) can remove the phosphate group, inactivating myos in activi ty. Seetext for more deta i l s . [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l ,2009.]

The lack of sarcomere s tructure in smooth muscle a lso enables polymerization of G-actin to F-actin thin fi laments to partly regulatecontraction. This process can form new thin fi laments at loca l areas in a smooth muscle where contraction or force generation i s required.Breakdown of exis ting F-actin thin fi laments can s top contraction. The exact role of actin polymerization and depolymerization in smooth and innonmuscle cel l s i s s ti l l being determined. Severa l ABPs involved in these changes are affected by ca lcium as wel l .

Smooth muscle cel l types are found throughout the human body and vary in function but a l l rely on the bas ic actin–myos in contracti lemechanism. Examples and their associated functions are shown in Table 12-1.

TABLE 12-1. Types and Functions of Smooth Muscle Cel l s

ENERGY PRODUCTION AND USE IN MUSCLESHumans use energy in the form of ATP to drive the necessary muscle contractions . During exercise, both aerobic and anaerobic energy-producing systems need to function because di fferent types of muscle fibers are activated, relying on one or both energy sources . The body i sable to move the energy-producing molecules back and forth between the aerobic and anaerobic pathways , depending on the type ofexercise/muscle contraction and duration of exercise. Adaptation between aerobic and anaerobic metabol i sm is a lso regulated by increasedtranscription and trans lation of factors such as hypoxia-inducible factor-1 α. Muscles mainly use one of three di fferent energy sources (Figure 12-10) to produce this ATP—muscle glycogen (glucose), creatine phosphate, or triglycerols/fatty acids from adipose ti ssue.

Figure 12-10. Sources of ATP in Muscles. ADP, adenos ine diphosphate; AMP, adenos ine monophosphate; ATP, adenos ine triphosphate.[Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Creatine phosphate, in essence a s torage form of energy for muscle, can use i ts high energy phosphate bond to readi ly produce ATP in areaction cata lyzed by the enzyme creatine kinase. Muscle glycogen i s a major s torage depot from which glucose-6-phosphate for the glycolyticpathway can be produced. Some types of muscle fibers rely primari ly on fatty acids from triacylglycerols (triglycerides) as fuel . Both glycogenand fatty acids can be metabol i zed via aerobic respi ration by types I and IIa muscle fibers and produce ATP by oxidative phosphorylation.Under aerobic conditions , the metabol i sm of glucose yields pyruvate and energy in the form of ATP. The pyruvate i s then completely oxidized toCO2 wi th the formation of addi tional 15 ATP for each pyruvate molecule. Additional ly, the nicotinamide adenine dinucleotide (NADH)generated from glycolys is can be processed by the malate–aspartate shuttle to generate additional three ATP per NADH. Hence, under aerobicconditions , each glucose molecule can yield up to 38 ATP.

In periods of low oxygen, the ci tric acid cycle and oxidative phosphorylation are l imited and anaerobic respi ration i s used. Types IIb and IIxmuscle fibers rely mainly on anaerobic ATP production. When this happens , pyruvate accumulates and i s then converted to lactic acid by theaction of the enzyme lactate dehydrogenase and use of an NADH molecule. In this way, NAD+ i s regenerated to susta in flux through glycolys is .

This point when lactate production i s greater than lactate uti l i zation i s known as the lactate threshold. This threshold usual ly occurs ataround 65% of a person’s maximal oxygen consumption. If muscle activation continues , then lactic acid accumulates in the muscle ti s sue andcan affect important metabol ic functions , giving the muscle pa in and the sensation of muscle fatigue. The surplus of lactic acid i s ul timatelytransported to the l iver or heart muscle where i t i s converted back into pyruvate and into the ci tric acid cycle.

McArdle’s disease: Glycogen s torage diseases are a series of disorders that impact on the body’s abi l i ty to produce, s tore, and/or breakdownthis important energy source. One type of glycogen s torage disease, type V or McArdle’s disease, resul ts from the absence ofmyophosphorylase, the muscle form of glycogen phosphorylase. Patients usual ly present in chi ldhood with muscle pa in, fatigue, cramps, andweakness with excess ive myoglobin in the urine, indicative of rhabdomyolys is (see above) during prolonged periods of exercise.Progress ive symptoms and muscle mass loss and weakness are usual ly evident as the patient ages . Diagnos is may be compl icated by a“second wind” fel t by patients . Al though once thought to be s imply due to the use of a l ternative energy sources such as fatty acids andproteins , evidence now suggests a poss ible role of increased numbers of GLUT4 receptors on the plasma membrane, a l lowing increasedglucose uptake for glycolys is ins tead of relying on glycogen s tores .

MICROTUBULE-BASED MOTILITYMicrotubules function as a s tructura l cytoskeleton but they a lso serve as a “track” system for transport within the cel l . With the use of motorproteins , intracel lular components such as organel les including mitochondria , ves icles , or granules can be moved within the cel l .Chromosomes can a lso be moved with unique attachment proteins . Joined together, microtubules form the s tructure of more complex cel lularmoti l i ty components such as ci l ia or flagel la . Centrioles , such as ci l ia and flagel la , are a lso made of microtubules . Centrioles come in pa i rsthat are oriented at right angles to each other and are respons ible for setting up the spindle that moves the chromosomes during mitos is .

Microtubules form the backbone of a l l of these s tructures . Microtubules are made of l inear polymers of a globular protein ca l led tubulin(Chapter 1) and have a polar arrangement within the cel l wi th the pos i tive end di rected toward the periphery and the negative end toward thecenter. Like actin, there are s l ightly di fferent forms of tubul in (α to ε), which form di fferent microtubule s tructures in di fferent parts of the cel land body. α- and β-tubul in form cytoplasmic microtubules , γ-tubul in forms centrosomes, and δ- and ε-tubul in are involved in centrioles andthe mitotic spindle apparatus .

Microtubule Polymerization and Cancer Therapy: Microtubule function i s essentia l for a variety of cel lular functions including severa l aspects ofmitos is , which involves polymerization and depolymerization of tubulin into microtubule polymers . The medications colchicine, nocodazole,vincristine, and colcemid a l l inhibi t polymerization by interfering with the tubul in monomer. The taxane fami ly of drugs , including taxol, breakdown exis ting microtubules . Al l lead to the dis ruption of mitos is and eventual death of the affected cel l . Al though these effects are used inthe treatment of gout (inhibi tion of microtubule-dependent, neutrophi l moti l i ty, as wel l as uric acid crysta l l i zation) and various otherinflammatory diseases , these agents have a lso emerged as important cancer therapies. Because cancerous cel l s often rapidly divide, agentsthat inhibi t microtubules have emerged in the treatment of cancer as wel l as other diseases . The inhibi tion of mitos is a ffects cancer cel l smore than normal cel l s , leading to a partly selective treatment agent. Unfortunately, s ide effects of these microtubule/mitos is inhibi torsare s ti l l apparent, l imiting thei r use and/or caus ing additional problems for patients .

The polar a l ignment of microtubules plays a role as part of the transport system within a cel l , us ing a convers ion of chemica l energy tokinetic energy via the motor proteins dynein and kines in, very s imi lar in mechanism to the interaction of actin and myos in. With thearrangement of the microtubule with the negative end toward the center and the pos i tive end toward the periphery, the dynein molecules arerespons ible for transport toward the nucleus and kines ins are respons ible for transport toward the plasma membrane.

Dynein, l i ke myos in, i s a large ATPase with two heads and uses the hydrolys is of ATP to move the components of a cel l by l i tera l ly walkinga long the microtubule network s tructure (Figure 12-11A). The polari ty of the microtubule plays a role in the di rection of the movement of thedynein to the negative end of the microtubule or toward the nucleus . Two forms of dynein exis t. Cytoplasmic dynein organizes intracel lularorganel les and moves ves icles within cel l s as wel l as chromosomes and the mitotic spindle during mitos is . Axonemal dynein functions in ci l iaor flagel la moti l i ty. The two heads of cytoplasmic dynein are joined together at a ta i l wi th intermediate and l ight cha ins , the latter of whichattaches the dynein molecule to the organel le or cargo being transported.

Figure 12-11. A–B. Microtubule Motors—Dynein and Kinesin. A. Structure and s ize of cytoplasmic dynein. B. Structure and s ize of kines in, includingi ts associated regulatory l ight cha ins and connection to i ts cargo ves icle. [Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review ofMedica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Kinesins are a lso microtubule-based motor proteins that function via the hydrolys is of ATP and, l ike dynein, are respons ible for movingsubstances around the ins ide of a cel l (Figure 12-11B). Like dynein, kines in molecules are a lso involved in mitotic spindle formation andchromosome movement during mitos is . The fi rs t kines in was discovered in 1985, with many more subfami l ies of kines ins discovered s incethen. Al l human kines ins are respons ible for intracel lular transport toward the pos i tive end of the microtubule network or toward the plasmamembrane. Kines in molecules resemble myos in II molecules with two heavy cha ins and two l ight cha ins . The heavy cha ins have two heads atone end, which bind to and transport a long the microtubules ; the ta i l portion of the heavy cha in attaches to the cargo organel le or ves icle viathe l ight cha ins .

In cilia and flagella, the microtubules are arranged in an organized s tructure (Figure 12-12). In ci l ia and flagel la , two microtubules are joinedtogether to form a doublet. The IF protein nexin (see Table 12-2) forms l inks a long the microtubules to hold them together. Nine of thesedoublets form a ci rcle around two microtubules in the center. Extending from the doublets are “arms” that connects neighboring doublets ,composed of axonemal dynein. Radial spokes a l so provide s tructure between the inner and outer microtubule doublets . The organization of themicrotubules a l lows the dynein to s l ide a long one tubule, whi le attached with i ts ta i l to another microtubule creating a bend in the ci l ium orflagel la . However, the nexin l inkages a l low for only l imited lengthwise movement and, therefore, create a bend in the ci l ium. The dyneinbridges are a lso regulated so the s l iding can lead to synchronized bending. A centriole/basal body s tructure at the base of the ci l ia or flagel laand composed of triplets of tubul in monomers helps to form and anchor these longer s tructures .

Table 12-2. Intermediate Fi lament Types and Functions

Figure 12-12. A–B. Structure of Cilia and Flagella. The s tructure of ci l ia and flagel la i s composed of (A) a centra l microtubule doublet surrounded bynine outer doublets with dynein arms between them. The dynein creates movement of the ci l ia or flagel la . Radia l spokes and nexin providestructure and l imits to this movement. The base of the main ci l ia or flagel la s tructure includes (B) a centriole/basa l body with a tripletmicrotubule s tructure, which serves as an anchor. See text for further deta i l s . [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas icHis tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Microtubules, Cilia, and Flagella—Roles in Disease Processes: Al though often rare, defects in ci l ia/flagel la , known as ci l iopathies , lead to severa ldiseases/syndromes including the fol lowing:

Kartagener Syndrome/Primary Ci l iary Dyskines ia—Defective ci l ia in the respiratory tract, Eustachian tube, and Fa l lopian tubes leading tochronic lung infections , ear infections and hearing loss , and inferti l i ty. Poss ible association with “s i tus inversus ,” a condition in whichmajor internal organs are “fl ipped” left to right.

Senior–Loken Syndrome/Nephronopthis i s—Eye disease and formation of cysts in the kidneys , leading to renal fa i lure.Bardet–Biedl Syndrome—Dysfunction of ci l ia throughout the body leading to obes i ty because of inabi l i ty to sense satiation, loss of eye

pigment/visua l loss and/or bl indness , extra digi ts and/or webbing of fingers and toes , menta l and growth retardation andbehaviora l/socia l problems, smal l and/or misshaped genita l ia (male and female), enlarged and damaged heart muscle, and kidneyfa i lure.

Als trom Syndrome—Chi ldhood obes i ty, breakdown of the retina leading to bl indness , hearing loss , and type 2 diabetes .Meckel–Gruber Syndrome—Formation of cysts in kidneys and bra in leading to renal fa i lure and neurologica l defici ts , extra digi ts , and

bowing/shortening of the l imbs .Increased Ectopic (Tubal ) Pregnancies/Male Inferti l i ty—Deficient ci l ia in Fa l lopian tubes or flagel la/sperm ta i l moti l i ty.Autosomal Recess ive Polycystic Kidney Disease—Much rarer than the autosomal dominant form, dys function of basa l bodies and ci l ia in

renal cel l s leads to a l terations of the lung and kidneys , resul ting in a variety of secondary medica l conditions and often death.Parkinson’s and Alzheimer’s Diseases—Although work i s s ti l l ongoing, researchers now feel that some forms of Parkinson’s and

Alzheimer’s diseases may resul t, in part, from damage to microtubules and associated proteins . Treatments a imed at s tabi l i zingmicrotubules may help sufferers from these speci fic proteins .

INTERMEDIATE FILAMENTSThe IF i s the name for a s ingle protein member of a fami ly of related cytoskeleta l l inear proteins (monomer forms) or the resul ting

fi laments that these proteins form. The fi laments forms are about 10 nm in diameter and, as the name impl ies , are between the s i ze of 6 nmactin microfi laments and ei ther 15–20 nm myos in thick fi laments or 25 nm microtubules (Figure 12-13). Al l IF monomer proteins have two headregions for binding and lengthy, connecting α-hel ica l portion. IFs are formed from the intertwining of the α-hel ica l section of pa i rs of the same(homodimer) or di fferent (heterodimer) monomers or pa i rs of pa i rs (homo- or heterotetramer) IFs can s tretch and compact because of thei rs tructure and, thus , can serve to transmit force or absorb mechanica l s tress . There are s ix bas ic types based on s tructura l s imi lari ties (Table12-2).

Figure 12-13. Comparison of Cytoskeletal Proteins. Relative s i zes of actin microfi laments , intermediate fi laments , and microtubules are shown.[Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

NONMUSCLE CELLSNonmuscle cel l s include skin fibroblasts ; platelets ; immune system lymphocytes and monocytes/macrophages (Chapter 15); neurons ,including photoreceptor cel l s of the retina (Chapter 19); chondrocytes and osteocytes (Chapter 13); various cel l s of the kidney, pancreas , andintestines (Chapter 11); as wel l as the thyroid and blood vessels . As such, nonmuscle cel l s are respons ible for diverse tasks such as woundheal ing, immune response, specia l i zed nerve functions , connective ti ssue and bone formation and heal th, digestion, regulation ofmetabol i sm, cardiovascular disease, and even embryologica l development. As noted previous ly, the contracti le apparatus in nonmuscle cel l si s far less s tructured than skeleta l , cardiac, or smooth muscle types but does preserve many of the same functional and regulatory elements .Actin thin fi laments and myos in II thick fi laments are major parts of nonmuscle cel l moti l i ty and interact in an analogous way. Other types ofmyos in (e.g., myos in I , myos in V, and others ) are a lso prominent in nonmuscle cel l moti l i ty that includes intracel lular transport (Figures 12-3Band 12-11B). Regulation of contraction i s much l ike smooth muscle where Ca 2+ regulate actin fi lament s tabi l i ty and length, aspects of actin–myos in head binding, and, fina l ly, myos in force generation.

REVIEW QUESTIONS1. What are the roles of the bas ic muscle components including how they may interact with each other to faci l i tate muscle function?2. What are the three domains of myos in and how is each involved in muscle contraction?3. From where and how is ca lcium released for skeleta l muscle contraction and what i s i ts role in the process?4. What i s the role of myos in ATPase in muscle contraction?5. How is ca lcium sequestered and concentrated in the sarcoplasmic reticulum after contraction cycles?6. What i s the role and mechanism of action of acetylchol ine in muscle contraction?7. What are the types and metabol ic characteris tics of the di fferent muscle fiber types?8. How does s tructure and contraction of cardiac muscle di ffer from skeleta l muscle?9. What are the types and functions of smooth muscle cel l s?

10. How do muscles generate energy for function in anaerobic versus aerobic conditions?11. What are the functions of microtubules and the associated roles of dynein and kines in in moti l i ty?12. What are the types and functions of intermediate fi laments?13. How is nonmuscle cel l contraction a l ike and di fferent from contraction by other muscle types?

CHAPTER 13CONNECTIVE TISSUE AND BONE

Editor: Jacques Kerr, BSc, MB, BS, FRCS, FCEMLead Consul tant in Emergency Medicine, Borders Genera l Hospi ta l , Melrose, Scotland, United Kingdom

Connective TissueComponents of BoneBone Growth and Remodel ingRegulation of Ca lcium LevelsMarkers of Bone Formation and ResorptionReview Questions

OVERVIEWConnective ti ssue provides a framework and support for a large variety of s tructures , including organs , blood vessel wal l s , as wel l as thebetter known functions of connecting muscle to bone and bone to bone. Chondrocytes are respons ible for the production of connective ti ssuecomponents , including the principa l protein, col lagen. The three types of connective ti ssues offer a wide variety of functions to ful fi l l the manyroles needed by the body. A multi tude of diseases resul t from abnormal i ties , deficiencies , or overproduction of connective ti ssues or thei rcomponents .

Bones provide a mechanica l s tructure for the human body and, in that role, a l so a l low effective muscle contraction and, therefore,movement. Bones offer protection for the body’s internal organs , especia l ly bra in, heart, lungs , l i ver, s tomach, and spleen. In the ear, bonestransduct sound waves from the ear drum to the inner ear. Osteoblasts , osteocytes , and osteoclasts produce, break down, remodel , and repairboth the organic and inorganic matrix that make up bones . In doing so, these cel l s , as wel l as severa l regulatory molecules , help to regulateca lcium and phosphate metabol i sms and levels in the body. Bones a lso have a synthetic function. Within thei r marrow, bones produce redand white blood cel l s as wel l as growth factors ; and s tore fatty acids as yel low marrow. Fina l ly, bones provide for the s torage of certa inminera ls , including ca lcium and phosphorus and, to a lesser extent, zinc, copper, and sodium. In an analogous role, bone can temporari lyabsorb and s tore toxic heavy meta ls to reduce their effects on the body.

CONNECTIVE TISSUEConnective tissue i s a fibrous ti ssue made mainly of col lagen (Chapter 1) and proteoglycans (Chapter 2) that forms, supports , and/or connectsvarious organs in the body, attaches muscles to bones (e.g., tendons) and bones to bones (e.g., l igaments ), forms the supportive matrix duringbone formation (see below), and makes up various s tructures such as parts of blood vessels and intestina l wal l s . One major example ofconnective ti ssue i s col lagen, which i s found in various forms throughout the body (Figure 13-1A–D).

Figure 13-1. A–D. Distribution of Cartilage in Adults. (A) There are three types of adul t carti lage dis tributed in many areas of the skeleton,particularly in joints and where pl iable support i s useful , as in the ribs , ears , and nose. Carti lage support of other ti s sues throughout therespiratory system is a lso prominent. The photomicrographs show the main features of (B) hya l ine carti lage, (C) fibrocarti lage, and (D) elas ticcarti lage. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Formation of connective ti ssue rel ies on chondrocytes, which both produce and mainta in the col lagen matrix. Chondrocytes di fferentiatefrom osteochondrogenic cel l s , which can a l ternatively develop into osteoblasts (see below). Al though the di fferent s tructures vary widely, threemajor fiber forms of connective ti ssue fibers are prominent—col lagenous fibers , elastic fibers , and reticular fibers .

Col lagen i s the principa l component of collagenous fibers, and the type of col lagen determines the s tructura l and functional qual i ties of thatparticular form. Col lagen i s bel ieved to represent about a quarter of the tota l protein in human body. Al though 29 types of col lagen have beendiscovered, each varying in i ts s tructure, location in parts of the body, and functions , four (types I–IV) make up a vast majori ty of connectivetissue. Structure and formation of a type I col lagen fiber i s i l lus trated in Figure 13-2A–B.

Figure 13-2. A. Structure of Collagen. I l lus tration of s tructura l arrangement of individual , overlapping col lagen molecules (1), col lagen fibri l s ,col lagen fibers (2 and 3), and col lagen fiber bundles (4 and 5). The overlapping nature of this s tructura l organization produces the requiredstrength and flexibi l i ty of this molecule. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12thedition, McGraw-Hi l l , 2010.] The fina l type of connective ti ssue fiber i s the elastic fiber (Figure 13-3C). Elas tic fibers are formed from smal lermicrofibrils, made mainly of the protein tropoelastin/elastin and the glycoprotein fibrillin, and cross -l inking polypeptides (Figure 13-4A–B). Themicrofibri l s are in the form of i rregular, random coi l s , wi th glycine, va l ine, a lanine, prol ine, and lys ine amino acids contributing to the s tablestructure. B. Formation of Collagen Fibers. Col lagen peptides are synthes ized by ribosomes in the lumen of rough endoplasmic reticulum (RER) as“preprocol lagen” molecules (not shown) with N-terminal s igna l peptides . Signal peptides are removed to produce “procol lagen” molecules .Prol ine and lys ine amino acid res idues are hydroxylated via prolyl hydroxylase and lysyl hydroxylase enzymes , which depend on vi tamin C as acofactor. Some amino acid res idues are a lso glycosylated. The procol lagen molecules form a left-handed triple hel ix within the RER lumen.The hel ica l molecules are transported to the Golgi apparatus , where they are processed and secreted via exocytos is to the cel l exterior. Theamino- and carboxy-terminal ends are removed by the enzyme procol lagen peptidase to form col lagen fibri l s with further cross -l inking ofhydroxylys ine and lys ine res idues on di fferent tropocol lagen molecules by the enzyme lysyl oxidase. Cross -l inked col lagen fibri l s formcol lagen fibers . Because there are many s l ightly di fferent genes for procol lagen α chains and col lagen production depends on severa lposttrans lational events involving severa l other enzymes , many diseases involving defective col lagen synthes is have been described.[Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Figure 13-3. A–C. Representative Types of Collagen-Based Connective Tissue. (A) Molecules of type I col lagen, the most abundant type, assemble toform much larger s tructures . Transmiss ion electron microscopy shows that fibri l s cut longi tudina l ly and transversely. In longi tudina l sections ,the fibri l s display a l ternating dark and l ight bands , which are further divided by cross -s triations , and in cross section, the cut ends ofindividual col lagen molecules can be seen. (B) Si lver-s ta ined sections of adrenal cortex i l lus trate a network of reticular fibers , which providesa framework for cel l attachment. Reticular fibers conta in type II I col lagen, which i s heavi ly glycosylated. (C) Elas tic fibers are composed of athi rd type of connective ti ssue, formed from microfibri l s of elastin and fibri l l in and add res i l iency to the connective ti ssue. They can be seenbetween layers of smooth muscles in the wal l of elastic arteries such as the aorta (pink, upper panel ) and in s tructures such as mesentery(dark magenta, lower panel ). [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Figure 13-4. A–B. Quaternary Structure of Elastic Fibers. Elas tin microfibri l s may form internal and external (between di fferent elastin fibers ) cross -l inks to form the cross -l ink s tructure “desmos ine,” which provides both s trength and elastici ty. I l lus tration of function of elastin fibersprovides s trength and elastici ty to a number of ti s sues (see text). 13-4A. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion,Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.] 13-4B. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text andAtlas , 12th edi tion, McGraw-Hi l l , 2010.]

Varying combinations of the di fferent types of col lagen can a l ter this bas ic s tructure for particular cel l functions . Most col lagenous fibersfol low the previous ly described, left-handed, triple-hel ix of type I (Figure 13-3A), with multiple glycine, prol ine/ hydroxyprol ine, andhydroxylys ine amino acids contributing to i ts s tructure.

The second type of connective ti ssue i s the reticular fiber, composed of type II I col lagen and forming an ordered, “reticulum” meshworkinstead of a l inear s tructure (Figure 13-3B). The reticular network l inks these proteins to carbohydrates , including glucose, ga lactose,mannose, and fucose (Chapter 3). Reticular fibers are found mainly in l iver, muscle, bone marrow, lymph system, and various other ti s sues andorgans , where i t offers a supportive framework.

Elastic fibers are found in severa l ti s sue types , but mainly in skin, the outer ear, larynx and epiglotti s , blood vessels , lungs , bladder, someintervertebra l discs , and as an attachment between teeth and the underlying bones . As the name suggests , elastic fibers can be eas i lys tretched (up to 1.5 × thei r origina l length). Diseases related to defects in elastin include Menkes disease, Hurler disease, Wi l l iam’ssyndrome, cutis laxa , Buschke– Ol lendorf syndrome, and pseudoxanthoma elasticum. Changes in the elastic fibers in the heart and/or arteriesmay a lso play a role in high blood pressure, as the abi l i ty to absorb the force of heart contraction with appropriate blood vessel s tretch andrebound i s diminished.

Diseases of connective ti ssues are usual ly associated with defects in the component col lagen molecule or deficiencies in essentia lnutrients required for thei r synthes is (e.g., vi tamin C) and secretion. A large group of autoimmune diseases of connective ti ssue a lso exis ts , inwhich the body’s immune system mistakenly attacks parts of the body. If too much col lagen i s produced, scleroderma may resul t, anautoimmune disease characterized by thickening and hardening of the skin and deleterious changes in blood vessels . Most of the connectivetissue diseases are diagnosed by phys ica l examination, looking for abnormal i ties in the organ(s ) involved that reflect changes in theconnective ti ssue. DNA studies and biops ies can help confi rm the condition(s ). Autoimmune connective ti ssue diseases are normal lydiagnosed by symptoms and by blood tests reveal ing diagnostic antibodies or reactive molecules . The col lagen types , functions , and relateddiseases are reviewed in Table 13-1.

Table 13-1. Summary of Col lagen Types

COMPONENTS OF BONEBone i s made up of the inorganic minera l hydroxyapatite, known more formal ly as ca lcium apati te [Ca 5(PO4)3(OH) or, in i ts crysta l -s tructureform, Ca 10(PO4)6(OH)2]. Carbonated hydroxyapati te i s a l so the main component of the enamel in teeth, and variable chemica l forms can causenatura l variations in teeth color. In addition, bone i s composed of an organic matrix, primari ly composed of type I col lagen and three primarycel l types—osteoblasts , osteoclasts , and osteocytes (Figure 13-5A-B).

Figure 13-5. A–B. Osteoblasts and Osteocytes. (A) The photomicrograph of developing bone shows the location and morphologica l di fferencesbetween osteoblasts (OB) and osteocytes (O). Rounded osteoblasts , derived from the mesenchymal cel l s nearby, appear as a s imple row ofcel l s adjacent to a very thin layer of l ightly s ta ined matrix covering the more heavi ly s ta ined matrix. The l ightly s ta ined matrix i s osteoid.Osteocytes are less rounded and are located within lacunae. In thin spicules of bone such as those seen here, canal icul i are usual ly notpresent. (B) Schematic diagram shows the relationship of osteoblasts with osteoid, bone matrix, and osteocytes . [Reproduced withpermiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Osteoblasts, a specia l i zed form of a fibroblast cel l , which produces bone, ari se from osteoprogenitor cells, whose growth and development areinfluenced by fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and bonemorphogenetic proteins (BMPs). FGF s ignal ing molecules bind to a receptor with tyro-s ine kinase (phosphorylation) activi ty, which ini tiates anumber of s igna l ing pathways inducing the express ions of selected genes .

TGF-β s ignal ing usual ly occurs by fi rs t combining two TGF-β proteins via a unique “cysteine knot” s tructure, in which nine, highly conserved,cysteine amino acids on each monomer form disul fide bonds with the analogous cysteine res idue. The TGF-β homodimer s tructure theninteracts with a dimer of type II receptors (Figure 13-6). The bound TGF-β) dimer/type II receptor dimer then recrui ts two type I receptors ,creating a four-protein (tetramer) receptor a long with two-protein (dimer) substrate complex. Binding of these s ix proteins causes aconformational change of the type II receptor dimer, which leads to serine kinase activi ty and phosphorylation of serine amino acids on thetype 1 receptor. These phosphorylated serine res idues can then bind to a variety of speci fic s igna l ing proteins (known as SMADs and SARAs ,the latter of which uti l i ze a zinc-finger binding moti f; see Table 9-1). These s ignal ing proteins combine and enter the nucleus , where they actas transcription-activating factors for a variety of gene products . BMP substrates s igna l in a s imi lar manner to TGF-β, uti l i zing the SMADproteins as transcription factors .

Figure 13-6. Mechanism of TGF-β Signaling. The TGF-β protein (l ight blue) forms a dimer via a series of eight disul fide bonds (represented by –S •S–). This dimer recrui ts two type I (TβR-1) and two type II (TβR-II) TGF-β receptors . Formation of tetramer receptor s tructure leads to serineamino acid phosphorylation on the type I cytoplasmic ta i l and subsequent s igna l ing via the SARA, RSMAD, and SMAD4 proteins , leading toincreased transcription in the nucleus . [Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion,McGraw-Hi l l , 2010.]

Osteoarthritis: Osteoarthritis (OA), sometimes ca l led “degenerative” or “wear and tear” arthri ti s , resul ts from the breakdown/loss of carti lageand bones in joints , resul ting in pa in, s ti ffness , and decreased movement of the affected areas . Al though a number of infective,inflammatory, and miscel laneous secondary causes are known, most cases have no identi fiable primary cause. However, the resul tingeffects are bel ieved to be due to decreas ing water content of the carti lage, caus ing a reduction in the proteoglycan content. With lessproteoglycans , the carti lage i s more prone to degradation that eventual ly leads to the gradual loss of the joint components , inflammationfrom these breakdown products , and, occas ional ly, reactive growth of bone “spurs .”

Treatment i s often multi factoria l , including pa in medications , moderate exercise, and, occas ional ly, surgery, a l though these often haveless than optimal effectiveness . A variety of a l ternative treatments have thus been developed, including acupuncture, herbs , supplements ,fi sh oi l s , and, probably most prominent, use of glucosamine and chondroi tin sulphate. Glucosamine, s imply glucose with an amino (NH2)group at the second carbon, i s a known component of glycosaminoglycans (GAGs) and proteoglycans . Chondroitin, a GAG with sul fate (SO3)groups , i s a l so a part of the proteoglycan s tructure of carti lage. As a resul t, some bel ieve that the ora l intake of these two supplements canhelp to restore the proteoglycan content of carti lage, leading to rel ief in OA symptoms. To date, research s tudies and analyses have notshown any objective improvement of OA, a l though they have a lso shown that no adverse effects resul t from their use and, more importantly,that a s trong placebo effect may actua l ly help many patients . Further s tudies are ongoing.

Fibrodysplasia Ossificans Progressiva (FOP) and BMPs: FOP i s a very rare, but potentia l ly, extremely debi l i tating disease, in which the repairprocess of fibrous ti ssues such as muscles , tendons , and l igaments i s changed to bone repair, resul ting in minera l depos i tion/bone matrixformation in the normal ly flexible ti s sue. Progress ion of the disease l i tera l ly locks the patient’s body parts in place, a process sometimesdescribed by the express ion “turned to s tone.” The disease i s bel ieved to be caused by an autosomal dominant mutation of BMP4. Recentresearch indicates that BMP4 is actua l ly the protein activin, a protein dimer better known for i ts role in the secretion of fol l i cle-s timulatinghormone (Chapter 20). Mutated activin erroneous ly affects i ts BMP type I receptor, promoting the activation of SMAD proteins , genetranscription, and bone matrix growth. Lymphocytes , which respond to the ini tia l damage to the fibrous ti ssue, are bel ieved to be thecarriers of the mutated protein. There i s no known cure for FOP, a l though i t i s poss ible that shark squalamine, a protein under tria l , whichprevents bone growth in carti lage, may provide some treatment.

Osteoblasts produce the protein osteoid, made primari ly of type I col lagen, and are a lso respons ible for laying down the hard minera lmatrix (Figure 13-7). Osteoid i s fi rs t synthes ized as approximately 300-nm long, tropocollagen “microfibri l s ,” which interdigi tate and bind viahydrogen and covalent bonding with neighboring microfibri l s to form a sheet-l ike col lagen matrix of para l lel fibri l s . Gaps betweentropocol lagen molecules offer s i tes for hydroxyapati te minera l i zation.

Figure 13-7. Mineralization in Bone Matrix. From their ends adjacent to the matrix, osteoblasts secrete type I col lagen, severa l glycoproteins , andproteoglycans . Some of these factors , notably osteoca lcin and certa in glycoproteins , bind Ca 2+ wi th high affini ty, thus ra is ing the loca lconcentration of these ions . Osteoblasts a lso release very smal l membrane-enclosed matrix ves icles with which a lka l ine phosphatase and

other enzymes are associated. These enzymes hydrolyze ions from various macromolecules , creating a high concentration of theseions loca l ly. The high ion concentrations cause crysta ls of CaPO4 to form on the matrix ves icles . The crysta ls grow and minera l i ze further withthe formation of smal l growing masses of hydroxyapati te [Ca 10(PO4)6(OH)2], which surround the col lagen fibers and a l l other macromolecules .Eventual ly, the masses of hydroxyapati te merge as a confluent sol id bony matrix as ca lci fi cation of the matrix i s completed. [Reproduced withpermiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

In addition to osteoid, osteoblasts a lso produce “ground substance,” composed primari ly of the GAG chondroitin sulphate (Chapter 2) andsevera l osteoblast-derived glycoproteins , including osteocalcin (function not completely understood, but bel ieved to regulate bone formation),osteonectin (binds ca lcium and col lagen and ini tiates minera l i zation process ), osteopontin, a l so known as bone sialoprotein [productionincreased by ca lci triol (Vi tamin D3), and thought to help anchor osteoclasts to the bone matrix for bone resorption, but may a lso s timulateformation of fi rs t hydroxyapati te crysta ls for new minera l i zation]. Newly formed osteoblasts a lso secrete the cytokine (Chapter 15) macrophagecolony-s timulating factor (M-CSF), another disul fide homodimer molecule, which binds to i ts receptor, leading to tyros ine phosphorylation.This activi ty promotes the growth and di fferentiation of osteoclasts (see below) to help mainta in the ba lance between bone formation andresorption, as wel l as ca lcium levels (see below).

Fol lowing laying down of the osteoid and ground substance matrix, osteoblasts secrete ves icles with the enzyme alkaline phosphatase, whichremoves phosphate groups from hydroxyapati te. The modi fied hydroxyapati te and remnant ves icles then act as centers for depos i t of ca lciumand phosphate crysta ls and, as a resul t, newly minera l i zed bones .

Collagen Synthesis and Scurvy: The proper production of col lagen i s essentia l for osteoid/bone formation, as wel l as other connective ti ssuesdiscussed below. Part of col lagen’s unique s tructure i s the presence of hydroxylated prol ine and lys ine res idues , which s tabi l i ze the triple-hel ica l s tructure essentia l for col lagen’s s tructure and function. Vitamin C (ascorbic acid) i s a required cofactor for these hydroxylationreactions , and the deficiency of this vi tamin causes the disease scurvy. Symptoms of scurvy include abnormal bleeding (from affectedcapi l laries ), skin discoloration, nonheal ing wounds and gum deterioration/loss of teeth (from connective ti ssue), and weakening of bones(from adverse effects on bone formation/remodel ing). Untreated scurvy can be fata l . The treatment for scurvy i s s imple supplementation ofvi tamin C in the diet. In Latin, “ascorbic” l i tera l ly means “no scurvy.”

Osteoclasts are modi fied forms of monocyte/macrophage cel l s that break down bones and are regulated in di fferentiation and growth by thebinding of receptor activator of nuclear factor kappa B (RANK) l igand and by the activi ty of the cytokine M-CSF (noted above). Binding of the RANKl igand to i ts receptor [a member of the tumor necros is factor (TNF) receptor fami ly] activates nuclear factor kappa B (NF-κB), an importanttranscriptional activator protein. M-CSF binds to a tyros ine kinase receptor, leading to a compl icated and not yet ful ly understood s ignal ingpathway that leads to osteoclast di fferentiation. These osteoclast effector molecules are produced by osteoblasts and surrounding cel l s , andboth RANK and M-CSF are required for osteoclast production. Di fferentiation of osteoclasts i s inhibi ted by the molecule osteoprotegerin (OPG)(see below), which inhibi ts RANK l igand binding to i ts receptor.

Osteoclasts perform the oppos i te function to osteoblasts , namely resorption and/or remodel ing of the minera l matrix. Bone remodel ing(see below) a l lows the body to change exis ting bone s tructure during growth and to repair microfractures that resul t from normal mechanica ls tress , as wel l as from pathologica l fractures due to injury. Remodel ing a lso provides a source of ca lcium as part of the regulation of ca lciumlevels in the body (see below).

Osteoclast resorption/bone remodel ing i s accompl ished by severa l mechanisms. Fi rs t, osteoclasts are able to attach to bone via a uniquepodosome s tructure, in which intracel lular actin fi laments attach to membrane integrin receptor molecules (Figure 13-8), which subsequentlybind to a speci fic arginine–glycine–aspartate amino acid sequence on the osteopontin protein (discussed earl ier). Once bound, an osteoclast-

derived carbonic anhydrase produces hydrogen ions via the reaction and pumps them out into the bonematrix by a specia l i zed ATPase pump. Osteoclasts a lso produce an Fe2+-conta ining glycoprotein, tartrate resistant acid phosphatase (TRAP),present in abundance in osteoclast lysosomes. TRAP’s Fe2+ binds with a phosphate in hydroxyapati te and cleaves the phosphate ester bondby nucleophi l i c attack of a hydroxyl group. Free phosphate and ca lcium ions that are released are ini tia l ly taken up by ves icles , but thensubsequently released into the blood s tream by exocytos is . TRAP i s a lso bel ieved to reduce osteopontin/bone s ia loprotein activi ty (seeabove) via dephosphorylation and a lso influences growth, di fferentiation, migration, and activi ty of osteoblasts , as wel l as the migration ofosteoclasts . TRAP a lso generates reactive oxygen species , which help to resorb bone. Severa l cathepsin enzymes , especia l ly the cathepsin Kenzyme, are a lso produced by osteoclasts , which degrade the col lagen matrix via selective cleavage of col lagen and other proteins . Thesecatheps ins have no apparent speci fici ty for particular peptide bonds , a l though some preference for amino acids with large hydrophobic s idechains has been noted. Other enzymes such as aspartate protease and matrix metalloprotease a l so a id in the breakdown of the inorganic andorganic matrices . Osteoclast activi ty i s regulated by severa l hormones , which are discussed below.

Figure 13-8. Integrin Cell Surface Matrix Receptor. By binding to a matrix protein and to the actin cytoskeleton (via ta l in) ins ide the cel l , integrinsserve as transmembrane l inks by which cel l s adhere to components of the extracel lular matrix (ECM). The molecule i s a heterodimer, with αand β cha ins . The head portion may protrude some 20 nm from the surface of the cel l membrane into the ECM, where i t interacts withfibronectin, laminin, or col lagens . [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion,McGraw-Hi l l , 2010.]

Osteoid Mineralization and Osteomalacia/Rickets: The process of minera l i zation of osteoid by osteoblasts i s cri ti ca l for the proper developmentof bones . When osteoblasts are unable to form hydroxyapati te or when sufficient ca lcium and/or phosphate are not ava i lable, i t resul ts inosteomalacia. In chi ldren, this condition i s known as rickets. Osteomalacia , l i tera l ly meaning “bone softness ,” exhibi ts normal amounts oforganic col lagen matrix, but deficient minera l i zation. This i s di fferent from osteoporos is (see below) in which the bone matrix i s normal ly

minera l i zed but decreased. As a resul t, patients suffering from osteomalacia have weak and eas i ly fractured bones . Most often,osteomalacia/rickets i s caused by deficiency of vi tamin D ei ther in the diet (i .e., poor intake or poor intestina l absorption) or secondary tolow sun exposure/absorption. Other causes may include kidney or l iver disease (or other disorders that affect vi tamin D metabol i sm and/orabsorption), decreased phosphate levels , cancers , and medication s ide effects (e.g., anticonvulsant medications). Symptoms and s igns ofosteomalacia include bone pa in (often s tarting in the lumbar region of the spine, pelvis , and legs ) and related muscle and nerveweakness/numbness . Laboratory tests show low ca lcium levels in serum and urine (often accompanied by low serum phosphate), highalka l ine phosphatase (see above) and parathyroid hormone (PTH) levels (see below), and radiologica l evidence of pseudofractures and/orbone loss . Treatment involves correction of the problem and replacement of ca lcium and vi tamin D; ful l recovery often takes 6 months orlonger.

Osteocytes , the thi rd and most abundant type of cel l conta ined in the organic bone matrix, are actua l ly a l tered forms of osteoblasts .Osteoblasts change into osteocytes when they become trapped ins ide the growing bone matrix. As part of this change, osteocytes l inktogether via specia l i zed “canalicular” extens ions conta ining gap junctions (Chapter 8), which a l low the interchange of nutrients and wasteproducts . Like osteoblasts , osteocytes are involved in bone formation, a l though their exact role and function(s ) are s ti l l unknown. Emergingresearch indicates a unique abi l i ty of osteocytes to “sense” s tra in and flow of fluid through the canal icul i channels . The mechanica l andfluid-derived s tra in appears to activate osteocytes to regulate bone remodel ing and growth via osteoclast-di rected s ignal ing to and betweenosteoblasts and osteocytes .

Osteoporosis/Mechanisms of Bisphosphonate Action: Osteoporosis i s a bone condition in which the amount of bone minera l i s s igni ficantlylowered, leading to an a l tered and weakened bone matrix and a markedly increased ri sk of fracture. These fractures are often seen invertebrae, leading to the s tooped-over posture of lordosis and hip fractures .

The cause of osteoporos is can be multi factoria l but can be genera l i zed as a condition in which bone resorption outweighs boneformation. Decreased estrogen s timulation resul ting in reduced bone formation as wel l as deficiency of ca lcium and vi tamin D are oftenthe principa l causes , especia l ly in postmenopausa l women of European or As ian descent. Heavy use of a lcohol , smoking, and somemedica l conditions (e.g., endocrine, hypogonadal , or certa in blood disorders ) are a lso known ri sk factors for the development ofosteoporos is . Chronic use of medications including glucocorticoids (e.g., adrenal insufficiency, severe asthmatics , transplant recipients ),levothyroxine, l i thium, certa in barbi turates and anti -seizure drugs , heparin and warfarin, the newer diabetes medication class ofthiazol idinediones , and proton pump inhibi tors can a lso contribute to osteoporos is by decreas ing ca lcium and/or vi tamin D metabol i smand/or osteoblast activi ty.

Al though increased ca lcium and vi tamin D intake a long with exercise and estrogen replacement can help promote bone formation, thedevelopment of bisphosphonates has a l lowed the di rected treatment of this condition (see below). Bisphosphonates are taken up by and ki l los teoclasts by ei ther replacing the terminal phosphate in adenos ine tri -phosphate (ATP), rendering the molecule inoperable(nonnitrogenous bisphosphonates), or inhibi ting 3-hydroxy-3-methylglutaryl -coenzyme A reductase enzyme farnesyl diphosphate synthes is(ni trogenous bisphosphonates) (Chapter 7). Al though required for cholesterol biosynthes is , this enzyme is a lso essentia l for modi ficationand correct trafficking of some membrane proteins and for particular cytoskeleta l functions of osteoclasts that enable them to resorbbones . Al ternative medications include calcitonin (inhibi tion of osteoclasts ), PTH (s timulation of osteoclasts ), and OPG (RANK receptorinhibi tion) analogues . Emerging treatments , including sodium fluoride and strontium, may offer addi tional treatment modal i ties .

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

BONE GROWTH AND REMODELINGThe growth of bone and i ts constant remodel ing in response to normal growth, microfractures (due to normal or abnormal mechanica ls tresses ), and fractures due to trauma is highly regulated and ba lanced between new bone formation via osteoblasts and bone resorption via

osteoclasts . The amount of “normal” remodel ing decreases markedly from complete bone turnover in infancy to only approximately 10% ofbone remodel ing in adults in any given year.

The ba lance of osteoblast and osteoclast activi ty i s dependent on severa l factors . Apart from FGF, PDGF, TGF-β, and BMPs noted above,osteoblast function i s s timulated by the pi tui tary-derived, polypeptide growth hormone, which serves both to augment bone matrix formationand to increase the retention of ca lcium in the body. Androgens and estrogens a lso promote osteoblast activi ty/bone growth. Estrogensfunction via promoting the increased secretion of the cytokine (Chapter 15) glycoprotein OPG, which competi tively binds to the RANK l ig-andTNF fami ly receptor (discussed earl ier). By blocking RANK l igand activi ty, OPG/osteoclastogenes is inhibi tory factor effectively s tops thedevelopment of osteoclasts from their monocyte/ macrophage precursor cel l s . PTH and vi tamin D a lso affect bone growth by increas ingca lcium levels (see below).

Osteoblasts can a lso s timulate the activi ty of osteoclasts . Increased PTH and vi tamin D from low serum ca lcium levels a long with s igna lsfrom osteocytes increase the production of RANK l igand and cytokine interleukin 6 (IL-6). Al though IL-6 i s better known for i ts role in fever andinflammation, when secreted by osteoblasts , i t a l so serves , together with RANK receptor activation, to promote the development and growth ofosteoclasts . IL-6’s receptor s igna ls via the Janus kinase–s ignal transducer and activator of transcription pathway, leading to phosphorylationof s igna l molecules and gene transcription (Chapter 8 and Figure 8-9C). Al though estrogen promotes osteoblast growth, i t can a lso block IL-6activi ty, thereby inhibi ting osteoclast growth. As noted above, M-CSF i s a lso involved in osteoclast activation by promoting the developmentand growth of osteoclasts from the monocytes/ macrophage precursor cel l s . M-CSF a lso decreases the secretion of the inhibi tory hormone,osteoprotegerin (see above).

Paget’s Disease: Paget’s disease of the bone, a lso known as osteitis deformans, i s a condition of excess ive bone turnover (breakdown andreformation), resul ting in bone deformities , pa in, decreased s trength, and arthri ti s and fractures . A genetic l inkage has been suggested asthe poss ible role of paramyxovirus , a l though no convincing evidence has been found. An emerging bel ief suggests involvement of anabnormal response to vitamin D and an increased response of the RANK ligand, poss ibly to the actions of IL-6. The disease s tarts withincreased, loca l i zed resorption by osteoclasts in long bones and the skul l . A resul ting increase of osteoblast activi ty resul ts in a quicklyla id-down and disorganized bone matrix, fibrous connective ti ssue, and new blood vessels rather than the organized and s turdy s tructure ofregular bones . The increased osteoclast and osteoblast activi ty eventual ly diminishes , leaving permanent and detrimenta l changes in thebone. Al though no longer used cl inica l ly, skul l X-rays show these changes as diagnostic “cotton wool spots .” Treatment i s normal ly viaca lcium and vi tamin D supplementation a long with bisphosphonates and, occas ional ly, ca lci tonin. Patients are monitored by fol lowingalka l ine phosphatase, ca lcium, and phosphate levels .

Bone growth in a fetus and a chi ld varies s l ightly from an adult and occurs via two s l ightly di fferent processes . Intramembranous ossificationoccurs in the skul l and requires the development of loca l i zed areas of bone growth, known as oss i fi cation centers . Ini tia l ly, newlydi fferentiated osteoblasts aggregate and secrete bone matrix around themselves to form bone spicules. As more bone matrix i s secreted, thespicules join together to form an open bone network ca l led trabeculae. The trabeculae join together, centers of bone marrow and a periosteumdevelops , and a more compact bone i s la id down. Intramembranous oss i fi cation i s a lso essentia l during the repair of bone fractures (seebelow). The a l ternative method of early bone formation i s endochondral ossification, respons ible for long bone formation, as wel l as some flatand i rregular bones . Endochondra l oss i fi cation i s a lso respons ible for continued growth during chi ldhood and parts of fracture heal ing. Thismode of bone formation s tarts with the formation of a carti lage matrix, fol lowed by the development of oss i fi cation centers where theminera l matrix i s la id down. A primary oss i fi cation center occurs in the center of the newly forming bone to form the diaphysis; a secondaryoss i fi cation center i s es tabl i shed near the ends of the growing bone as part of the epiphyseal plate, which i s completely replaced by inorganicbone matrix near the end of the second decade of l i fe (epiphyseal closure).

Bone Fractures and Healing: A fracture of a bone ini tiates a series of three main phases of heal ing-reactive, reparative, and remodeling, whichare respons ible for both quick s tabi l i zation of the fracture and a s lower restoration of the damaged bone to i ts near-origina l s tate.Immediately fol lowing a fracture, the reactive phase begins , in which blood vessels ini tia l ly constrict and a clot forms, which, a long withfibroblasts , form an ini tia l matrix of fibrous connective ti ssue with a blood vessel supply. A few days after the fracture, the reparative phasebegins , with development of chondroblasts and osteoblasts from fibroblast cel l s within the periosteum. These cel l s produce an ini tia lwoven bone “callus,” composed of quickly and haphazardly organized hya l ine carti lage (type II col lagen and chondroi tin sulphate), whichunites the broken bone fragments and provides some support for the fracture. This hya l ine carti lage matrix i s replaced by a regular andpara l lel array of type I col lagen lamellar bone matrix via endochondra l oss i fi cation. The growth of a more permanent blood vessel supplymodi fies the lamel lar bone to trabecular bone, wi th a lmost a l l of the origina l s trength of the bone restored. The fina l phase, remodeling, cantake from severa l weeks to months to complete, with selected resorption by osteoclasts and redepos i tion by osteoblasts unti l a fina lcompact bone matrix i s produced, closely mimicking the origina l bone.

Adapted with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.

REGULATION OF CALCIUM LEVELSBecause bone represents a large s torage depot of readi ly ava i lable ca lcium, the regulation of ca lcium levels in the body influence the

balance of bone formation and breakdown. Thus , a multiorgan, regulated process involving the intestines , kidneys , and bones works insynchrony to absorb new ca lcium, s tore new and old ca lcium, and, when necessary, free s tored ca lcium for the body’s use. The normal dieta l lows an addition of approximately 5 mmol of ca lcium per day. The kidneys excrete a net of 5 mmol of ca lcium, thereby negating any dietaryga ins . Bone, which conta ins approximately 99% of the body’s tota l ca lcium, therefore, serves as both a s torage depot and a source for ca lcium.Intestina l ca lcium absorption i s s trongly influenced by vi tamin D, which has the important role of increas ing the number of ca lcium-bindingproteins , known as calbindin, on the cel l s of the smal l intestine as wel l as the kidney. Increased amounts of ca lbindin present in the intestinedirectly increase the amount of ca lcium absorbed and may a lso s timulate an ATP-dependent ca lcium pump, which transports ca lcium into theblood s tream. Kidney reabsorption of ca lcium is a lso increased by vi tamin D; the kidney i s a lso respons ible for converting 25(OH) vi tamin D3

into the active form of ca lci triol , 1,25(OH)2 vi tamin D3 (Chapter 3). In addition, PTH and calcitonin play prominent roles in the regulation ofintestina l absorption, kidney excretion/ reabsorption, and bone turnover of ca lcium. PTH is secreted by the parathyroid glands in response tolow serum ca lcium levels (sensed by specia l receptors on the parathyroid cel l s ) and/or increased serum phosphate, which binds to anddecreases the level of free ca lcium ions . Increased ca lcium ions lead to a reduction in PTH secretion. PTH i s respons ible for increas ing Ca 2+

concentration via the action of a Gq protein, which s timulates phosphol ipase C to produce inos i tol 1,4,5-tri sphosphate (IP3) and, therefore,increase ca lcium release (Chapter 8). Additional ly PTH acts via Gs to increase protein kinase A activi ty.

Calcitonin i s a polypeptide hormone produced by the parafol l i cular C-cel l s of the thyroid gland and i s respons ible for decreas ing ca lciumlevels . Ca lci tonin levels are increased when ca lcium levels are high, as wel l as by gastrin secretion (Chapter 11). Al though the exactmechanism of ca lci tonin function i s s ti l l being determined, i t i s known to act not only via a Gq-protein receptor mechanism but a lso via a Gs-protein receptor (adenyl cyclase/cycl ic adenos ine monophosphate) mechanism. Interaction with these G proteins i s known to inhibi tosteoclast resorption by quiescence (Q-effect) and retraction (R-effect) of the osteocyte membrane. Ca lci tonin receptors are mainly found onosteoclasts and a lso on ovaries and testes . The actua l importance of ca lci tonin in the regulation of ca lcium in humans i s s ti l l in question.The effects of PTH and ca lci tonin are i l lus trated in Table 13-2.

TABLE 13-2. Summary of PTH and Ca lci tonin Effects on Target Organs

PTH-Related Peptide and Cancer: A common consequence of some cancers can be increased levels of ca lcium (hyperca lcemia) with secretion ofPTH-related peptide (PTH-rP). PTH-rP has s igni ficant homology with PTH, but only in the amino-terminal 13 amino acids , which make up thereceptor-binding region. Because PTH-rP i s not secreted by the parathyroid glands , hyperca lcemia produces no feedback regulation of i tsproduction. The ri se in ca lcium levels due to PTH-rP i s termed humoral hypercalcemia of malignancy (HHM). Cancers usual ly associated withHHM include breast and lung cancers and multiple myeloma.

A summary of ca lcium regulation in the body i s shown in Figure 13-9.

Figure 13-9. Control of Blood Calcium and Phosphate Levels. Overview of the regulation of ca lcium (Ca 2+) and phosphate (P) levels by the coordinatedactions of active vi tamin D [1,25(OH)2D3], parathyroid hormone (PTH), ca lci tonin, and estradiol (E2). See the text above and Table 13-2 forfurther deta i l s . [Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

MARKERS OF BONE FORMATION AND RESORPTIONOn the bas is of various mechanisms involved in the formation of bone matrix, a number of chemica l markers have been characterized, whichal low cl inicians to measure and monitor bone formation and breakdown. Ca lcium can be measured as ei ther total calcium or ionized calcium,wi th ca lcium bound to serum proteins (e.g., serum a lbumin) affecting the measurement of ca lcium freely ava i lable to the body. Abnormallevels of a lbumin (hypoalbuminemia or hypera lbuminemia) wi l l a l ter the amount of bound ca lcium, and therefore, a lbumin levels are oftentested as wel l , so a corrected calcium level can be ca lculated. Vi tamin D levels , speci fica l ly the active 1,25(OH)2 D3 (ca lci triol ) form, wi l l a l soimpact on ca lcium metabol i sm and may provide ins ight into a number of related disease s tates . Phosphate levels are a lso important inrelation to the binding and excretion of ca lcium and are, thus , often measured. The important role of PTH in ca lcium, phosphate, and vi taminD regulation and particular diseases may a lso prompt a cl inician to order measurement of i ts level . Ca lci tonin’s importance in ca lciumregulation usual ly precludes i ts measurement, except as a tumor marker for medul lary thyroid adenocarcinoma. Fina l ly, a lka l inephosphatase, the enzyme that promotes minera l i zation of newly forming bone, i s sometimes measured in patients suffering from bonedisorders . As a lka l ine phosphatase has three di fferent i soforms [intestina l , placenta l , and nonspeci fic (e.g., l i ver/bone/kidney)] and i s foundin a l l ti s sues in the human body, particular i soenzymes need to be i solated and their separated level (s ) careful ly cons idered. Alka l inephosphatase i s often measured in conditions such as Paget’s disease of bone, osteosarcoma, cancers that have metastas ized to bone, bonefractures , osteomalacia (rickets ), achondroplas ia , congenita l hypothyroidism (previous ly known as cretinism), renal osteodystrophy,hypophosphatas ia , osteoporos is/estrogen use, and/or vi tamin D deficiency. However, a lka l ine phosphatase levels are more often used tomeasure blockage of l iver bi le ducts than for genera l bone and ca lcium studies .

REVIEW QUESTIONS1. How would you describe the three main types of connective ti ssue and how do they di ffer?2. What are the functions of chondrocytes?3. What i s the bas ic pathway for the formation of col lagen?4. What are the major components of bone (inorganic and organic)?5. How would you describe the three types of cel l s in the organic matrix, including their function(s ) and regulation?6. How would you describe the processes of bone formation and resorption, including their regulation?7. How would you describe the mechanisms regulating ca lcium levels and bone formation, including the roles of ca lcium, vi tamin D,

parathyroid hormone, and ca lci tonin?8. What are the major markers of bone formation/resorption?

CHAPTER 14BLOOD

Co-Authors/Editors: Matthew Porteus, MD, PhDDivis ions of Cancer Biology, Hematology/Oncology, and Human Gene Therapy, Stanford Univers i ty, Stanford CA

Tina MantanonaUnivers i ty of Texas Southwestern Medica l Center, Da l las , TX

Bas ic Components of BloodRed Blood Cel l (RBC) FunctionsDiseases Associated with Inadequate Synthes is of Hemoglobin ComponentsOxygen BindingPhys iologic Response to Inadequate O2 Del ivery

Sickle Cel l Disease (SCD)IronClottingReview Questions

OVERVIEWBlood, including red cel l s , white cel l s , platelets , and a col lection of specia l i zed proteins , serves an essentia l phys iologic function as i t carriesmolecules from one part of the body to another. This chapter discusses the biochemica l underpinnings of how blood transports oxygen fromthe lungs and i ron to the ti ssues that need them without caus ing damage to the ti ssues that do not. This transport system a lso insuresadequate waste removal from the human body. In addition, the biochemica l properties of clotting, the system that mainta ins the integri ty ofthe blood vasculature, are discussed. In describing the normal way in which blood carries out these functions , the discuss ion wi l l i l lus trate,from a biochemica l perspective, the ways diseases may resul t when these processes go awry.

These three important roles , however, are just a smal l fraction of the cri tica l functions that blood carries out to mainta in the properperformance of the human body. The core biochemica l principles of these three systems—the role of a l lostery in regulating l igand binding inthe oxygen–hemoglobin system, the specia l i zed functions of proteins for i ron transport and s torage, and the importance of biochemica lcascades as i l lus trated by the clotting system—apply broadly to other functions of the blood and other organ systems in genera l . Thus , byunderstanding these fundamenta l biochemica l mechanisms, a genera l foundation for understanding the normal phys iology andpathophys iology of other systems can be establ i shed.

BASIC COMPONENTS OF BLOODHematology, the s tudy of blood, has been of centra l importance in medicine throughout his tory. For much of his tory, hea l th and i l lness werecons idered a reflection of di fferent “humors” in the body: black bi le, yel low bi le, phlegm, and blood. Good heal th was atta ined when a l l thehumors were in harmonic ba lance and i l lness was cons idered as the resul t of an imbalance in the humors . Thus , the goal of hea lers was torestore the ba lance of the humors in those who were i l l . To manipulate blood, healers employed methods such as applying leeches , cupping,and bloodletting in hope of achieving heal th through creating proper ba lance of humors .

A human adult conta ins approximately 5 l i ters (l ) of blood, with the enti re volume ci rculating through the body every 1–2 min. Centri fugationseparates blood into a cel lular layer, which col lects at the bottom end of the col lection tube, and a noncel lular layer, located at the top of thetube (Figure 14-1). If whole blood clots before centri fugation, the remaining noncel lular component i s ca l led serum. On the other hand, i fanticlotting additives are added to the test tube, the noncel lular layer i s ca l led plasma. The cel lular and noncel lular layer each account forapproximately 50% of the tota l blood volume.

The cel lular compartment cons is ts of red blood cel l s (RBCs), the most abundant cel l in the body, white blood cel l s , and platelets . Thegeneration of new RBCs (erythrocytes) from bone marrow is termed erythropoiesis, and i t rel ies on the kidney-secreted hormone erythropoietin(Epo) (see below). White blood cel l s , which primari ly serve to fight infections , can be further divided into speci fic subtypes and are discussedin Chapter 15. Platelets are smal l cel l s that primari ly participate in clotting. The clotting system cons is ts of both cel lular and noncel lularcomponents and wi l l be discussed below. Simi lar to the cel lular layer, the noncel lular layer can be divided into various components , each ofwhich carries out essentia l functions for mainta ining heal th. For instance, within the noncel lular component are carrier proteins , whichtransport other proteins or smal l molecules . Important examples include hemoglobin (Hgb), which carries oxygen (O2) and carbon dioxide (CO2),and transferrin, which transports i ron and regulates i ron metabol i sm. Other carrier proteins transport smal l peptides , l ipophi l i c hormones(Chapter 3), and even drugs .

Figure 14-1. Separation of Blood into its Basic Components. Upon centri fugation, blood separates into a noncel lular “plasma” layer at the top of thetube; a middle “buffy coat,” composed of platelets and white blood cel l s ; and a bottom layer of mostly red blood cel l s . Because this sampleconta ins anticlotting additives , the noncel lular layer i s ca l led plasma. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas icHis tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

The most abundant noncel lular protein in the blood i s albumin (3.5–5 g/dl ). Albumin i s secreted by l iver cel l s (hepatocytes , discussed inChapter 11), serves as a carrier protein, and, most importantly, serves to mainta in oncotic pressure. Oncotic pressure i s the term used todescribe the force that keeps fluid from leaking out of a conta iner through a di ffus ible barrier. When a lbumin levels become low(hypoalbuminemia), such as with l iver fa i lure (inadequate a lbumin i s made) or in protein-los ing diseases (where too much a lbumin i s los tthrough ei ther urine or s tool ), the oncotic pressure of the blood fa l l s . This change in pressure causes noncel lular fluid to leak from the bloodinto the ti ssues , resul ting in edema (swel l ing). Edema can be l i fe-threatening, particularly pulmonary edema, where lungs become fluid fi l ledand O2 exchange i s impaired. Pulmonary edema from pure hypoalbuminemia i s extremely rare, and the most common causes of pulmonaryedema are heart fa i lure and inflammation. Immunoglobulins (antibodies ) form the second most common type of protein in the blood (2.3–3.5g/dl ). Al though immunoglobul ins play a minor role in mainta ining blood oncotic pressure, the most serious compl ication from lowimmunoglobul in levels i s an increased ri sk of infection (Chapter 15). The blood a lso transports hormones, smal l bioactive molecules thataffect the function of one or multiple target organs and ti ssues (Chapters 1 and 3). In addition to protein components , serum conta ins anincredible variety of nonprotein components , including minera ls , electrolytes , l ipoprotein particles , and essentia l nutrients (such as glucoseand glutamine).

RED BLOOD CELL (RBC) FUNCTIONSRBCs are smal l cel l s (6–8 μm in diameter) whose primary function i s to transport O2 from the lungs to the periphera l ti s sues . A secondaryfunction of RBCs i s to carry CO2, generated by the periphera l ti s sues , back to the lungs for el imination by exhalation (Chapter 17) through themouth and nose. Hgb i s the O2-carrying molecule in RBCs . Hgb cons is ts of four heme molecules with four globin cha ins , giving the molecule i tsname. Adult Hgb i s a tetramer of four globin cha ins : two α-globin subunits and two β-globin subunits (Figure 14-2A–C and 14-3A–B).

Figure 14-2. A–C. Basic Components of a Red Blood Cell. (A) Each erythrocyte (red blood cel l ) conta ins many molecules of (B) hemoglobin (Hgb), afour-subunit molecule. Each subunit of Hgb conta ins (C) one heme molecule. [Adapted with permiss ion from Murray RA, et a l .: Harper’sI l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Each heme molecule cons is ts of two parts : a protoporphyrin molecule and iron. The loca l envi ronment created by each globin subunitmainta ins i ron in the ferrous (Fe2+) s tate. In this form, i ron has s ix “coordination s i tes” that interact with the protoporphyrin ring and othermolecules . The fi rs t four of the coordination s i tes bind to the protoporphyrin ring a long i ts plane, suspending the i ron in the middle of thering (Figure 14-4A). The fi fth s i te forms a s trong, cova lent l inkage to Hgb through his tidine (ca l led the “proximal his tidine”) of the globin cha in(Figure 14-4A). Si tes 1 through 5 are fixed. The s ixth s i te, however, i s not fixed and accounts for the versati l i ty of Hgb binding. It i s the s ixth s i tethat binds and releases O2 as wel l as other gases . The deoxygenated protoporphyrin ring has a dome-l ike s tructure because of s terichindrances and other forces from the surrounding amino acids (Figure 14-4B).

Anemia: Anemia occurs when the quanti ty of RBCs i s lower than normal . In severe forms of anemia, the capaci ty of the blood to carryadequate O2 to the periphera l ti s sues i s compromised. Anemia can be caused by ei ther the fa i lure to produce enough RBCs (e.g., iron

deficiency or aplastic anemia), a loss of RBCs (bleeding), an increased destruction of RBCs that cannot be compensated for by the bone marrow(e.g., sickle cell anemia or autoimmune hemolytic anemia), or by sequestration, in which RBCs are hidden away (usual ly in the spleen). The mostcommon cause of anemia, and the most common nutri tional deficiency in the world, i s i ron deficiency anemia. In this condition, there i sinsufficient dietary i ron for erythropoies is . The fa i lure to meet ongoing, pers is tent demands of the bone marrow to create new RBCs resul tsin vague symptoms of weakness and fatigue. Serious cases can mani fest as shortness of breath and even death.

Figure 14-3. A–B. Synthesis and Structure of Hemoglobin (Hgb). (A) Heme is synthes ized via multiple enzymatic s teps (indicated in blue), whichoccur in both the mitochondria and cytosol . Increased levels of heme negatively regulate ALA synthase, the fi rs t enzyme in the syntheticpathway. The fina l s tep, cata lyzed by ferrochelatase, i s respons ible for the addition of Fe2+ to the finished heme molecule. Defective enzymesin a l l of the remaining synthetic s teps lead to a series of diseases noted in red. (B) A heme molecule cons is ts of a protoporphyrin ring with acentra l ly suspended i ron molecule (see the text below). This i ron atom binds oxygen (O2); therefore, one Hgb molecule can bind a maximum offour O2 molecules . CoA, coenzyme A; PLP, pyridoxa l phosphate. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, JaypeeBrothers Medica l Publ i shers (P) Ltd., 2009.]

Carbon Monoxide (CO) Poisoning: CO i s a dangerous gas not only because i t i s undetected by our sense of smel l but a lso because i t has anaffini ty 200 times greater than that of O2 for Hgb’s s ixth coordination pos i tion. As a resul t, CO wi l l displace O2 for binding to Hgb. Thedisplacement of O2 by CO severely decreases O2 del ivery to the periphera l ti s sues and decreases the cel l ’s abi l i ty to carry out oxidativephosphorylation. The detection of CO poisoning can be di ffi cul t. Notably, a patient does not have to appear blue to be in di re need of O2;patients are just as pink when Hgb i s saturated with ei ther O2 or CO. The treatment for CO poisoning i s to fi rs t remove the patient from thesource of CO exposure and, in extreme cases , to put the patient in a hyperbaric O2 chamber. In a hyperbaric O2 chamber, the equi l ibriumbetween Hgb binding O2 and CO is changed and the high-pressure O2 di splaces the CO molecule from the s ixth coordination pos i tion of theHgb molecule.

Figure 14-4. A–B. Binding of Oxygen (O2) to Hemoglobin (Hgb) Molecule. (A) Binding of O2 by the Fe2+ a toms’ fi fth (5) coordination s i te his tidine withadditional regulation by the s ixth coordinate s i te (6) his tidine. Coordination s i tes 1–4 are symbol ica l ly indicated in the plane of the porphyrinring. (B) When the heme molecule’s s ixth coordination s i te i s empty, the i ron molecule projects outward from the center of the protoporphyrinring, caus ing the molecule to assume a domed formation. Upon O2 binding, the heme molecule assumes a planar conformation (right).[Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

DISEASES ASSOCIATED WITH INADEQUATE SYNTHESIS OF HEMOGLOBIN COMPONENTSSevera l disease s tates are associated with the inadequate synthes is of any of the fina l Hgb components . Of the four components of Hgb (theprotoporphyrin ring, α-globin, β-globin, and i ron), cel l s must synthes ize a l l but i ron, which i t absorbs from the intestina l tract. These diseasesare summarized in Table 14-1.

TABLE 14-1. Summary of Hemoglobin Diseases

OXYGEN BINDING

TENSE AND RELAXED HGB

Each Hgb uni t i s in equi l ibrium between two di fferent s tructura l s tates : tense (T) or relaxed (R) (Figure 14-5). Hgb in the T s tate has a low affini tyfor O2 and i s more l ikely to surrender O2 molecules i t i s holding. Hgb in the “R” s tate has an approximately 100-fold increase in affini ty for O2

as compared with the T s tate; R s tate Hgb i s less l ikely to surrender O2 to ti s sues . A mutation in a globin gene or a smal l molecule thatstabi l i zes the T s tate (or destabi l i zes the R s tate) wi l l resul t in a higher proportion of O2-binding uni ts being in the T s tate. An overa l l shi ft tothe right in the O2 di ssociation curve wi l l occur (lower affini ty for O2) (Figure 14-6). Conversely, mutations or smal l molecules that destabi l i zethe T s tate (or s tabi l i ze the R s tate) wi l l resul t in a higher proportion of O2-binding uni ts being in the R s tate. An overa l l shi ft in the O2

dissociation curve to the left (higher affini ty) wi l l occur.

Figure 14-5. Hemoglobin Binding of Oxygen (O2) Causes a Change in the Shape of the Hemoglobin (Hgb) Molecule. Hgb molecules exis t in an equi l ibriumof deoxy/tense and oxy/relaxed s tates . Deoxy Hgb (left) has a low affini ty for O2 and i s associated with the tense (T) s tate and the release ofO2 to the ti ssues . Binding of O2 causes an approximately 15° rotational change in the quaternary s tructure to form oxy Hgb (right). Oxy Hgb has ahigh affini ty for O2 and i s associated with the R s tate. Oxy Hgb wi l l bind O2 more than release O2. [Reproduced with permiss ion from MurrayRA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Figure 14-6. The Oxygen (O2) –Hemoglobin (Hgb) Dissociation Curve. The O2–Hgb dissociation curve depicts the saturation of Hgb with O2 as afunction of the partia l pressure of O2 (p O2). p O2 i s a measure of the concentration of O2 in the ti ssues . The pO2 of metabol ica l ly active ti ssues

is approximately 40 mmHg. At mmHg, the affini ty for O2 s teeply decl ines and Hgb surrenders O2 to the ti ssues . [Adapted withpermiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

ALLOSTERIC BINDING OF O2 BY Hgb

Each of the four subunits of Hgb conta ins one protoporphyrin ring, thereby a l lowing a tota l of four O2 molecules to bind per Hgb molecule.However, O2 binding i s not a s imple, l inear process . The R and T s tates exis t in equi l ibrium with each other and this equi l ibrium is whatdetermines the overa l l sigmoid or “S” shape of the O2 di ssociation curve (Figure 14-6). The binding of the fi rs t O2 to a s ingle Hgb s i te sets inmotion a series of conformational changes of the globin, polypeptide cha ins that increase the probabi l i ty that the other three s i tes wi l l adopt

the “R” s tate (the high affini ty s tate). Binding of subsequent O2 molecules a lso increases the binding of the other s i tes (a l though much lessthan the binding of the fi rs t O2). In addition, more relative R s tate than T s tate shi fts the curve to the left. Conversely, more T than R wi l l shi ftthe curve to the right.

The role of Hgb i s to transport O2 from higher partia l pressure of O2 (pO2) envi ronments (such as lungs) to lower pO2 envi ronments (such asti ssues). Because the l igand affini ty of Hgb i s increased by success ive binding, O2 i s a positive allosteric effector (Chapter 5). The S shape of theO2–Hgb dissociation curve depicts a positive cooperativity relationship between the percent saturation of the Hgb molecule and pO2 (Figure 14-6). The pos i tive cooperativi ty of O2 binding i s an essentia l characteris tic of Hgb that makes i t both an efficient O2 carrying and del iverymolecule. This characteris tic a lso a l lows more O2 del ivery to ti s sues with high metabol ic demands (low pO2). Correspondingly, ti s sues thatneed less O2 because of fewer metabol ic demands have higher pO2 and extract, appropriately, less O2.

REGULATION OF O2 BINDING

An important concept in understanding Hgb function i s that Hgb must be able to carry out two seemingly oppos ing functions : (1) to becomeful ly saturated with O2 in the lungs and (2) to be able to efficiently unload O2 in the ti ssues . That is, O2 binding does not guarantee O2 delivery. Infact, i f Hgb binds O2 too tightly, ti s sues would not be able to extract O2 from the Hgb molecule, and functional ly i t would be equiva lent tothere being no O2 a t a l l . It i s the release of O2 from Hgb that drives ti s sue oxygenation. Negative allosteric regulators such as increased

temperature, increased CO2, and decreased pH (increased H+ concentration) wi l l cause the curve to shi ft to the right and help Hgb unload O2 inthe ti ssue (Figure 14-7).

Figure 14-7. Effects of Temperature and pH on Hemoglobin (Hgb) Binding of Oxygen (O2). (A) Increas ing temperature (e.g., 10, 20, 38, and 43°C) causesreduced binding of O2 by Hgb, reflecting reduced affini ty for O2 (T s tate > R s tate). In this higher temperature envi ronment, Hgb wi l l morereadi ly release O2 as noted by the right shi ft of the curves . (B) Increas ing pH (e.g., 7.2, 7.4, and 7.6) causes increased binding of O2 by Hgb. Asthe curve shi fts left, Hgb has an increased affini ty for O2 (R s tate > T s tate). [Adapted with permiss ion from Kibble JD and Halsey CR: The BigPicture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

2,3-bisphosphoglycerate (2,3-BPG) (a l so known as 2,3-diphosphoglycerate) i s a smal l molecule created as a s ide product of glycolys is from 1,2-BPG (Chapter 6). It i s present in the same concentration as Hgb in the RBC (~2 mM) and i s the key molecule in assuring that Hgb does not bindO2 too tightly (insuring del ivery of O2 to the ti ssues). The biochemica l mechanism of 2,3-BPG action i s that i t speci fica l ly fi ts into a cleftpresent in the T s tate, which i s not present in the R s tate (Figure 14-8A). This s tabi l i zes the T s tate through a series of noncovalentinteractions in the globin cha in, thus shi fting the O2 di ssociation curve to the right (Figure 14-8B). Because 2,3-BPG binds at a s i te separatefrom the l igand-binding s i te (in this case, the l igand i s O2) and through that binding affects the affini ty of l igand (O2) binding, 2,3-BPG is anallosteric effector (Chapter 5) of Hgb function.

Acid–base ba lance in the body i s regulated by an integrated combination of mechanisms found in the lungs (Chapter 17), kidney (Chapter18), and blood (Figure 14-9). The regulation of Hgb binding of O2 by acid–base s tatus , in particular H+ and CO2 (both regarded as acids ), i s

ca l led the Bohr effect. H+ di rectly faci l i tates the formation of sa l t bridges that s tabi l i ze the T s tate (the low-affini ty s tate). CO2 di rectlys tabi l i zes the T s tate by binding to the amino terminal ends of the globin cha ins to generate a T s tate s tabi l i zing carbamate moiety. Inaddition, CO2 indi rectly s tabi l i zes the T s tate when i t i s converted by carbonic anhydrase into carbonic acid. Carbonic acid generates extra H+,

which then act as described above. Metabol i sm creates H+ and CO2, and the more demanding the ti ssue, the more H+ and CO2 are created.When more acid i s created (a decrease in pH), this causes a right-shi ft in the O2–Hgb dissociation curve. A right-shi fted O2 binding curvemeans that, for a given pO2, the affini ty of Hgb for O2 wi l l be decreased and the probabi l i ty that O2 wi l l be del ivered to the ti ssue wi l l beincreased.

Figure 14-8. A–B. Binding of 2,3-BPG to Deoxyhemoglobin. (A) 2,3-BPG (in red) fi ts into a cleft and interacts with the amino terminal va l ine (Va l )’sNH3

+ group plus the pos i tively charged R-group s idechains of lys ine (Lys ) and his tidine (His ) on both β-globin cha ins . These same interactionsdo not occur in the relaxed (R) s tate of oxy Hgb. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.] (B) These noncovalent bonds s tabi l i ze the tense (T) [low oxygen (O2) a ffini ty] s tate and change the normal binding(blue, sol id l ine), promoting the release of any bound O2 molecules to ti s sues . Thus , increas ing BPG (red, dotted l ine) resul ts in a right shi ft(lower binding of O2) and decreas ing BPG (green l ine) resul ts in a left shi ft (higher binding of O2) of the binding curve. 2,3-BPG, 2,3-bisphosphoglycerate. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l ,2009.]

Hypoxia, Altitude Sickness, and Acetazolamide: Altitude sickness can occur when people move from low a l ti tudes to high a l ti tudes , resul ting ininadequate O2 to the periphera l ti s sues , especia l ly the bra in. The main symptom of a l ti tude s ickness i s headache and nausea that usual lyresolve without any intervention. In certa in ci rcumstances , a l ti tude s ickness can cause l i fe-threatening pulmonary edema (fluid fi l l ing thelungs , caus ing severe shortness of breath and di ffi cul ty in breathing) and cerebra l edema (swel l ing of the bra in, caus ing severe headaches ,disorientation, confus ion, coma, and poss ibly death). The only treatment i s to move the patient to lower elevations as rapidly as poss ible.To prevent the onset of symptoms, acetazolamide, a carbonic anhydrase inhibitor, i s prescribed prophyl lactica l ly to those prone to a l ti tudes ickness . Inhibi ting carbonic anhydrase before moving to higher a l ti tude causes an a lka l inization of the urine and consequent acidi fi cationof the blood. The increased H+ in the blood shi fts the O2 di ssociation curve to the right through the Bohr effect and increases O2 unloadingin the periphera l ti s sues , including the bra in.

Figure 14-9. Fate of Carbon Dioxide (CO2) in the Red Blood Cell (RBC). Upon entering the RBC, CO2 i s rapidly hydrated to H2CO3 by carbonic anhydrase.

H2CO3 i s in equi l ibrium with H+ and i ts conjugate base HCO3–. H+ can interact with deoxy hemoglobin, whereas HCO3

– can be transported

outs ide of the cel l via band 3, an HCO3–/ Cl – anion exchanger in the RBC membrane (green ci rcle). In effect, for each CO2 molecule that enters

the RBC, there i s an additional HCO3– or Cl – in the cel l . [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l

Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

In summary, the relative s tabi l i zation of the T s tate by three factors (H+, CO2, and carbonic acid) in the ti ssue immediately shi fts the O2

dissociation curve to the right loca l ly, thus increas ing the amount of O2 del ivered. A lower affini ty means that Hgb wi l l more readi ly release O2

to ti s sues with high H+ (low pH) and/or high CO2 levels . Metabol ica l ly active ti ssues (e.g., muscle cel l s ) produce high amounts of thesemolecules . By decreas ing the affini ty of Hgb for O2, the Bohr effect insures the abi l i ty of Hgb to surrender O2 to the ti ssues where i t i s neededmost. In addition, metabol ica l ly active ti ssues generate heat, ra is ing the temperature of the ti ssue loca l ly. This increase in temperature a lsodecreases the affini ty of Hgb for O2 and faci l i tates the unloading of O2 in ti s sues that are generating heat through their metabol ic activi ties .When the RBCs return to the normal pH and temperature of the lung capi l lary bed, the excess CO2 i s exhaled (Chapter 17) and the normalequi l ibrium between the R and T s tates i s restored.

How Does the Fetus Get Enough O2? The s igmoid shape of the O2 di ssociation curve expla ins how Hgb can bind O2 in the lungs and del iver O2

to the ti ssues . But how does the developing fetus , a period of high macromolecular synthes is , cel l growth, and consequent O2 requirement,get enough O2 to grow? The fetus does not breathe a i r. Ins tead, i t must extract O2 from the mother’s RBCs across the placental vasculature andthen del iver sufficient O2 to i ts own developing ti ssues . The fetus solves this problem by us ing a di fferent set of globin genes as part of i tsHgb molecule. In contrast to the mother, whose Hgb molecule cons is ts of two α- and two β-globin cha ins creating HgbA, the fetus uses twoα- and two β-globin cha ins creating fetal Hgb (HgbF). HgbF has a lower affini ty for 2,3-BPG than HgbA because of a serine at pos i tion 143 inthe γ-globin gene instead of his tidine (see Figure 14-8A for his tidine pos i tion). This decreased affini ty for 2,3-BPG resul ts in HgbF having anincreased affini ty for O2 and a shi ft in the O2 di ssociation curve to the left. Thus , HgbF i s able to extract O2 from HgbA in the placenta andthen del iver the O2 to the feta l ti s sues . The shi ft to the left i s precise, however, because a l though i t a l lows the feta l Hgb to “s tea l” O2 fromthe maternal Hgb, i t i s not so s igni ficant that the feta l ti s sues cannot remove the O2 for thei r own use.

PHYSIOLOGIC RESPONSE TO INADEQUATE O2 DELIVERY

The inadequate del ivery of O2 to ti s sues (hypoxia) induces a complex variety of normal and pathophys iologic compensatory mechanisms. Cel l smust turn to anaerobic respi ration (Chapter 6), which produces lactic acid and lowers the pH. As described above, a low pH has an immediateeffect on O2 del ivery and extraction through the Bohr effect. The next level of compensation to decreased O2 del ivery i s to increase theconcentration of the a l losteric molecule, 2,3-BPG. The presence of 2,3-BPG lowers the O2 a ffini ty of Hgb, resul ting in a rightward shi ft of the O2

binding curve and increased O2 del ivery to ti s sues in low O2 conditions . The compensatory increase in 2,3-BPG concentration takes about 24hrs to occur.

A s low compensatory mechanism of inadequate O2 del ivery i s via the production and secretion of erythropoietin (Epo) to create more RBCs .Kidney cel l s have high O2 demands because they need abundant adenos ine triphosphate to power multiple channels and pumps thatregulate electrolyte ba lance. Thus , these cel l s are idea l ly sui ted to be sensors of O2 del ivery. When there i s inadequate del ivery of O2 tokidney cel l s , a transcription factor ca l led hypoxia-inducible factor i s not degraded and i s able to activate the transcription of multiple genes ,including the blood hormone Epo, leading to an increased level of Epo in the blood s tream. Epo migrates through the blood to the bonemarrow (where RBCs are made), binds to the Epo receptor, and activates a Janus kinase (JAK) and Signal Transducer and Activator of Transcription(STAT) intracel lular s igna l ing cascade (Chapter 8). Activation of this cascade does not s timulate RBC precursor prol i feration; rather, theJAK/STAT pathway protects RBC precursors from their normal ly programmed cel l death (apoptos is ). Increased RBC surviva l accounts for anincrease in hematocri t (the amount of RBCs in the blood, see Appendix I I). Ul timately, the increased numbers of mature RBCs augment the O2-carrying capaci ty of the blood. It takes approximately 1 week for an RBC to be made and, thus , the ful l effect of this s low response requiresabout a month to mani fest i tsel f; however, beneficia l effects can be found in just a few days . Patients with kidney disease do not secreteenough Epo to mainta in an adequate quanti ty of RBCs and, thus , are given supplementa l Epo as treatment to prevent severe anemia.

SICKLE CELL DISEASE (SCD)Normal RBCs have a biconcave shape that maximizes thei r abi l i ty to pack Hgb and del iver O2. RBCs are a lso flexible and deformable so thatthey can squeeze through the capi l laries . SCD i s caused by a recess ive, s ingle-nucleotide mutation in the β-globin gene of Hgb, inheri ted fromboth parents (i .e., homozygous). This mutation resul ts in the substi tution of the normal glutamic acid (negative charge) with a va l ine (hydro-phobic) at amino acid in pos i tion 6. SCD i s associated with early morta l i ty (the average l i fespan i s 40–45 years in the United States and muchshorter in developing nations) and l i felong medica l problems.

Sickle RBCs bind O2 normal ly (Figure 14-10A, left panel ), and the oxygenated s ickle RBCs have a normal biconcave shape. However, when thes ickle Hgb unloads i ts O2, the normal conformational change exposes the va l ine at pos i tion 6 to the surface creating a “hydrophobic (s ticky)patch” (Figure 14-10A, middle panel ). The hydrophobic patch on one deoxygenated Hgb molecule can interact with the hydrophobic patch on asecond Hgb molecule (Figure 14-10A, right panel ), creating s ti ff Hgb polymers (Figure 14-10B). These internal polymers cause the RBC to s ti ffenand adopt abnormal shapes , including a crescent or “s ickle” shape that gives the disease i ts name (Figure 14-10C). These s ti ffened RBCs losetheir pl iabi l i ty and cannot eas i ly squeeze through normal capi l laries , thereby forming blockages . Dehydration can worsen this effect becausesmal l changes in the hydration s tatus of the RBC can have dramatic effects on s ickle cel l Hgb polymerization. When Hgb rebinds O2, theresul ting conformational change hides the hydrophobic patch, a l lowing separation into monomers and resumption of a normal , biconcaveform. After multiple rounds of excess ive Hgb polymerization (s ickl ing) and recovery, erythrocyte membranes lose their elastici ty, adopt apermanently s ti ffened form, and are ul timately destroyed and broken down (hemolys is ).

Figure 14-10. Sickle Cell Disease. (A) A s ingle nucleic acid change in the β-globin subunit gene (oxy HbA, left panel ) causes a substi tution of theamino acid va l ine for the normal glutamate, creating a hydrophobic “s ticky patch” on the exterior (Oxy HbS, middle panel ). Release of O2 fromthe s ickle cel l hemoglobin (Hgb) (deoxy HbS, right panel ) creates a compl imentary conformational change on the α-subunit that can bind tothe s ticky patch and lead to (B) polymerization. [Adapted with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.] (C) Polymers of s ickle Hgb deform the natura l biconcave shape of the red blood cel l into a s ickle shape. [Reproducedwith permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Inheri tance of the mutation from only one parent (heterozygous) i s termed sickle cell trait. Patients with s ickle cel l tra i t are typica l lyasymptomatic, unless placed under extreme conditions (low O2 or severe dehydration). Sickle cel l tra i t a l so provides relative res is tance todiseases such as malaria and i s bel ieved to have been a pos i tive evolutionary tra i t in areas of the world where malaria i s endemic. Forexample, in Sub-Saharan Africa , there i s such severe selective pressure that nucleotide change has been selected for multiple times . Otherless common variants of SCD a l l have at least one mutated copy of the β-globin gene.

SCD and Hydroxyurea: SCD leads to a number of l i felong, cl inica l consequences due to a l terations in the biophys ica l properties of the RBC.Veno-occlus ive cri ses (a lso known as “pa inful cri ses”) resul t from obstructions of blood vessels due to the inabi l i ty of tough, inflexibleRBCs to squeeze through capi l laries . This obstruction leads to organ i schemia (diminished blood flow) and may resul t in i rrevers ibledamage and necros is of vi ta l bra in regions (s troke) and chronic injury to organs such as the spleen and kidney. The l i fespan of a s ickle RBCis only about 10 days , s igni ficantly shorter than the approximately 120-day l i fespan of a normal RBC, because they are destroyed by thespleen and l iver because of thei r abnormal shapes (hemolys is ). This markedly shortened l i fespan leads to s igni ficant anemia (the Hgbconcentration in the blood i s ~50% of normal ). The bone marrow is unable to compensate for the increased RBC destruction (hemolys is ).The pers is tent severe anemia and the pers is tent microvasculature blockages can ul timately lead to chronic endorgan damage inessentia l ly every organ of the body over time. Increased ri sk of infections by encapsulated organisms such as Streptococcus (caus ing seps is )or Salmonella (caus ing osteomyel i ti s ) are a lso a common problem for SCD patients .

One form of treatment i s the use of hydroxyurea, which causes an increase in the express ion of γ-globin, a variant globin gene that canreplace β-globin in the Hgb molecule. Hgb molecules that conta in the γ-globin gene do not have a va l ine at pos i tion 6 and they block theformation of Hgb polymers , reducing the RBC deformity problems.

IRONBlood uses carrier proteins to transfer essentia l nutrients as part of each person’s metabol i sm. One important example i s i ron that i sinvolved in a vast array of important biologic reactions :

1. Binds O2 as part of Hgb molecule. An adult human has approximately 4 g of i ron, of which about two-thi rds i s employed in the O2-carryingrole of Hgb.

2. Mediates a wide variety of oxidative–reductive reactions by serving as an essentia l cofactor for many proteins via oxidation between ferric(Fe3+) and reduced Fe2+ s tates .

3. Iron i s important for many microorganisms. Keeping i ron sequestered from these invaders i s an important part of the immune system.

IRON METABOLISM

In most wel l -rounded Western diets , meats and green, leafy vegetables provide adequate i ron to prevent i ron deficiency. Certa in i ron-poordiets , such as vegan diets , however, can resul t in i ron deficiency i f not supplemented. Once i ron i s ingested, i t i s converted from the Fe3+ s tateto the Fe2+ s tate by intestina l ferric reductase. Fe2+, not Fe3+, i s transported into the intestina l epi thel ia l cel l through the divalent metaltransporter (DMT1) protein. Fe2+ i s transported out of the intestina l epi thel ia l cel l s into blood through a second transporter, ferroportin (Figure14-11).

Figure 14-11. Overview of Iron Transport. Intestina l lumen ferric (Fe3+) reductase reduces Fe3+ to Fe2+. Fe2+ i s transported from the lumen into theintestina l epi thel ia l cel l through heme transporter (HT), endosomes, and/or diva lent meta l transporter 1 (DMT1). Fe2+ can be converted back toFe3+ and bound to transferrin within the intestina l cel l or can be transported into the blood by ferroportin (FP) and hephaestin (HP). The Fe2+

oxidized to Fe3+, which binds to plasma transferrin, i s carried through the ci rculation to the ti ssues . [Adapted with permiss ion from Kibble JDand Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

TRANSFERRIN

Once in the blood, the Fe2+ i s quickly oxidized back to Fe3+and bound by the i ron carrier protein, transferrin. The affini ty of transferrin for Fe3+ a tpH 7.4 i s 10–23, which means that transferrin wi l l bind Fe3+ even when i ts concentration i s 10–23 (10 yoctomolar or 0.01 zeptomolar). This a ffini tysuggests that in the enti re 5 l blood volume of an adult, there would be only approximately five free molecules of Fe3+ a t a time. Thisexceedingly high affini ty i s the biochemica l mechanism that the human body has adapted to prevent any free i ron from exis ting in the bloodstream (Figure 14-12).

Figure 14-12. Iron Transportation by Transferrin. Transferrin transports i ron in the blood to the bone marrow to make hemoglobin and red bloodcel l s (erythropoies is ). Transferrin a lso carries i ron to the l iver and heart for s torage in ferri tin molecules , as wel l as to other parts of the bodyfor various enzymatic and other functions .

The i ron bound transferrin ci rculates through the blood s tream unti l i t binds to a transferrin receptor on the surface of a cel l . Cel l s thatexpress transferrin receptors have high i ron demands . These include developing RBCs , dividing cel l s , and microorganisms. The transferrin–i ron–transferrin–receptor complex i s endocytosed through the class ic clathrin-coated pi t pathway. Fol lowing endocytos is , pH of the endocyticves icle i s reduced to 5, l iberating i ron from i ts carrier protein and making i ron ava i lable for biologic reactions .

FERRITIN

Although Hgb i s the most abundant protein that uses i ron, ferri tin i s the most important protein for i ron s torage. Ferri tin cons is ts of a 24-uni tmultimer of heavy (H) and light (L) cha ins that create a hol low shel l . Iron i s imported into the shel l as Fe2+ but i s converted to Fe3+ wi thin theferri tin core by the H chain. A ful ly loaded molecule of ferri tin conta ins 4500 atoms of i ron. It i s for this reason that the ferri tin molecule i smetaphorica l ly referred to as a “bag of rust.” A pathologic form of i ron depos i tion i s ca l led hemosiderin. This i s a nonstructured conglomerateof intracel lular i ron and i s often a pathologic consequence of prolonged inflammation. Prolonged depos i tion of hemos iderin can causefibros is (scarring) of ti s sues .

The importance of i ron i s highl ighted by the fact that the human body has no natura l way to excrete i t. Unl ike other ions , such as sodium,potass ium, and ca lcium, kidneys do not excrete excess i ron in the urine. Indeed, the phys iologica l defaul t i s to conserve i ron. Iron deficiencyis much eas ier to treat than i ron overload. Accumulated i ron i s toxic to a variety of ti s sues , especia l ly the heart and l iver. Infus ion of an i ronchelator, such as deferoxamine or deferasirox, provides the only means of reducing some of the i ron accumulation. Chelation works by the drug-binding free i ron in the blood fol lowed by excretion of the i ron–drug complex. The body does lose a smal l amount of i ron each day by thes loughing of skin and epi thel ia l cel l s and in women through their menstrua l flow. However, the same reactivi ty that makes i t a va luablecofactor for proteins a lso means that free i ron can be dangerous because i t can eas i ly generate damaging O2 free radica ls . Thus , the body hasa powerful system to control the metabol i sm of this precious and peri lous meta l .

REGULATION OF IRON AVAILABILITY BY HEPCIDIN

Although the human body has no natura l way of excreting excess i ron, i t i s able to regulate the uptake and ava i labi l i ty of i ron through theprotein hepcidin. Hepcidin i s synthes ized as an 84-amino acid precursor, which i s then processed to the 25-amino acid active form. Hepcidinacts as a negative regulator of ferroportin, blocking the abi l i ty of cel l s to export i ron from the cytoplasm into the blood. In the intestina l cel l ,this inhibi tion resul ts in decreased i ron absorption from the diet. In reticuloendothelial cells, the primary s torage depot for i ron, this resul ts in adecreased abi l i ty of i ron to be mobi l i zed from storage pools to cel l s that need i t.

Hepcidin i s made in the l iver, and the levels of hepcidin in the blood are control led by a variety of di fferent s timul i , including the tota l i ronstores in the body, the erythropoietic demands of making RBCs , hypoxia , and inflammation. When i ron s tores are high, the level of hepcidin i sincreased and the amount of i ron absorbed i s decreased. When the demand for making RBCs i s increased, for example, in response to acuteblood loss , the level of hepcidin decreases and the amount of bioava i lable i ron i s increased. Simi larly, when ti ssues do not receive sufficientO2 (hypoxia), i t s igna ls that more RBCs are needed, and hepcidin production i s decreased. Fina l ly, inflammation, such as s igna led through theinflammatory cytokine interleukin-6 (IL-6), causes an increase in hepcidin production (Figure 14-13). This inflammatory regulation i s thought toreflect a host defense mechanism because i t sequesters i ron away from infectious organisms, l imiting thei r growth. If the inflammatory s tatepers is ts , however, hepcidin sequesters i ron away from both the microorganism and the human cel l s . There i s insufficient i ron to support thedemands for RBC production, and a s tate of anemia of inflammation or anemia of chronic disease develops .

Figure 14-13. Action of Hepcidin in an Inflammatory Response. Response to inflammation (e.g., an infection) leads to activation of monocytes andlymphocytes , often by IL-6. The resul ting immunologica l s igna ls el i ci t responses from severa l organs , including increased hepcidin synthes isin the l iver, inhibi tion of erythropoietin from the kidneys , inhibi tion of red blood cel l (RBC) production in the bone marrow, and increasedmacrophage phagocytos is of RBCs . Increased hepcidin a lso decreases i ron absorption from dietary sources . Al l of these effects decrease theavai labi l i ty of i ron for infective organisms, which a ids in thei r destruction and clearance. IL-6, interleukin-6.

Iron Deficiency Anemia: Iron deficiency anemia i s the most common nutri tional disorder in the world, bel ieved to affect 1 bi l l ion people. Inchi ldren, in the developed world, the most common cause of i ron deficiency anemia i s the excess consumption of cow’s milk. Excess cow’smi lk can cause inflammation damaging the intestina l l ining, resul ting in blood loss as wel l as a diminished capaci ty to absorb i ron.

The fi rs t mani festation of i ron deficiency i s an anemia that s tems from an inadequate i ron supply to susta in erythropoies is . Thesymptoms of i ron deficiency anemia include pa l lor, weakness , and lethargy. Severe and prolonged i ron deficiency can resul t inneuropsychologica l problems. The diagnos is of i ron deficiency i s usual ly made by laboratory s tudies that demonstrate a microcytic anemia(smal ler, pa le RBCs), reflective of poor Hgb production. Blood s tudies a lso show low ferritin level and low transferrin saturation (only 10%conta in i ron as compared with the normal 30–40%). Because ferri tin i s an acute phase reactant that increases in times of s tress and i l lness ,sometimes i t can be paradoxica l ly high even in i ron-deficient s tates .

The treatment for i ron deficiency i s to give i ron. The most bioava i lable dietary i ron i s in red meat, but often the patient i s unable orunwi l l ing to pursue this method. In this s i tuation, ora l elementa l i ron i s given. Because i ron i s transported by DMT1 as Fe2+, not Fe3+, somephys icians wi l l advise thei r patients to s imultaneous ly drink orange juice or take ascorbic acid (vitamin C). Ascorbic acid reduces Fe3+ i ron tothe Fe2+ s tate, faci l i tating the absorption of elementa l i ron. In treating i ron deficiency anemia in chi ldren, caused by excess ive mi lkconsumption, i t i s important to both adminis ter elementa l i ron and dramatica l ly decrease mi lk consumption. In rare cases of adul t and

chi ld anemia, such as poor compl iance or anatomic or genetic defects in i ron absorption, i ron i s adminis tered intravenous ly. Supplementa li ron i s given unti l the microcytic anemia i s resolved and normal levels of ferri tin and transferrin saturation are achieved.

CLOTTINGBlood vessels are subjected to occas ional trauma (e.g., cuts and lacerations) and a lso to routine microtraumas by seemingly benign activi tiessuch as running or knocking on a door. Without clotting, these events would cause hemorrhage (bleeding), which i s the continuous flow of RBCsfrom the intravascular space into the extravascular space and ti ssues . Running would resul t in bleeding into the knee joint (hemarthroses), andknocking on a door would leave ecchymoses (bruises ) over the knuckles . To prevent hemorrhage, i t i s essentia l to form clots at trauma s i tes inthe vessels . The clots must be formed quickly to minimize blood loss . Clotting a lso needs to occur in a control led and loca l i zed fashion,nei ther occluding the vessel nor caus ing clots to form at remote s i tes that have no need for clot formation. The key principles of clotting are asfol lows:

1. Clots cons is t of platelets , the protein fibrin, and RBCs . Fibrin assumes a netl ike matrix that spans the ful l thickness of the clot, ensnaringRBCs throughout the thickness of the mesh-work. Ensnaring the RBCs in the fibrin network creates the clot that s tops hemorrhage.

2. The formation of a clot occurs in two phases :

a. Platelet plug formation—Temporary repair unti l a proper clot can form.b. Clot formation—Longer term and s tronger than a platelet plug. Clot formation s tops hemorrhage long enough for ti s sues to repair.

PLATELET PLUG FORMATION CONSISTS OF ADHESION, AGGREGATION, AND ACTIVATION OF PLATELETS

Damage to the endothel ia l l ining of blood vessels exposes col lagen on the basement membrane (Figure 14-14A). Damage a lso exposes vonWillebrand Factor (vWF), normal ly located between the endothel ium and the basement membrane (Figure 14-14B).

Figure 14-14. A. Formation of the Platelet Plug. Platelet plug formation resul ts from the ini tia l response of platelets to a s i te of blood vessel wal linjury. Control of formation of the ini tia l plug i s via ni trous oxide and prostacycl ins , as noted in the text. NO, ni tric oxide. [Reproduced withpermiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.] B. Mechanism of Clot Formation byPlatelets. Fol lowing the ini tia l formation of a platelet plug, partly a ided by exposed von Wi l lebrand factor (vWF) at the injury s i te (see thefigure), platelets continue to aggregate via fibrin l inking of thei r Gp IIb/II Ia receptors . Adhes ions by these receptors lead to activation of the

platelets and degranulation, which releases adenos ine diphosphate (ADP), thromboxane A2 (TXA2), and other factors , a l l of which furtherincrease the formation of ini tia l platelet plug/clot. As noted in Figure 14-14A, ni tric oxide and antiplatelet prostacycl in (PGI2) l imit plug andclot formation beyond the injury. The clotting cascade noted in the figure i s described further below. EC, endothel ia l cel l . [Reproduced withpermiss ion from Simmons ML and Decker JW: Br Heart J, McGraw-Hi l l , 1995.]

1. Adhesion of platelets to the endothel ium: Col lagen i s highly thrombogenic and platelets wi l l adhere to i t. vWF, conta ined within plateletsand endothel ia l cel l s , enhances platelet adhes ion by increas ing the number of l inks between platelets and col lagen fibri l s .

2. Aggregation of platelets to one another: Platelets s tick to one another by fibrin l inking the glycoprotein IIb/IIIa (Gp IIb/IIIa) of one platelet tothe Gp IIb/II Ia of another.

3. Activation: The adhes ion s tep causes platelets to release their s tored granules (activation) that conta in the fol lowing, which a l l act toenhance plug formation and l imit bleeding:

a. Adenosine diphosphate (ADP)—increases express ion of Gp IIb/II Ia on platelets and causes them to swel l .b. Prostaglandin Thromboxane A2 (TXA2) (Chapter 3) activates a G-coupled protein receptor (Gq). TXA2 increases vasoconstriction (to decrease

blood flow and l imit hemorrhage) and the aggregation of platelets .c. PLA2 increases platelet adhes ion to fibrin via Gp IIb/II Ia .

d. ADP and Ca 2+ lead to additional fibrin depos i tion.

THE CLOTTING CASCADE

The Clotting cascade cons is ts of a series of ordered enzymatic s teps whose end resul t i s the formation of a s turdy clot. There are two l imbs ofthe clotting cascade: the extrinsic pathway and the intrinsic pathway (Figure 14-15). Factors II–XII (named in order of discovery, not in order ofactivation) are inactive serine proteases that are sequentia l ly activated by enzymatic cleavage of the serine protease that immediately precedesi t (the “a” fol lowing a factor number des ignates the active form).

Aspirin (Acetylsalicylic Acid) Prevents Platelet Aggregation: Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inactivating or inhibi tingcyclooxygenase (COX) activi ty (Chapter 3). Aspirin noncompeti tively and i rrevers ibly inhibi ts the COX enzyme via acetylation. Other NSAIDssuch as indomethacin and ibuprofen are revers ible, competi tive inhibi tors of COX. In genera l , NSAIDs inhibi t the synthes is of moleculesrespons ible for various aspects of pa in and inflammation: prostaglandins , prostacycl ins , and thromboxanes . Aspirin i s the only NSAID thatblocks TXA2 production, l imiting platelet plug formation (see the text). Thus , low-dose aspirin i s used to treat potentia l prothromboticdiseases and to reduce the ri sk of heart attack and s troke (which are in part caused by thrombus formation in the wrong vessels ) insusceptible patients . Because aspi rin inhibi ts platelet plug formation, people taking aspi rin are more prone to bruis ing and prolongedbleeding after injury.

The intrins ic pathway i s triggered when Factor XII, an inactive serine protease, encounters exposed collagen and other polyanions from aninjured blood vessel . A protein complex i s formed, which transforms Factor XII into an activated serine protease, XIIa . XIIa cleaves inactiveFactor XI into activated Factor XIa . Factor XIa i s a serine protease that cleaves the inactive form of Factor IX into IXa . Factor IXa cleaves inactiveFactor VIII to VIIIa . High-molecular-weight kininogen and prekallikrein a l so trigger the intrins ic pathway by activating Factor XII. Injury triggers theactivation of Factor I I I and sets off the extrins ic pathway. Factor I I I cleaves inactive Factor VII (the most abundant of the clotting factors ) toactive VIIa .

Figure 14-15. Extrinsic and Intrinsic Pathways of the Clotting Cascade. See the text for deta i l s of both extrins ic and intrins ic pathways . [Reproducedwith permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

The extrins ic and intrins ic l imbs of the clotting cascade operate independent of one another and converge into a common pathway,beginning with the cleavage of Factor X into Xa. Subsequent activi ty of Factor Xa and Va convert prothrombin into thrombin. (Factor Xa a lsopos i tively regulates Factor VII of the extrins ic pathway.)

Al though i t i s the aggregation and cross -l inking of fibrin molecules that di rectly resul t in clot formation (see below), thrombin formation i sthe committed s tep in clot formation. Thrombin pos i tively regulates the intrins ic pathway factors Factor XI as wel l as Factor VIII , the terminals tep of the intrins ic l imb of the clotting cascade. The activation of Factor VIII perpetuates coagulation, caus ing more and more clot to form. Iti s this widespread ava i labi l i ty of fibrinogen and the thrombogenic versati l i ty of thrombin that a l low for a prompt response to hemorrhage, nomatter where i t occurs .

THE FIBRIN MESHWORK

Unl ike other clotting factors , proteolys is does not transform fibrinogen into an active serine protease. Rather, the cleavage of fibrinogen resul tsin the formation of fibrin monomers . Fibrin monomers cross -l ink with one another, forming a netl ike fibrin mesh that ensnares RBCs , puttingan end to the spi l l ing of RBCs by forming a clot.

Fibrinogen, a large (340 kD), soluble hexamer (s ix subunits ), i s formed by two trimers made of α, β, and γ, cha ins (Figure 14-16A). The twoheterotrimers are l inked through disul fide bonds . The secondary and tertiary s tructures (Chapter 1) form three globular domains : one betweenthe two heterotrimers and one on each end of the fibrinogen molecules . The N termini of the α and β cha ins project from the center globulardomain and are the s i tes where thrombin cleaves off cysteines (Figure 14-16B).

Figure 14-16. A–B. Conversion of Fibrinogen to Fibrin. (A) Fibrinogen, a soluble hexamer cons is ting of two α, two β, and two γ cha ins , which arecross -l inked by disul fide bonds (note l ines between s ingle peptide chains above), undergoes cleavage at α (FPA) and β (FPB) cha ins to formfibrin. (B) Thrombin action on FPA or FPB to produce the α- or β-fibrin cha ins . FP, fibrinopeptide. [Adapted with permiss ion from Murray RA, eta l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Fol lowing proteolys is , fibrin aggregates to form a soft clot, which connects the centra l globular domains to the C-terminal domains of other(di fferent) fibrin molecules . Factor XIIIa s tabi l i zes the soft clot into a hard clot. Factor XIIIa cata lyzes the formation of a cross -l ink betweenone end of a globular domain and a di fferent fibrin’s globular domain, s tabi l i zing the soft clot into a hard clot (Figure 14-17A–B).

Figure 14-17. A–B. Formation of a Hard Clot. (A) Fibrin molecules aggregate to form a soft clot. Factor XIII cata lyzes cross -l ink formation (red l ines )among fibrin molecules , transforming the soft aggregate into a hard clot. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus tratedBiochemistry, 28th edi tion, McGraw-Hi l l , 2009.] (B) Scanning electron microscopy image of a clot, i l lus trating the meshwork of fibrin,erythrocytes , and platelets in various s tates of degranulation. [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Textand Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

DIFFERENCE BETWEEN PLATELET PLUG FORMATION AND CLOT FORMATION

Hemophilia refers to a group of diseases characterized by frequent hemorrhages , both spontaneous and traumatic. This may mani fest ashemarthroses , easy bruis ing, and, in genera l , an increased amount of time needed to form a clot. Hemophi l iacs require more time to formclots due to clotting factor defects. Hemophilia A (80% of hemophi l ia patients ) resul ts from Factor VIII deficiency and Hemophilia B (20% ofhemophi l ia patients ) from Factor IX deficiency. Patients with hemophi l ia have less than 5% of the normal factor levels as compared withnon-hemophi l ia patients , and patients with severe hemophi l ia have less than 1% of normal levels (usual ly having no functional Factor VIIIor Factor IX). Patients with hemophi l ia require regular intravenous infus ions of puri fied Factor VIII or Factor IX protein to prevent smal lbleeds from turning into l i fe-threatening hemorrhages .

von Willebrand’s Disease (vWD) i s a mi ld, inheri ted bleeding disorder that mani fests as easy bruis ing, bleeding gums, and frequentnosebleeds . Despi te multiple, heredi tary, and acquired forms, the condition s tems from a deficiency, in quanti ty and/or qual i ty, of vWF.Bound to Factor VIII , vWF is respons ible not only for platelet adhes ion, but a lso for the surviva l of Factor VIII . Without vWF, Factor VIII i squickly degraded and patients experience increased bleeding times . Patients with vWD, therefore, have a deficiency of both platelet plugformation and clot formation. Treatment i s often not necessary unless control of bleeding i s required (e.g., excess ively long menses ,surgery), and cons is ts of giving ei ther addi tional Factor VIII or by the medication desmopressin, which helps to ra ise the vWF level .

Platelet plug formation i s a temporary repair unti l a proper clot forms. Deficient platelet plug formation resul ts in epis taxis (bloody nose) andpurpurae (tiny red/purple spots of bleeding) on the mucosa (especia l ly l ips ) and underneath the skin. Deficient clot formation, on the

contrary, resul ts in rebleeding. For instance, were one to have a molar tooth removed, a platelet plug would s top the bleeding temporari ly. Afew hours later, however, the gums wi l l rebleed as the plug i s only a temporary fix and the individual lacks the permanent fix of a clot.

REGULATION OF CLOT FORMATION

Fibrinogen and other clotting components freely exis t in the ci rculation. Without regulatory mechanisms, thrombin would continue to activateFactors V, VIII , and fibrinogen, and clots would form indiscriminately and at inappropriate times . Three major regulators serve to control clotformation: anti -thrombin II I (ATIII), protein C, and protein S. A deficiency in ATIII , protein C, and/or protein S leads to a hypercoagulable s tate,making patients vulnerable to inappropriate and potentia l ly dangerous clot formation.

Vitamin K, Clotting, and Warfarin: Synthes ized by gut bacteria , vi tamin K (Chapter 10) i s a cofactor for γ-glutamyl carboxylase, the enzyme thatcarboxylates (activates ) prothrombotic Factors I I , VII , IX, and X and anti thrombotic regulatory proteins C and S. Vitamin K epoxide reductase i srequired to return oxidized vi tamin KO back to i ts active form, vi tamin KH2 (see figure below). Warfarin, a medication used to l imit clotting,blocks epoxide reductase, leading to lowered levels of active vi tamin KH2 and, therefore, active clotting factors .

Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.The ba lance between the body’s normal clotting level and i ts inhibi tion by warfarin i s not s imple, and patients uti l i zing this drug must beconstantly monitored to insure both efficacy and safety of i ts use. If vi tamin K i s deficient, clot formation may be hindered and chronicbleeding may resul t. Vi tamin K deficiency can a lso be caused by antibiotics that destroy the gut flora , thereby decreas ing the amount ofvi tamin K ava i lable for absorption. Vi tamin K deficiency i s treated by giving supplementa l vi tamin K ei ther ora l ly or intravenous ly.

ATIII inactivates Thrombin, IXa , Xa , and XIa . ATIII ’s activi ty i s a l so enhanced many thousands of times by the molecule heparin (Chapter 2).Heparin exis ts in a wide range of molecular s i zes , determined by i ts variable number of ol igosaccharides . The number of ol igosaccharideunits determines heparin’s anticoagulation effects on ATIII . For instance, in order to inhibi t thrombin, ATIII requires a heparin with more than18 saccharide uni ts . If ATIII encounters the pentasaccharide form (five saccharide uni ts ) of heparin, then ATIII inhibi ts Factor X.

Protein C and protein S work together to degrade and inactivate Factors Va and VIIIa . Protein C i s activated as a membrane-bound complexcons is ting of the proteins thrombomodulin, thrombin, and calcium (Ca2+) plus phospholipids (see Figure 14-18). As protein C requires protein S as acofactor, both protein C and protein S are regarded as coagulation inhibi tors .

Figure 14-18. Regulation of the Fibrinolytic System by Protein C. u-PA, urokinase-type plasminogen activator. [Reproduced with permiss ion fromBarrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

PLASMIN AND CLOT DISSOLUTION

Breakdown of the fibrin-based meshwork and dissolution of clots rel ies on the serine protease plasmin, which i s formed via activation of i tszymogen plasminogen. The convers ion of plasminogen to plasmin occurs via tissue plasminogen activator (tPA), urokinase plasminogen activator(uPA), the peptidase kallikrein, or coagulation Factor XII (a l so known as Hageman factor). Activation occurs via cleavage of plasminogen betweenarginine 560 and va l ine 561 (Figure 14-19). Further cleavage of plasmin can produce the molecule angiostatin, which l imits the new growth ofblood vessels . Research i s currently attempting to use this molecule for cancer and other medica l treatments .

Figure 14-19. Activation of Plasmin. Plasmin i s formed from plasminogen by the cleavage of an arginine (Arg)–va l ine (Va l ) bond via any one ofsevera l plasminogen activators . The two chains of plasmin are held together by a disul fide bridge. [Reproduced with permiss ion from MurrayRA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Factor V Leiden: Factor V Leiden polymorphism changes arginine to glutamine in the Factor V clotting factor. The arginine change i s present inthe cleavage s i te that protein C uses to degrade and regulate Factor V. Thus , in the Factor V Leiden variant, protein C i s less efficiently ableto regulate Factor V, leading to higher than normal levels of active Factor Va. High levels of active Factor Va lead to excess ive thrombinproduction, fibrinogen activation, and clot formation. This higher level of Factor Va activi ty creates a hypercoagulable s tate. People with theFactor V Leiden variant are prone to developing blood clots in the legs (deep vein thrombosis, DVT), which can embol ize (break off and travelin the ci rculation). These embol i can lead to subsequent emergencies , including right-s ided heart clots , pulmonary embol ism, trans ientischemic attacks (temporary s trokes), and, in pregnant women, a smal l increase in miscarriages .Clot Dissolution and D-dimers: Clot formation (embolism) or breakage of an exis ting clot (thrombus) i s one major cause of myocardial infarction(heart attack) and cerebral vascular accidents (s trokes). Treatment of these “thromboembol ic events” i s via a number of “clot-busting”medications , which include streptokinase, uPA, and anyone of severa l genetica l ly produced tPAs (see figure). Al l clot busters lead to thebreakdown of clots (thrombolys is ) by activating plasminogen and have a l lowed cl inicians to effectively treat early presentations of heartattacks , s trokes , and other medica l disorders . Free plasmin, released from a dissolved clot, i s subsequently bound to α2-antiplasmin tostop i ts actions .

Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.Fibrin breakdown products (see the figure), of which one major type i s “D-dimers” (named as such because their molecular s tructure

appears as two connected “Ds”), offer a method to di rectly measure the activi ty of plasmin. In medica l conditions such as DVT or pulmonaryembolism, measurement of the level of D-dimers a l lows cl inicians to both confi rm and grade the extent of an exis ting clot by relying on thiss ide product of the body’s efforts to breakdown clots .

REVIEW QUESTIONS1. What are the bas ic components of the blood and their functions?2. What are the functions of red blood cel l s?3. What i s the genera l s tructure of hemoglobin?4. How do defects in the α-globin or β-globin genes lead to the various types of tha lassemias?5. What are the functions of proximal his tidine, i ron, and the heme group in hemoglobin in relation to oxygen binding?6. How does carbon monoxide poisoning resul t in an anaerobic-l ike s tate?7. How does the oxygen–hemoglobin binding curve expla in how hemoglobin can be saturated by O2 in the lungs , but only partia l ly saturated

in periphera l ti s sues?8. What i s the Bohr effect and what i s the mechanism by which i t promotes O2 di ssociation at ti s sues that require O2?

9. What i s the role of hemoglobin in acid–base ba lance?10. What are the roles of transferrin, ferri tin, and hepcidin in i ron homeostas is?11. What i s the most common cause of nutri tional anemia and why may this be particularly preva lent in chi ldren and females of

chi ldbearing age?12. What i s the role of von Wi l lebrand Factor in blood clot formation?13. What are the three parts of platelet plug formation?14. What are the di fferences between the intrins ic and extrins ic pathways of the clotting cascade?15. What are the events associated with the common pathway of the clotting cascade?16. What i s the role of proteolys is in the clotting cascade?17. What are the roles of protein C, protein S, and anti thrombin II I in l imiting clot formation (anticoagulation) and how does a deficiency of

any of these components lead to a hypercoagulable s tate?

CHAPTER 15THE IMMUNE SYSTEM

Editor: Eric L. Greidinger, MDStaff Phys ician, Miami VAMC Rheumatology Section and Associate Professor, Divis ion of Rheumatology and Immunology, Leonard M. Mi l ler

School of Medicine, Univers i ty of Miami , Miami , FL

Overview of the Immune SystemAntigenAntibodyCel l s Associated with the Immune SystemCytokinesInnate ImmunityComplement SystemHypersens i tivi ty ReactionsReview Questions

OVERVIEWThe immune system, composed of antigens , antibodies , and severa l specia l i zed leukocyte (white blood cel l ) types , offers the human bodyadaptable protection aga inst infections and invas ion by foreign molecules and cel l s . Bes ides responses by antibodies and cel l s , the immunesystem is a lso composed of a complement-activated pathway that a l lows an a l ternative to respond to infectious organisms.

Al though able to attack these pathogens , the immune system is a lso able to recognize the host human cel l s and molecules and canselectively not respond to “sel f.” Uncontrol led immune responses can lead to ti ssue damage or death.

OVERVIEW OF THE IMMUNE SYSTEMThe human immune system is respons ible for generating a protective response to infective organisms (e.g., bacteria , vi ruses , and paras i tes )and foreign cel l s (e.g., tumor and transplant) whi le both recognizing the host body and l imiting damage to i tsel f (sel f–nonsel f theory). Asattacks are a lways evolving, the immune system must be capable of adapting to each new chal lenge. The immune system a lso provides aninflammatory response to trauma. When ti ssue or cel l s are damaged, molecules that act as endogenous danger signals can be released. Thesedanger s igna ls may include molecules such as uric acid (produced by purine metabol i sm, Chapter 4), which, i f present at high concentrations ,can form crysta ls that innate immune sensors recognize and thus activate immune and inflammatory responses .

Innate immunity refers to preformed host defense systems that can provide immediate host defense activi ties without fi rs t being tra ined todis tinguish sel f from invader. Elements of the innate immune system often conta in s tructura l recognition moti fs that a l low them to identi fyl ikely pathogens to target. Molecules that are found in microbes without s tructura l homologs in human cel l s , such as flagel l in orunmethylated deoxyribonucleic acid (DNA), are other examples of danger signals that induce innate immune activation when their s tructure i srecognized by innate immune sensors .

The adaptive immune system i s capable of generating a response very speci fic to the s tructure of an invading organism or molecule (antigen,see below) as wel l as the abi l i ty to remember that s tructure via memory cel l s (immunological memory). The repeat activation of the immunesystem by an antigen that has previous ly induced an immune response i s normal ly much quicker and s tronger. The invading pathogen servesas a target, which el ici ts a response speci fic for that antigen from immune cel l s . This response may include the production of a speci ficantibody aga inst that pathogen (humoral immune system) or the activation of immune cel l s that ei ther attack and ki l l the offending organism ororchestrate this activi ty, the cell-mediated immune system. These cel l types include lymphocytes (including T and B lymphocyte types),monocytes , macrophages , dendri tic cel l s (DCs), neutrophi l s , eos inophi l s , and basophi l s .

Passive immunity involves antibody molecules that are transferred to the baby from the mother’s active immune system through theplacenta. Immunoglobulin (Ig) G (see below) i s the s ingle antibody type that binds to a neonatal Fc receptor and i s endocytosed via pinocytosis.Newborns a lso receive IgA antibodies (see below) via breast mi lk, which provides ini tia l protection aga inst pathogenic microorganisms.Pass ive immunity i s cons idered to be a short-term system, which i s taken over in the fi rs t year or two of l i fe by the adaptive immune system.

Immunodeficiencies: Medica l conditions known as immunodeficiencies are characterized by the absence or mal function of a component of theimmune system, leading to the inabi l i ty to fight infectious disease. More than 120 congenita l immunodeficiencies have been recognized.External causes of immunodeficiency include poor diet, the effects of immunomodulating drugs , and infectious diseases including humanimmunodeficiency vi rus (HIV)/acquired immune deficiency syndrome.

ANTIGENAntigen, origina l ly termed from the phrase “antibody generator,” i s a molecule (normal ly protein or carbohydrate in source) that can s timulatethe immune system to make antibodies and/ or ini tiate a cel l -mediated response. Sel f-antigens are present on a l l cel l s of the host organism.Sel f-reactive immune cel l s are typica l ly deleted or mainta ined in s tates of impaired reactivi ty to prevent the development of autoimmunity.Many pathogenic microbes and cancer cel l s can use some of these same mechanisms to evade immune attack. Other “opportunis tic”organisms may induce aggress ive responses from intact immune systems (that effectively ki l l the organism) but can s ti l l lead to disease inimmunocompromised hosts .

The speci fic s tructura l surface recognized by cel l s of the adaptive immune system is known as an epitope. Severa l antibodies may recognizevarious parts of one antigen (various epi topes). To be recognized by T cel l s in the cel l -mediated immune system, antigens must be presentedon the cel l surface of particular immune cel l s (see below).

ANTIBODYAn antibody i s a large, Y-shaped protein (Figure 15-1) respons ible for immunologica l identi fi cation and binding of antigens and, ul timately, forprotection aga inst invading pathogens . Another name for an antibody i s immunoglobulin, normal ly abbreviated as “Ig.”

The functions of antibodies are summarized in Figure 15-2.

Figure 15-1. Basic Structure of an Antibody Molecule. The antibody molecule i s a Y-shaped, tetramer protein, composed of two heavy cha ins andtwo l ight cha ins , held together by hydrophi l i c/hydrophobic forces and disul fide bonds . The body of “Y” i s composed of a constant proteinstructure and a highly variable end, which defines the affini ty of a particular antibody for one antigen epi tope. The Fc region of the moleculemay bind to the surface of receptors of severa l cel l types . [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text andAtlas , 12th edi tion, McGraw-Hi l l , 2010.]

Figure 15-2. Summary of Antibacterial Antibody Functions. Antibodies can el ici t a number of reactions resul ting in the el imination of bacteria ,including (A) recognition and binding to bacteria , which a l lows recognition by other immune system components to include (B) opsonization,leading to recognition and phagoyctys is by macrophages , and (C) binding leading to complement activation (see below), leading to bacteria llys i s . [Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, Mc Graw-Hi l l , 2009.]

Antibodies exis t in five types : IgA, IgD, IgE, IgG, and IgM. The bas ic s tructure, source, and function of each are summarized in Table 15-1.

TABLE 15-1. Summary of Antibody Molecules

CELLS ASSOCIATED WITH THE IMMUNE SYSTEMCel l s of the immune system, col lectively ca l led leukocytes or white blood cel l s , can be categorized as lymphocytes , monocytes , neutrophi l s ,eos inophi l s , and basophi l s (Figure 15-3).

Figure 15-3. White Blood Cells. Overview of white blood cel l s , including those derived from granulocytes and agranulocytes . A laboratory ful lblood count usual ly includes tota l white blood cel l s and a di fferentia l measurement of the five major types of white blood cel l s . Normalva lues are as fol lows: neutrophi l s (45%–60% of tota l ), l ymphocytes (25%–35% of tota l ), monocytes (3%–7% of tota l ), eos inophi l s (1%–3% oftota l ), and basophi l s (<1% of tota l ). See Appendix I I for further deta i l s . [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas icHis tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

T LYMPHOCYTES

T lymphocytes conta in a T-cell receptor (TCR) and CD4 or CD8 coreceptor on their plasma membrane (Figure 15-4A–B). Each TCR recognizes amolecule’s dis tinct epi tope s tructure and a l lows the speci fici ty needed for a control led immune reaction (Figure 15-4A). T lymphocytes with aCD8 coreceptor recognize antigen presented by Class I major his tocompatibi l i ty complex (MHC) molecules on the surface of a wide variety of

cel l s , and frequently are effectors of di rect cel l ki l l ing (cytotoxic T cel l s ). T lymphocytes with a CD4 coreceptor (helper T cel l s or Th cel l s )recognize antigen presented by Class I I MHC molecules on the surface of specia l i zed antigen-presenting cel l s (APCs , such as macrophages ,DCs , and some B cel l s ), and frequently orchestrate the responses of other immune cel l s . This process of antigen presentation i s summarizedin Figure 15-4B.

Figure 15-4. A. T-Cell Receptor Recognition of Antigen. Bas ic s tructure of the heterodimeric T-cel l receptor (bottom), showing the α- and β-subunitsand the constant and variable region that recognize the epi tope of the antigen fragment presented by an antigen-presenting cel l (top). ECF,extracel lular fluid; MHC, major his tocompatibi l i ty complex. B. Binding of T Cells to MHC Complex. CD4 (helper T) cel l s have a T-cel l receptor (TCR)and CD4 coreceptor that recognize antigen (blue ci rcle) bound to MHC Class I I receptor on an antigen-presenting cel l (left panel ). CD8 (cytotoxicT) cel l s bind via thei r TCR to antigen (blue ci rcle) presented by MHC Class I molecules (right panel ). MHC, major his tocompatibi l i ty complex;TCR, T-cel l receptor. [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, Mc Graw-Hi l l ,2010.]

Recognition of a speci fic antigen in the antigen–MHC complex by the TCR of the T lymphocyte cel l s (Figure 15-5, left) activates a s igna l ingprocess , involving a periphera l membrane leukocyte-specific tyrosine kinase (Lck), which i s tethered to the ins ide of the plasma membrane byl ipid res idues l inked to i ts amino-terminal end. Lck a lso conta ins two highly conserved amino acid sequences , known as sarcoma homologydomains, and binds to the cytoplasmic ta i l s of ei ther the CD4 or CD8 coreceptor molecules . Upon activation, Lck phosphorylates a subunit of theTCR at a highly conserved tyros ine separated from a leucine or i soleucine by two other amino acids . A double repeat of this four amino acidssequence, the two separated by 7–12 amino acids , i s known as an immunoreceptor tyrosine-based activation motif (ITAM) and i s found onsubunits of the TCRs as wel l as B-cel l receptors (see below). Phosphorylation of the two tyros ines of the ITAM a l lows binding of others ignal ing proteins resul ting in subsequent phosphorylation of another tyros ine kinase ca l led zeta-chain-associated protein kinase-70 (ZAP-70).ZAP-70 activation eventual ly leads to phosphorylation and activation of phosphol ipase C-γ 1; secretion of ca lcium from the endoplasmicreticulum; and a l tered gene express ion by the actions of nuclear factor of activated T cel l s , nuclear factor kappa B (NF-κB), and activatorprotein 1 (AP-1) transcription factors , among others , in the cel l nucleus . Cytotoxic T cel l s produce perforin and granulysin proteins , which createopen channels in the plasma membrane of infected cel l s , and granzymes , serine protease enzymes , both of which lead to cel l death.Cytokines including interleukin-2 (IL-2) are a lso expressed, leading to susta ined repl ication and activi ty of the lymphocytes .

Figure 15-5. Comparison of Cell-Mediated and Humoral Immunity. Cel l -mediated immunity (left) involves the presentation of antigen derived fromingested foreign molecules (e.g., bacteria) for T helper (Th) lymphocyte recognition and Th1 activation of cel l s such as macrophages , natura lki l ler, and cytotoxic T cel l s (see the text). Humoral immunity (right) resul ts from recognition of the foreign molecule by an antibody, which i sa l ready expressed on a B lymphocyte fol lowed by interaction with a Th2 cel l and di fferentiation into memory B and plasma cel l s , the latter ofwhich express large amounts of antibody aga inst the offending molecule. MHC, major his tocompatibi l i ty complex; NK, natura l ki l ler. [Adaptedwith permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, Mc Graw-Hi l l , 2009.]

As the name suggests , helper T lymphocytes ass is t a number of other immune cel l s including subsequent activation of B lymphocyte antibodyproduction (see below), macrophage phagocytos is (see below), and activation of cytotoxic T cel l s . Th cel l s are a major producer of cytokines . Thcel l s known as Th1 cells produce the cytokines interleukin-12 and interferon-γ (IFN-γ) via Janus kinase (JAK)–signal transducer and activator oftranscription (STAT) signaling (Chapter 8), leading to both di fferentiation and production of more Th1 cel l s . Additional ly, Th1 cel l s cause (a)increased express ion of MHC Class I molecules on normal cel l s , (b) production of adhes ion molecules that promote white blood cel lmigration to s i tes of infection, (c) decreased production of Th2 cel l s (see below), (d) increased lysosomal activi ty and antigen presentation bymacrophages , (e) increased natura l ki l ler (NK) cel l activi ty (see below), and (f) increased production of ni tric oxide, which can ass is t inbacteria l and vi ra l ki l l ing. In sum, these s ignals activate multiple cel l s to fight infecting organisms whi le downregulating other T-cel lpathways . One a l ternative T-cel l set known as Th2 cells induces B lymphocytes and produces interleukin-4, a cytokine that a lso induces DNArepl ication, via the JAK–STAT s ignal ing pathway (Chapter 8) that leads to the di fferentiation/production of more Th2 cel l s . The TCR and CD4coreceptor of Th cel l s bind to an antigen–MHC II complex on APCs but Lck s igna l ing resul ts in the production and release of severa l types ofcytokines (see below), including IL-2. IL-2 i s a l so expressed to promote prol i feration of the lymphocytes and prolongation of thei r response.

An additional set of T lymphocytes , suppressor T or regulatory T cells, acts to l imit activation of the immune system as wel l as phagocytos is .Their functions help to shut off immune responses to mainta in the essentia l ba lance between an activated and surveying immune systemand one that i s tolerant to i ts own sel f-antigens . Pathways of action of some regulatory T cel l s include (a) upregulated express ion oftransforming growth factor-β (TGF-β), a cytokine that upon binding to i ts receptor activates SMAD proteins that act as transcriptional regulatorsimportant in immune effector function and cel l cycle control (see Chapters 9 and 20); (b) upregulated interleukin-10, which decreases activationof Th cel l s and blocks NF-κB s ignal ing pathways ; and (c) induction of indoleamine 2,3-dioxygenase, an enzyme that depletes tryptophan in theloca l area preventing optimal function of T cel l s , which depend on a ready supply of this amino acid.

As the precursor to a T-cel l response, invading bacteria , vi ruses , and cel l s are processed and presented to T cel l s . Internal ly derivedpathogens (e.g., a vi rus repl icating ins ide a cel l ) are digested into smal l peptides within cel l s by a proteosome, a specia l i zed center of protein-cleaving enzymes within the cel l a l so respons ible for breaking down damaged or mis folded proteins and processes such as cel l cycle control .Peptides digested from cel lular proteins (including those of invading organisms) are taken up by TAP transporters (Transporter associated withAntigen Processing) found in the endoplasmic reticulum and sorted into ves icles , which travel to the cel l surface where they can associate withClass I MHC molecules for presentation to CD8+ (cytotoxic) T cel l s . Pathogens outs ide the cel l (e.g., bacteria , paras i tes , and/or toxic molecules )are normal ly taken up by specia l i zed APCs including DCs . APCs express Class I I MHC molecules that can present peptides to CD4+ (helper) Tcel l s . Dendri tic cel l s are specia l i zed to take up the exogenous pathogen and digest them via enzymes in lysosomes. The resul ting smal l

peptide fragments from ei ther the proteosomes or lysosomes are coupled with the appropriate class of MHC molecule and moved to the cel lsurface. Peptides binding to Class I MHC are typica l ly no more than 8 amino acids long, whereas Class I I MHC accommodates longer peptidesand can present cha ins of up to 14 amino acids in length.

The antigen–MHC complex i s presented by the APCs and recognized by ei ther cytotoxic T or Th cel l s a long with the appropriate CD4 or CD8molecule. This interaction involves very speci fic recognition of primary amino acid sequence of the antigen a long with any secondary s tructurethat may exis t. This antigen s tructure i s a lso presented in the context of the MHC and i l lus trates how remarkably exact the generation andpresentation of minute di fferences in protein s tructure must be. A recognized antigen wi l l el i ci t T-cel l activation and can lead to an immuneresponse; an unrecognized antigen wi l l be s imply ignored by the T lymphocyte. To induce an immune response, activated T cel l s must receivesecond s ignals from APCs that promote an immune response. The second s ignal l igands on APCs are expressed on activated APCs , typica l ly inresponse to a danger s igna l . In the absence of an appropriate second s ignal , activated T cel l s wi l l undergo programmed cel l death(apoptos is ) or lose the abi l i ty to respond to subsequent TCR l igation (develop anergy).

B LYMPHOCYTES/PLASMA CELLS

B lymphocytes are the antibody-producing cel l s of the immune system. Antibody-dependent immune response i s known as humoral immunity.Each B cel l has an antibody molecule on i ts surface, which serves as a type of receptor aga inst a particular pathogen molecule. When a B-cel lreceptor recognizes and binds to i ts antigen, the B cel l becomes activated. Activated B cel l s secrete antibody molecules andimmunostimulatory cytokines (Figure 15-5, right).

Some activated B cel l s can a lso act as APCs ; the antibody–antigen complex can be internal i zed and the antigen processed and displayed viaan MHC Class I I molecule on the B cel l surface. B cel l s can respond to T cel l s and danger s igna ls by switching the kind of antibody moleculesthey produce (class switching) from larger, less efficient IgM forms to smal ler, more efficient IgG forms. Other specia l i zed forms of antibodymolecules are a lso produced in association with mucosa l immunity (IgA) and in association with a l lergic-type responses (IgE). Theseantibodies ci rculate and bind to the target pathogen for attack and removal by the complement system, T cel l s , and phagocytic cel l s includingneutrophi l s (see below). Fol lowing an immune response, a smal l number of the activated B lymphocytes and some T lymphocytes becomememory cel l s to mainta in recognition of the offending antigen in the case of future infection. Memory cel l activation resul ts in a faster andstronger immunologica l response. During an active immune response, cytokine s timulation can induce some B cel l s to di fferentiate intoplasma cel l s , a population of long-l ived cel l s that produce susta ined high levels of antibodies . The protective immunity benefi ts of vaccinesare mediated in part by the induction of pathogen-speci fic antibody-producing plasma cel l s .

NATURAL KILLER (NK) CELLS

NK cells are another type of lymphocyte-l ike immune cel l . They di ffer from other lymphocytes in that they do not express a TCR or surfaceantibodies . As in cytotoxic T cel l s , NK cel l s attack infected cel l s and tumors by the production and insertion of perforin/granulysin and granzyme,which lead to thei r death. The activi ty of NK cel l s i s closely regulated by various cytokines (see below), activating and inhibi tory receptors ontheir cel l surface, and an antibody-binding Fc receptor that provides a target for NK activi ty. In contrast to cytotoxic T cel l s that attack cel l spresenting a foreign peptide in a proimmune context, NK cel l s are capable of attacking cel l s that fa i l to express surface molecules thatidenti fy the cel l s as sel f.

MONOCYTES AND MACROPHAGES

Monocytes are a major type of immune cel l respons ible for the ingestion or phagocytos is of invading organisms and foreign/toxic molecules .Monocytes and cel l s di fferentiating from them are capable of recognizing danger s igna ls and producing large quanti ties of proinflammatorycytokines such as interleukin-1 (IL-1) and tumor necros is factor-α (TNF-α) to produce or perpetuate immune responses . To a l low for moreefficient phagocytos is , monocytes require the opsonization or coating of the ingested targets by ei ther antibodies or complement (see below),a l though they can a lso recognize the outs ide surface of certa in pathogens . Monocytes respond within 8–12 h of infection, attracted bycytokines and danger s igna ls . Monocytes further di fferentiate into macrophages in ti s sue, where they serve as loca l defenders of immunity.Monocytes can a lso di fferentiate into DCs, which serve a primary function as an APC.

NEUTROPHILS

Neutrophils are the most abundant type (45%–60%) of leukocyte (white blood cel l ) in the human body and are one of the fi rs t immune cel l s toarrive at the s i te of a new infection and/or inflammation. As such, neutrophi l s are a primary marker of acute infection/inflammation.Neutrophi l s can phagocytose invading organisms or particles and release reactive oxygen species such as superoxide that can ki l l microbia land host cel l s . However, the ki l l ing of engul fed bacteria and other particles i s bel ieved to be more rel iant on a number of digestive enzymesconta ined in primary, secondary, and tertiary granules as wel l as lysosomes. Neutrophi l s can a lso ki l l bacteria l and fungal pathogens byforming neutrophil extracellular traps (NETs) composed of a unique s tructure of extracel lular protein fibers and DNA chromatin. Bes ides thephys ica l barrier, NETs a lso conta in a high concentration of antimicrobia l proteins and serine proteases .

EOSINOPHILS

Eosinophils mainly function in paras i tic infections and a l lergies/asthma. Al though usual ly compris ing less than 5% of ci rculating leukocytes ,their number can ri se dramatica l ly during an active response. They s tay in the ci rculation for up to 2 weeks , serving as a marker of such aninfection or inflammatory disorder. Degranulation of eos inophi l s releases a wide variety of molecules , including the fol lowing: His tamine—increases chemotaxis and blood vessel permeabi l i ty to a l low entrance of other leukocytes into the s i te of infection. Major basic protein—highly toxic protein to paras i tes . Inflammatory eicosanoids and leukotrienes (Chapter 3). Growth factors and cytokines—lead to continued growth and prol i feration of immune cel l s to continue the immune response.

Histamine: Histamine i s a multi functional s igna l ing molecule produced by the decarboxylation of histidine by the enzyme L-histidinedecarboxylase. His tamine i s a lso found in fermented and/or spoi led beverages and foods . Defects in the breakdown of his tamine by theenzyme diamine oxidase or acetaldehyde dehydrogenase i s fel t to be respons ible for the flushing seen during a lcohol intake, especia l ly incerta in As ian populations , and some cases of food poisoning. His tamine’s roles include that of a neurotransmitter and a s timulant ofparieta l cel l s to produce gastric acid. However, i ts function in a l lergic inflammatory reactions and immune response are better known.

Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

Four receptors are known for the receptor molecule. The histamine1 (H1) receptor acts via Gq protein, which activates the phosphol ipaseC/phosphatidyl inos i tol pathway, leading to di lation of blood vessels (flushing), contraction of smooth muscles in bronchi (breathingproblems), contraction of endothel ia l cel l s (hives and leakage of fluid), induction of unmyel inated C fibers (pa in and i tching), and evensome forms of motion s ickness . Most “antihis tamine” medications [e.g., diphenhydramine (Benadryl ), loratidine (Clari tin), fexofenadine(Al legra), and ci ti ri zine (Zyrtec)] are H1 blockers , as are promethazine (Phenergan) and mecl i zine (Dramamine or Antivert) fornausea/motion s ickness . Nonsedating H1 blockers di ffer from the other members of this group by their inabi l i ty to cross the blood–bra inbarrier and inhibi ting his tamine receptors in the centra l nervous system.

The H2 receptor, located on parieta l cel l s , i s the target of the class of reflux medications termed H2 blockers [e.g., cimetidine (Tagamet),rani tidine (Zantac), and famotadine (Pepcid)]. H3 receptors are of the Gi receptor class , inhibi ting formation of cycl ic adenos inemonophosphate, which leads to the inhibi tion of release of severa l neurotransmitters , including acetylchol ine, dopamine, γ-aminobutyricacid (GABA), noradrenal ine, and serotonin. The H4 receptor, another Gi-l inked receptor, i s found in bone marrow and basophi l s/mast cel l s ,which harbor a majori ty of the body’s his tamine in the nasa l and ora l mucosa, intestina l/l iver/ spleen and lung/trachea mucosa andthymus, testes , and tons i l s . The immune response, often activated by IgE antibody, leads to the release of his tamine from these sources(mainly mast cel l s and basophi l s ) increases mast cel l chemotaxis and capi l lary permeabi l i ty to a l low other immune cel l s access to fight aninvading pathogen. The effects are summarized below:Nose/s inuses : Blood vessel di lation and swel l ing (congestion), fluid leakage (runny nose), neura l s timulation (sneezing), for example,“Hay fever.”Eyes : Fluid leakage (watery eyes) and s timulation of unmyel inated C fibers (i tching and a l lergic conjunctivi ti s ), for example, “Hay fever.”Ears : Swel l ing of Eustachian tubes leading to blockage (ful lness ).Pharynx/lungs : Neura l s timulation (coughing), bronchi contraction (wheezing, shortness of breath), fluid leakage (throat swel l ing), forexample, “anaphylaxis .”Skin: Swel l ing and fluid leakage [hives (urticaria) and rash], for example, wasp or bee s tings .Gastrointestina l tract: Swel l ing, fluid leakage, neura l s timulation (pa in, bloating, nausea and vomiting, and diarrhea).Bra in: Effects on posterior hypothalamus (s leep problems).

BASOPHILS

Basophils comprise only about 0.01%–0.03% of white blood cel l s in plasma and are recrui ted to s i tes of inflammation and/or infection,especia l ly by a l lergies or paras i tes . Upon activation, basophi l s release a number of anti -inflammatory molecules , including his tamine(promoting blood flow), heparin (anticlotting); proteolytic enzymes such as elastase and lysophosphol ipase; and leukotrienes (Chapter 3).

DENDRITIC CELLS (DCs)

DCs are specia l i zed to sample molecules from their surroundings and present these as antigen to potentia l ly el i ci t an immune response(Figure 15-6). Many bel ieve them to be a key to cel l -mediated immunity involving T lymphocytes . Al though research i s s ti l l ongoing, DCs appearto ari se from cel l s in the bone marrow and travel to a variety of s i tes in the body where they di fferentiate into severa l subtypes with di fferingfunctional characteris tics . Developed DCs are mainly found in areas where they are exposed to the external envi ronment such as skin [i .e.,Langerhans cel l s (LCs )], the respiratory tract, and the digestive tract. They are a lso found in blood and lymph ti ssue. DCs s tart as ova l “vei l”cel l s but upon di fferentiation, they form severa l arm-l ike processes s imi lar to dendri tes of a neuron, which give them their name. They areproducers of severa l interleukin molecules and IFNs (see Table 15-2) involved in primary immune response. DCs have been impl icated in theini tia l s tages of vi ra l infection, including HIV and severe acute respiratory syndrome, and in autoimmune diseases . DCs may a lso play a primerole in the innate immune system by recognizing molecules from bacteria or vi ruses (danger s igna ls ) through Toll-like receptors and otherdanger s igna l sensors .

Figure 15-6. Dendritic Cell Presentation of Antigen to a T Cell. Activation occurs via MHC antigen presentation to the T-cel l receptor (s igna l 1) andbinding of costimulatory molecules including CD80/86 to CD28 (s ignal 2) as wel l as a number of other binding pa i rs , which s trengthen theinteraction, leading to a robust immune response. CTLA-4, cytotoxic T-lymphocyte antigen 4; ICAM-1, intercel lular adhes ion molecule 1; LFA,lymphocyte function-associated antigen 1; MHC, major his tocompatibi l i ty complex; TCR, T-cel l receptor. [Adapted with permiss ion from KatzungBG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, Mc Graw-Hi l l , 2009.]

CYTOKINESCytokines are proteins , sometimes glycoproteins , which are secreted by particular immune cel l types as part of intracel lular s igna l ing andactivation/inhibi tion of immune processes . Inter-leukins (involved in leukocyte s ignal ing) and IFN molecules are examples of cytokines . Thefunctions of cytokines vary widely per the cel l type with which they interact (see Table 15-2).

TABLE 15-2. Selected Immune Cytokines and Their Activi ties

Cytokine receptors can be ei ther membrane bound or soluble and fa l l under s ix genera l categories , depending on their s tructure. (1) Type Iand (2) Type II receptors, which include many of the major cytokines and IFNs , both function via a Janus Kinase (JAK) mechanism (Chapter 8). Theclass ca l led (3) Ig receptors, including the IL-1 receptor, activate phosphol ipase C and subsequent NF-k B gene activation. The (4) TNF receptorgroup uti l i zes an intermediary protein such as TNF receptor type 1-associated death domain, which transmits conformational changes from thereceptor to binding partners such as TNF receptor-associated factor 2, which interacts with severa l other s igna l ing peptides to mediateinflammation or programmed cel l death (apoptos is ) via NF-kB gene activation or by AP-1, a transcription factor related to c-Fos and c-Jun. The

(5) chemokine receptor group functions via Gq proteins , leading to the release of intracel lular ca lcium that el ici ts di rected chemotaxis . The fina lgroup, (6) tumor growth factor β-receptors are serine–threonine kinases that phosphorylate SMAD proteins (see Chapter 13) that can trans i t to thenucleus and act as DNA transcription inducers and/or suppressors .

Warts: Warts (verruca) from human papillomavirus (HPV) infection affect mi l l ions of people and can las t and/or recur for years despi teaggress ive medica l care. Even surgica l removal i s not 100% effective. More than 100 s tra ins of HPV are known, caus ing skin warts as wel l ascervical and other cancers of the reproductive tract. HPV i s an especia l ly di ffi cul t vi rus to treat, regardless of i ts location, because of severa limmune evas ion mechanisms i t has evolved.

Fi rs t, HPV infects keratinocytes, a cel l that i s a l ready “destined to die” as layers of growing skin progress to the surface and are shed.These cel l s are less prone to regular immunologica l survei l lance than other cel l types , and they are less apt to respond to danger s igna lswith cytokine release that would activate dendri tic and other immune cel l s—a vi ra l death i s seen no di fferently than a preprogrammeddeath. Second, vi ra l infection normal ly invokes an IFN-a response (see below). Unfortunately, HPV and s imi lar vi ruses s top IFN-α productionby di rectly inhibi ting i ts gene express ion or i ts JAK–STAT s igna l ing pathways . Fina l ly, external vi ra l s tructures normal ly activate LCs,specia l i zed DCs of the skin respons ible for detecting and responding to loca l i zed infections . HPV, though, di rectly inhibi ts the activation ofLCs and, therefore, any immune response aga inst them. Treatments for skin warts ranging from medications (sa l icyl i c acid, s i lver ni trate,podophyl lum, imiquimod, fluorouraci l , and even the reflux medication cimetidine) to freezing (cryotherapy) to duct tape to apple cidervinegar have proven success ful in some patients . Al l are based on an attempt to activate the immune system to recognize and respond tothe vi ra l infection.

INNATE IMMUNITYThe innate immune system includes the phys ica l barriers of skin (including the continued shedding of skin cel l s ), mucosa l l inings , mucus , andnormal bacteria found in the intestina l tract and other locations , tearing and sa l ivary production, as wel l as a variety of white blood cel l s (NKcel l s , phagocytic/macrophages , neutrophi l s , and DCs and ti ssue eos inophi l s , basophi l s , and mast cel l s ). The complement system (see below)is a lso part of this innate system. Al l provide an a lmost immediate response to infective microbes by bringing immune cel l s and inflammatorymediators to the s i te of invas ion, activating biochemica l cascades di rected aga inst them, and, in most cases , inducing the phys ica l removal ofthese offending agents .

Autoimmune Diseases and Immunosuppressants: The abi l i ty of the immune system to di fferentiate between foreign “non-sel f” and host “sel f”antigens i s important to control immune response and the resul ting cel l ki l l ing. A loss of this control and the subsequent attack on thehost body can lead to autoimmune diseases . These diseases include Graves ’ disease and Hashimoto’s thyroidi ti s , type 1 diabetes , vi ti l igo,pernicious anemia, rheumatoid arthri ti s , sys temic lupus erythematosus , multiple scleros is , Sjogren’s syndrome, polymyos i ti s , myastheniagravis , and Goodpasture’s syndrome. A mainstay of treatment of autoimmune disorders i s immunosuppress ive drugs . These, unfortunately,genera l ly place the patient at increased ri sk for infections by nonspeci fica l ly impeding immune functions . Many immunosuppressant drugscan cause other s ide effects . Main categories of these medications include the fol lowing:

1. Glucocorticoids (e.g., corti sol , prednisone, and dexamethasone)—alter cel l gene transcription with effects including reducing cytokineexpress ion to l imit cel l -mediated and humoral immunity; and inhibi ting phosphol ipase A2–arachidonic acid interaction, therebyblocking inflammatory pathways .

2. Cytostatics (e.g., cyclophosphamide, azathioprine, and mycophenolate)—inhibi t cel l divis ion of T and B lymphocytes .3. Immunophi l in inhibi tors (e.g., cyclosporin and tacrol imus)—block production of cytokines (e.g., IL-2) and or cel l cycle proteins that

promote T lymphocyte growth, prol i feration, and activi ty.4. TNF-α inhibi tor—proteins or monoclonal antibodies that block the proinflammatory effects of TNF-α, including induction of IL-1 and

interleukin-6.5. Biologica l therapies—typica l ly monoclonal antibodies that interfere with a defined aspect of immune function, such as by ki l l ing cel l s

that express thei r antigen target (e.g., ri tuximab/B cel l s ), blocking second inflammation-associated receptor s igna l to T cel l s (a lefacept,abatacept, ipi l imumab, efa l i zumab, see the figure), or preventing immune cel l migration to s i tes of inflammation (nata l i zumab) byblocking integrins .

Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

COMPLEMENT SYSTEM

The complement system is a pathway of immunologica l protection composed of more than 25, mainly l iver-synthes ized polypeptides .Complement can be activated through three separate paths : class ica l , a l ternative, and lectin (Figure 15-7). In each case, these pathways leadto the proteolys is and activation of complement component C3, and the same downstream inflammatory effects , including recrui tment andactivation of cel lular immune responses , induction of loca l ti s sue warmth, swel l ing and pa in, and formation of a membrane attack complex(MAC), which creates a transmembrane pore (Chapter 8) that can participate in ki l l ing bacteria and abnormal cel l s (including vi ra l ly infectedcel l s ). The early complement components that induce C3 activation are each examples of danger s igna l sensors .

Figure 15-7. The Classical and Alternative Complement Pathways. Representation of the two major complement pathways and analogousmechanism of the mannose-binding lectin pathway. The left figure i l lus trates the various components and interactions of the class ic pathway(top) and the a l ternative pathway (bottom). The right figure shows various functions of components of both pathways . See text for furtherdeta i l s . [Reproduced with permiss ion from Naik: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

The classical pathway i s ini tiated by binding of the C1 complex (composed of one C1q, two C1r, and two C1s subunits ) to IgM or IgG/IgMantibodies or binding of one C1q to an infective organism. Binding and a resul ting conformational change of C1q activates a serine proteaseenzyme on C1r, which cleaves C1s . C1s a lso has serine protease activi ty and cleaves complement proteins C4 and C2, forming C4a and C4b andC2a and C2b, respectively. The C4b and C2a proteins next combine to form a C3 convertase, which cleaves C3 into C3a and C3b. A fina l C5convertase complex i s then formed from C4b, C2a, and C3b, which cleaves the C5 protein into C5a and C5b. C5b subsequently binds to C6, C7, C8,and C9 proteins to form the MAC complex.

The alternative pathway i s activated by the cleavage of a thioester bond in the C3 protein to form C3a and C3b. The C3b protein i s able to binddirectly to the plasma membrane of an infective agent after which i t i s joined by the protein factor B to form a C3bB complex. This binding,therefore, requires no antibody but i s restricted to only certa in types of infective organisms. Factor D, a tryps in-l ike enzyme, then cleaves factorB into Ba and Bb (an active serine protease), resul ting in C3bBb, the a l ternative pathway C3 convertase. The addition of one more C3b proteinby the action of the newly formed C3 convertase leads to subsequent cleavage of C5 and formation of the MAC complex as described above.Decay accelerating factor and another protein, factor H, inhibi t the a l ternative pathway by competi tively inhibi ting factor B binding to orcaus ing i ts dissociation from surface-bound C3b. Another molecule, factor I , cleaves C3b into an inactive form to inhibi t the reaction process .

The lectin pathway, known more formal ly as the mannose-binding lectin pathway, i s activated by binding of the speci fic glycoproteins (Chapter2) to mannose sugar res idues on the surface of the infective agent. This binding turns on two mannan-binding lectin-associated serine proteases,MASP-1 and MASP-2, which are analogous to the class ica l pathway proteins C1r and C1s , respectively. MASP-1 and MASP-2 fol low the class ica lpathway in cleaving C2 and C4 with progress ion to C3 cleavage and activation.

Individual complement proteins a lso serve additional functions . C3b binds to infective organisms and promotes phagocytic ingestion bymacrophages and other phagocytic cel l s . C5a, the other part of the C5 protein, helps to attract immune cel l s to the source of infection. C3a andC5a can activate mast cel l s to degranulate and increase the permeabi l i ty of blood vessels and smooth muscle contraction as part of animmune response. Bb can a l ternatively increase the repl ication of preactivated B lymphocytes (see above), whereas Ba inhibi ts this process .Kupffer cel l s in the l iver are a lso involved in clearing infective cel l s that are coated with complement proteins .

Complement Deficiency and Disease: The importance of the complement immune system is i l lus trated by the resul ts of deficiencies or harmfulmutations of i ts components . Al though conclus ive evidence i s s ti l l lacking, problems with the complement system have been impl icated indiseases such as asthma, sys temic lupus erythematosus, paroxysmal nocturnal hemoglobinuria, forms of angioedema, macular degeneration,hemolytic uremic syndrome, and membranoproliferative glomerulonephritis. The importance of the MAC complex in protecting aga inst Gram-negative bacteria i s a l so shown by the marked increase in meningi ti s in complement-deficient patients .

HYPERSENSITIVITY REACTIONSHypersens i tivi ty reactions occur when the immune system reacts excess ively, resul ting in undes i red damage to the body’s ti s sues and, insome cases , death. Al l hypersens i tivi ty shares the common moti f of presens i ti zation or pre-exposure to an antigen with a prior immuneresponse. Four major types of hypersens i tivi ty reactions have been described, which are summarized in Table 15-3.

TABLE 15-3. Review of Hypersens i tivi ty Reaction Types

Transplant Rejection: The rejection of transplanted organs occurs when the recipient immune system identi fies the new ti ssue as foreign andmounts an immune attack aga inst i t. Despi te efforts to adequately serotype the donor and recipient to insure as close an immunologica lmatch as poss ible (histocompatibility), this problem plagues transplant patients and the cl inicians who care for them.

Transplant rejection resul ts from the processes a l ready described and are normal ly categorized as hyperacute rejection, acute rejection,and chronic rejection. Hyperacute rejection processes occur within minutes and are caused by the complement immune system respondingimmediately to the donor organ because of pre-exis ting or cross -reacting antibodies . Certa in ti s sue types are especia l ly prone tohyperacute rejection (e.g., kidney, some ti ssues from other species ), whereas others a lmost never react (e.g., l i ver). Hyperacute rejection i sa lso respons ible for the adverse reaction seen during some blood transfus ions . Acute rejection occurs within a week after transplant andcan las t for up to 3 months . It i s usual ly because of mismatch of human leukocyte antibody, which i s found in a l l cel l s , which lead to a Tlymphocyte response (i .e., cell-mediated immunity). Aga in, certa in ti s sue transplants (e.g., kidney and l iver) are more prone to this mode ofrejection. Chronic rejection, occurring months to years after transplant, i s usual ly secondary to gradual breakdown and scarring of bloodvessels in the transplant. The exact mechanism is not wel l understood.

Treatment i s via immunosuppressant medications (see above) and/or, in some cases , bone marrow transplantation, a procedure with i tsown ri sks in patients who are a l ready probably i l l .

REVIEW QUESTIONS

1. What are the bas ic features of innate, pass ive adaptive, humoral , and cel l -mediated immunities?2. What are the bas ic features and di fferences between the sel f–nonsel f and danger s igna l theories of immunologica l activation?3. What are the bas ic s tructures and functions of antigens and antibodies , including the five bas ic types of antibody molecules?4. What are the di fferent types of T lymphocytes , and their roles in immunity and mechanism of immune activation?5. What are the functions of B lymphocytes and natura l ki l ler cel l s?6. What are the bas ic functions of monocytes/macrophages , neutrophi l s , eos inophi l s , and basophi l s?7. What are the bas ic functions of cytokines , including the mechanism of the five types of cytokine receptors?8. What are the s imi lari ties and di fferences of the class ic, a l ternative, and lectin pathways of complement activation?9. How do the four types of hypersens i tivi ty reactions compare?

CHAPTER 16THE CARDIOVASCULAR SYSTEM

Editor: Ralph V. Shohet, MDDirector of Cardiovascular Research, John A. Burns School of Medicine (JABSOM), Univers i ty of Hawai i at Manoa, Honolulu, HI

Cardiac Muscle Structure and FunctionSinoatria l and Atrioventricular NodesThe Cardiac CycleBlood VesselsEndogenous Cholesterol/Lipoprotein Metabol i sm and TransportAtheroscleros isBiochemica l Mechanisms Associated with Heart AttackReview Questions

OVERVIEWThe cardiovascular system, including the heart and blood vessels , i s respons ible for the ci rculation of blood to a l l ti s sues of the body whi lecarrying away carbon dioxide and waste products . Cardiac muscle shares many attributes with skeleta l and smooth muscles but represents aseparate muscle group with dis tinct s tructure, function, and regulation. Key in this function i s the sel f-activating nature of particular cardiaccel l s that normal ly provide an ordered contraction of the cardiac chambers . Blood vessels conta in smooth muscle cel l s regulated by a varietyof s igna l molecules and a lso play an important role in maintenance of blood pressure and oxygen/nutrient dis tribution. Diseases of theseblood vessels , especia l ly hypertens ion and atheroscleros is , cause much of the i l lness and most of the deaths in the developed world.

CARDIAC MUSCLE STRUCTURE AND FUNCTIONThe heart i s derived from the mesoderm and provides the force that propels oxygenated blood to a l l cel l s of the body and returnsdeoxygenated blood back to the lungs (Figure 16-1). The heart comprises specia l i zed s triated muscle termed cardiac muscle.

Figure 16-1. Sectional View of Heart. Bas ic anatomy of the heart i s indicated, including chambers , major blood vessels , and conducting system(yel low), including the s inoatria l node, atrioventricular node, and Purkinje fibers . [Reproduced with permiss ion from Barrett KE, et a l .:Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Cardiac muscle di ffers from skeleta l and smooth muscle in severa l important ways :

1. Increased numbers of mitochondria a l low continuous high-level production of adenos ine triphosphate (ATP) to support continuous functionwithout fatigue. During periods of low oxygen, anaerobic respi ration can provide enough energy for contraction to continue.

2. Uti l i zes l ipids [fatty acids and triglycerides (60%)] as the major energy source fol lowed by carbohydrates (35%), amino acids , and ketonebodies (5%).

3. Conta ins fewer but larger T tubules and intercalated discs that a l low the rapid and synchronous spread of action potentia ls between adjacentcel l s , a l lowing the coordinated contraction of cardiac muscle.

4. Rel ies on the rapid release and re-uptake of intracel lular ca lcium stores to convert the electrica l s igna l of the action potentia l into themechanica l work of contraction.

Because cardiac cel l s and smooth muscle cel l s rely s trongly on an L-type (“longlasting”) calcium channel for contraction, the medication class

of ca lcium channel blockers (CCBs) are used to treat high blood pressure and certa in cardiac disorders . In the heart, CCBs decrease the tota lca lcium released and both the heart rate and force of the contraction, thereby reducing oxygen demand. This effect of CCBs i s particularlyuseful in helping to control abnormal heart rhythms such as atria l fibri l lation. In blood vessels , the decreased ca lcium-induced contractionresul ts in di lation of the blood vessels (by decreas ing the abi l i ty of the smooth muscle to contract) and lowers the res is tance of thosevessels . Additional cardiovascular uses of CCBs include reducing the outflow gradient in hypertrophic cardiomyopathy and reducing pulmonaryarteria l res is tance in pulmonary hypertens ion. CCBs are a lso used in the treatment of epi lepsy via thei r effect on neurons .

CCBs are often separated into dihydropyridines, most commonly used for hypertens ion because of greater affini ty for receptors in vascularsmooth muscle, and non-dihydropyridines, which are relatively cardiac selective and less l ikely to cause reflex tachycardia . Benzothiazipines suchas di l tiazem have intermediate characteris tics . These three classes are summarized in Table 16-1 and Figure 16-3.

TABLE 16-1. Ca lcium Channel Blocker Classes and Actions

SINOATRIAL AND ATRIOVENTRICULAR NODESParticular cardiac cel l s located in the sinoatrial (SA) node (Figure 16-1) have the abi l i ty to ini tiate an action potentia l (Chapter 12), which thenspreads through the muscle cel l s in the upper part of the heart (right and left atria ) via the interca lated disks , resul ting in a contraction ofthese two chambers . In normal s inus rhythm, the SA node ini tiates action potentia ls at 60–100/min that are conducted to the atrioventricular(AV) node, which coordinates the sequentia l contraction of the upper (atria l ) and then lower (ventricular) chambers of the heart. If the SA nodefa i l s to provide action potentia ls , the AV node can independently generates action potentia ls at 40–60 beats per minute. Subsequenttransmiss ion of the action potentia l to the ventricles i s via specia l i zed heart cel l s (cardiomyocytes ) in the right and left bundle branches andPurkinje fibers that spread throughout the ventricles . These conduction cel l s conta in increased numbers of sodium ion channels andmitochondria whi le conta ining fewer cardiac muscle fibers . If both the SA and the AV nodes fa i l to provide an action potentia l , the Purkinjefibers or ventricular myocytes can a lso produce a coordinated contraction at rates s lower than the AV node.

Al though the SA and the AV nodes and Purkinje fibers can independently ini tiate and continue action potentia ls , which provide heartcontractions , the vagal nerves (parasympathetic nerves caus ing a decreased SA node rate and force of contraction) and thoracic nerves T1–4(sympathetic nerves resul ting in increased SA node rate as wel l as force of contraction) a lso convey neurologica l influences on heart rate.Hormones/neurotransmitters such as epinephrine (adrenaline) and dopamine increase heart rate and/or the amount of blood pumped percontraction (s troke volume). Epinephrine works via binding to α1-,α2-,β1 and β2-adrenergic, G-protein-coupled receptors. The receptor s igna ltransduction includes activation of phosphol ipase C to increase production of inos i tol phosphates/diacylglycerol , protein kinase C activi ty,and the release of Ca 2+ (α1-/Gq protein). Additional ly, there wi l l be inhibi tion (α2-/Gi protein) or activation (β1 and β2-/Gs protein) of adenylylcyclase to decrease or increase cycl ic adenos ine monophosphate (cAMP) production and protein kinase A activi ty, respectively (see Chapter 8for review of G-protein activi ties ).

Figure 16-2. Mechanism of Calcium Activation of Smooth Muscle Cells. Ca lcium ions (Ca 2+) activate intermediary s igna l ing molecules such asca lmodul in and myos in l ight-chain kinase (MLCK) to phosphorylated regulatory myos in l ight cha ins , leading to contraction of vascular smoothmuscle cel l . Ca lcium channel blockers inhibi t the influx of ca lcium, leading to inhibi tion of this process and resul ting vascular smooth musclerelaxation. One effect of β-agonis ts/blockers leads to relaxation of vascular smooth muscle as shown. ATP, adenos ine triphosphate; cAMP,cycl ic adenos ine monophosphate; cGMP, cycl ic guanos ine monophosphate. [Reproduced with permiss ion from Katzung BG, et a l .: Bas ic andCl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Dopamine: Dopamine i s a neurotransmitter that not only affects the centra l nervous system but a lso el ici ts dose-dependent effects on bloodvessels and cardiac muscle via Dopamine (D) receptors and α1- and β1-adrenergic receptors . It i s frequently used in the intens ive care uni tto support blood pressure. At low doses (2–5 μg/kg/min), binding i s primari ly to D1 and D5 receptors found on blood vessels , especia l ly inthe kidneys , activating a Gs-receptor (adenyl cyclase/cAMP), resul ting in increased urine production. Dopamine when del ivered at a rate of5–10 μg/kg/min binds to β1-adrenergic receptors in cardiac muscle, producing increased contraction rate and force for treatment ofshock/heart fa i lure patients . Dopamine provided at 10–20 μg/kg/min binds to α1-adrenergic receptors , resul ting in contraction of bloodvessels and increased res is tance/blood pressure.

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

Figure 16-3. Representatives of Calcium Channel Blocker Classes. Ca lcium channel blocker medications can be divided into three bas ic classesdepending on their mechanism, including phenyla lkylamines (e.g., verapami l ), dihydropyridines (e.g., ni fedipine), and benzothiazepines (e.g.,di l tiazem). See text and Table 16-1 for further discuss ion. [Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica lPharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

THE CARDIAC CYCLEThe Cardiac cycle i s an autonomous process that resul ts in the organized, sequentia l , contraction of the chambers of the heart, tempora l lycoordinated to produce efficient pumping throughout the body of blood, which then returns to the heart and lungs for re-oxygenation. Thecardiac cycle includes s teps that resul t in heart sounds , which produce the characteris tic electrica l s igna ls seen on an electrocardiogram (ECG)tracing, including the P-wave (atria l depolarization), QRS-complex (ventricular depolarization), and T-wave (ventricular repolarization).

B-type Natriuretic Peptide (BNP): BNP i s a 32-amino acid polypeptide (Chapter 18) that i s secreted by the ventricles when cardiac cel l s areexcess ively s tretched. BNP binds to atrial natriuretic peptide receptors, activating a guanylyl cyclase producing cycl ic GMP (cGMP), and, via acGMP-dependent protein kinase, producing relaxation of blood vessels . Rises in BNP levels are seen in congestive heart fa i lure (CHF) where i treduces the systemic res is tance and blood volume, thereby reducing the work of the a i l ing heart. Measurement of BNP i s useful to bothassess and fol low patients with CHF.

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

β-Blockers: β-Blockers (a l l ending in “-lol”) represent a class of medications used for the treatment of high blood pressure, i rregular heartbeat, chest pa in (angina), and heart attack therapy. β-Blockers inhibi t β1- and β2-adrenergic receptors in the heart and blood vessels .Stimulation of β1-receptors on cardiac cel l s by molecules such as epinephrine normal ly leads to increased heart rate and augmentedcontraction that increases myocardia l oxygen requirements . Blockade of these receptors reduces oxygen demand and can a lso help theheart reta in a normal geometry as i t hea ls . Stimulation of β2-receptors on vascular smooth muscle relaxes (di lates ) blood vessels (seeFigure 16-2). So, vasoconstriction, for example, resul ting from certa in drugs of abuse, may be exacerbated by β-blockers .

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.Like CCBs , β-blockers a lso have “classes” related to thei r relative effect(s ) on cardiac muscle or vascular smooth muscle. Relatively

speci fic β1-blockers such as metoprolol a ffect mainly cardiac muscle, decreas ing heart rate and the force of contraction and reducing work ofthe heart and oxygen requirements . This effect i s a key in the treatment of heart attack/angina victims . Nonspeci fic β-blockers , of which thearchetype i s propranolol, inhibi t β2-receptors as wel l .

β-blockers a lso interact with β-adrenergic receptors in other organs (e.g., eye, bra in, kidney, and lung) and may decrease intraocularpressure (glaucoma) and may improve migra ine headaches , anxiety/PTSD. They a lso reduce renin release which l imits convers ion ofangiotens inogen to angiotens in 1. This reduces angiotens in I I wi th subsequent reduction in a ldosterone from kidneys (Chapter 18),reducing blood volume. Both effects lower blood pressure. Their effect on the lung may worsen bronchia l constriction in asthma attacks . β-blockers are a lso used in many cardiac arrhythmias , CHF (via thei r relaxing effects on the heart but a lso thei r effect on renin/a ldosterone),mitra l va lve prolapse, porta l hypertens ion and any resul tant bleeding of esophageal varices , pheochromocytoma, and hypertrophiccardiomyopathy. However, the cross -reactivi ty of some β-blockers for di ffering receptors and ti ssues and the abi l i ty of some β-blockers toboth inhibi t and s timulate the same receptors depending on concentration leads to a number of unwanted s ide effects that must becareful ly understood, monitored, and managed by the cl inician.

The ini tiation of cardiac muscle contraction rel ies on voltage-dependent calcium channels (VDCCs). The predominant VDCC on cardiac cel l s i sthe L-type channel , which i s composed of an α1 subunit with s ix transmembrane α-hel ices that form a membrane channel or pore (Chapter 8),

but which a lso responds to vol tage changes by a l terations in conformation that blocks or a l lows Ca 2+ passage. Along with the α1 pore, thischannel includes other subunits . The disul fide-l inked (Chapter 1) α2δ subunits anchor the α1 protein in the membrane and augment i tsresponse to vol tage changes . A β subunit both enhances α1 express ion on the membrane and conta ins guanylate kinase activi ty that cata lyzesthe reaction ATP + GMP 4 ADP + GDP. The convers ion of GMP to GDP serves as a regulatory mechanism to augment the vol tage sens i tivi ty for theα1-pore (i .e., β-subunit activi ty a l lows smal ler depolarizations to resul t in channel opening). Fina l ly, a γ-subunit i s bel ieved to be of l i ttles igni ficance in cardiac muscle, a l though i t may play an important regulatory role in skeleta l muscle contraction. Sarcoplasmic/endoplasmicreticulum Ca2+-ATPase (SERCA) a l so plays a prominent role in cardiac cel l contraction (see Chapter 12).

The sel f-ini tiating action potentia l of the SA, AV, or Purkinje fibers provides the ini tia l s timulus for contraction. In response to an actionpotentia l propagated down the conduction system to the T-tubules , VDCCs open by depolarization of the cel l membrane and mechanica ls tretching of the cel l s (i .e., propagated from other nearby cel l s ), G-protein s igna l ing, or autonomic nervous system activation. This actional lows Ca 2+ into the cardiac cel l s (myocytes ). Ca 2+ that enter into the cardiac muscle cel l s bind to specia l i zed calcium release channels via aryanodine receptor, which releases much larger internal Ca 2+ s tores from the sarcoplasmic reticulum. This cytoplasmic ca lcium binds to theprotein troponin C, which causes a conformational change of the inhibi tory troponin I, which in turn a l lows tropomyos in to reveal myos in-binding s i tes on the actin thin fi laments . Propagation from one cardiac cel l to the other resul ts from the passage of Ca 2+ via interca lateddisks , specia l i zed cardiac gap junctions , completing depolarization of a l l the cardiac muscle. Ca lcium is then taken back into the sarcoplasmicreticulum through the ATP-requiring action of SERCA, the sarcoplasmic endoplasmic reticulum ATPase, restoring the cel l to a s tate where i t canbe depolarized aga in.

BLOOD VESSELSBlood vessels , cons is ting of arteries, veins, and capillaries provide the conduit for blood to be carried to and from every ti ssue in the human bodywith del ivery of oxygen and nutrients and the removal of carbon dioxide and waste materia l (Figure 16-4). The average human body hasapproximately 60,000 mi les of blood vessels , of which the vast majori ty are capi l laries that let only a s ingle l ine of red blood cel l s to pass .Arteries and veins are composed of three layers each of cel l s and/or polysaccharide matrix and/or connective and elastic ti s sues and/orsmooth muscle cel l s a long with their own system of blood vessels (vasa vasorum) and nerves , which feed and regulate thei r s tructure and,therefore, activi ty. Capi l laries , respons ible for the actua l transfer of molecules between the blood and the cel l s , usual ly are only made of as ingle layer of cel l s with some, interconnected connective ti ssue.

Figure 16-4. Blood Vessels. Blood vessels include the artery (left), vein (right), and connecting capi l lary (center) each of which conta insspecia l i zed layers of cel l , elas tic, and connective and muscular ti s sue surrounding a centra l lumen. The vaso vasorum, which provides nutrientsto these ti ssue layers . [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l ,2010.]

The abi l i ty of arteries and, to a lesser extent, veins to di late and constrict depends on the di rected action of the autonomic nervous systemon the smooth muscle in thei r layers . This abi l i ty to change their internal diameter i s essentia l for the regulation of blood pressure, bodytemperature, del ivery of blood and nutrients to various areas of the body, conservation of blood in times of bleeding, and so forth.Vasoconstriction i s normal ly due to the actions of peptide hormones such as vasopressin, angiotensin II, and endothelin or via neurologica ltransmitter molecules such as epinephrine. These molecules affect vascular smooth muscle via the Gq receptor, which el ici tsphosphatidyl inos i tol/protein kinase C and ca lcium secretion, leading to contraction of blood vessel smooth muscle layers (Chapter 8; see a lsoeffects on urinary system, Chapter 18). The effects of vasopress in are mainly seen in the arteries , kidneys and bra in, and a l l act to mainta inand increase the arteria l blood pressure. Angiotens in and epinephrine affect both arteries and veins . Al l serve as a potentia l compensatorymechanism during periods of blood loss/hemorrhage to avoid shock. Al though vasopress in and angiotens in each have their own receptors ,the smooth muscle constriction effects of epinephrine work mainly via activation of the α1-adrenergic receptors. Epinephrine can a lso causevasodi latation and/or vasoconstriction via α2- and β2-receptors, respectively, (a l though these actions are usual ly speci fic to a particular ti s sueand not a l l blood vessels ) and increased heart rate and contracti le force via β1-receptors, a l l via the Gi or Gs, adenylyl cyclase/cAMP messengersystem (Chapter 8).

The process of vasodilation rel ies on molecules such as nitric oxide (NO), which di ffuses through the plasma membrane of blood vessel cel l sto bind to a soluble guanylate cyclase (Figure 16-5). cGMP produced by this enzyme activates protein kinase G, which leads to dephosphorylationand inactivation of smooth muscle myos in molecules via inactivation of MLCK (Figure 16-5A). Even though the average l i fetime of NO in thebody i s only a few seconds , i ts pharmacologica l effects are seen in medications such as sublingual nitroglycerin (Figure 16-5B) for chest pa in viadecrease in cardiac workload, treatment of neonata l patients suffering from pulmonary hypertens ion in an intens ive care setting, and evenmodern, erecti le disorder drugs (e.g., s i ldenafi l / Viagra), which prolong the l i fetime of cGMP and, therefore, vasodi lation of vessels in thepenis , promoting erection (Figure 16-5A).

Figure 16-5. A–B. Nitroglycerin and Mechanism of Action of Nitrous Oxide. (A) Relaxation of smooth muscle in blood vessels by ni trous oxide occursvia activation of guanylyl cyclase producing cGMP, which promotes protein kinase G dephosphorylation of myos in l ight cha ins via inactivationof MLCK. (B) Ni trates such as ni troglycerin (a lso know as glyceryl trini trate) can a lso cause vessel relaxation via the same pathway and, as aresul t, i s used for the rel ief of symptoms of myocardia l infarction. cGMP, cycl ic guanos ine monophosphate; eNOS, endothel ia l ni tric oxidesynthase; GTP, guanos ine triphosphate; MLCK, myos in l ight-chain kinase; PDE, phosphodiesterase. [Reproduced with permiss ion from KatzungBG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Blood vessel permeabi l i ty i s important not only for normal human phys iologica l processes but a lso for pathology. The s ingle-cel l layer ofcapi l laries a l lows the exchange of oxygen and carbon dioxide (Chapter 14) as wel l as nutrients and waste. In disease, inflammation of theendothel ia l layer of blood vessels can lead to increased permeabi l i ty and leakage of plasma and other blood components , contributing tothe four class ic s igns of inflammation: ca lor (warmth), tumor (swel l ing), rubor (redness ) and dolor (pa in) (see a lso Chapter 3). Moleculesleading to increased blood vessel permeabi l i ty include histamine, working via the his tamine-1 receptor (Gq/phosphatidyl inos i tol/ca lciumsecond messenger pathway), prostaglandins (Gs/adenylyl cyclase/cAMP), and by interleukin-1 (IL-1) via a Janus kinase (JAK) receptor(phosphorylation of tyros ine res idues/cGMP) (Chapter 8). The blood vessel wal l i s a l so damaged in diseases such as atheroscleros is andimmunemediated vascul i ti s .

ENDOGENOUS CHOLESTEROL/LIPOPROTEIN METABOLISM AND TRANSPORTAbnormal ly elevated levels of l ipids in l ipoproteins , termed hyperl ipidemia, i s one of the most important ri sk factors for the development ofatheroscleros is (see below). The metabol i sm and transport of cholesterol and l ipoproteins occur in two genera l ways . Fi rs t, process ing ofcholesterol and triacylglycerols synthes ized by the l iver (Chapter 11) i s termed as the endogenous pathway of cholesterol/l ipoprotein andbegins with l iver secretion of nascent very-low-dens i ty l ipoprotein (VLDL) and high-dens i ty l ipoprotein (HDL). Second, process ing of ingestedcholesterol and triacylglycerols , the exogenous pathway, occurs in the blood s tream and begins with secretion of chylomicrons after eating byintestina l epi thel ia l cel l s ini tia l ly into the lymphatic system. Both are summarized in Figure 16-6 and discussed below.

Figure 16-6. Overview of Exogenous and Endogenous Pathways of Cholesterol/Lipid Transport and Metabolism. The exogenous pathway carries dietaryl ipids via chylomicrons for uptake by target ti s sues (e.g., adipose and muscle) and/or clearance and s torage in the l iver. Endogenous VLDLparticles carry l iver-derived fatty acids primari ly to adipose and muscle, IDL (VLDL remnants ) carry cholesterol primari ly to l iver, and LDL takescholesterol to periphera l ti s sues as wel l as the l iver. Both pathways uti l i ze HDL as a source of apo C and apo E to a l low maturation of thenascent chylomicrons and VLDL to thei r mature form (see Figures 16-7 and 16-8). A-1, LCAT activator apol ipoprotein; apo, apol ipo-protein; C,cholesterol ; CM, chylomicron; CMR, chylomicron remnant; HDL, high-dens i ty l ipoprotein; IDL, intermediate-dens i ty l ipoprotein; LCAT, leci thincholesterol acyl transferase; LDL, low-dens i ty l ipoprotein; PL, phosphol ipid. [Reproduced with permiss ion from Naik P: Biochemistry, 3rdedition, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

Further deta i l s of chylomicron metabol i sm are provided in Figure 16-7.

Figure 16-7. Metabolic Fate of Chylomicrons. Nascent chylomicrons are assembled in the smal l intestine from dietary triacylglycerols (TG) andcholesterol (C) a long with apol ipoproteins A and B-48. The addition of apol ipoproteins C and E from HDL forms a chylomicron, which usesapol ipoprotein C to activate l ipoprotein l ipase (LPL) for periphera l fat depos i tion. LPL in capi l lary wal l s hydrolyzes the triacylglycerolsreleas ing fatty acids into the extrahepatic ti s sues (e.g., adipose ti ssue). The remaining chylomicron remnant remains in the ci rculation andreturns apol ipoproteins E and C to HDL and then continues to the l iver where i t binds the apol ipoprotein E receptor for subsequent clearance.apo, apol ipoprotein; HDL, high-dens i ty l ipoprotein; HL, hepatic l ipase; LRP, LDL receptor-related protein; PL, phosphol ipid. [Reproduced withpermiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

VLDL

The triacylglycerol -rich l ipoprotein produced by the l iver i s the VLDL, respons ible for the transport of triacylglycerol from the l iver to ti s suessuch as adipose and muscle as wel l as transporting Cholesteryl es ters in the form of low-dens i ty l ipoproteins (LDL) to periphera l cel l s . Thetriacylglycerol i s formed from free fatty acids that are ei ther mobi l i zed from adipose cel l s or synthes ized in l iver from acetyl coenzyme A (CoA)derived from nonl ipid precursors , primari ly from glucose (Chapter 7). Cholesteryl es ter that i s a l so packaged in VLDL i s derived mostly fromdirect synthes is in the l iver from acetyl -CoA derived from β-oxidation of saturated fats (Chapter 7) or from dietary sources carried to the l iver bychylomicron remnants (Chapter 11). Apolipoprotein (apo) B100, a long with some apoC and apoE, are synthes ized in the l iver and are required forthe proper assembly and export of the nascent VLDL. Subsequently, apo C and apo E are transferred from HDL to the nascent VLDL in theci rculation, thereby creating the mature VLDL particles (Figure 16-8).

Figure 16-8. Metabolic Fate of Very-Low-Density Lipoprotein (VLDL). Nascent VLDL particles are assembled in the l iver from triacylglycerols (TG);cholesterol (C); and apol ipoproteins B-100, C, and E. The addition of apol ipoproteins C and E from HDL forms a VLDL particle, which usesapol ipoprotein C to activate l ipoprotein l ipase (LPL) for periphera l fat depos i tion. LPL in capi l lary wal l s hydrolyzes the triacylglycerolsreleas ing fatty acids into the extrahepatic ti s sues (e.g., adipose ti ssue). Once the TGs are hydrolyzed, apol ipoprotein C returns to HDL. Theremaining VLDL remnant, known as intermediate-dens i ty l ipoprotein (IDL), remains in the ci rculation and ei ther continues to the l iver where i tbinds the apol ipoproteins B-100 or E receptor for subsequent clearance or the complete removal of TG from the IDL resul ts in the formation ofLDL, which binds to LDL receptors on the l iver or other periphera l ti s sues , primari ly via apo B-100 receptors to faci l i tate uptake/clearance. LDLcan a lso become oxidized and be taken up by “scavenger receptors” on macrophages , which then invade artery wal l s , el i ci ting cholesterol

depos i tion and s tarting the process of atheroscleros is . apo, apol ipoprotein; HDL, high-dens i ty l ipoprotein; PL, phosphol ipid. [Adapted withpermiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Simi lar to the case of chylomicrons (Chapter 11), triacylglycerols in VLDL undergo l ipolys is in a reaction cata lyzed by lipo-protein lipase (LPL) torelease fatty acids for use by adipose and muscle (Figure 16-3). LPL i s a cel l surface enzyme on capi l lary wal l s that hydrolyzes triacylglycerolsin chylomicrons (and VLDL) to fatty acids that are taken up into cel l s . LPL i s most often found in capi l laries that supply heart, skeleta l muscle,adipose ti ssue, and mammary cel l s . In muscle, the released fatty acids are oxidized for energy; in adipose, they are re-esteri fied for s torageas triacylglycerols . LPL i s activated by apo C, bound to phosphol ipids on the surface of the chylomicron. In the fed s tate, insul in increases theactivi ty of LPL, whereas in s tarvation, the activi ty decl ines .

INTERMEDIATE-DENSITY LIPOPROTEIN (IDL) AND LDL

After repeated rounds of LPL action, apo C and apo E (not i l lus trated) are transferred back to HDL from the shrinking VLDL (Figure 16-8). Thisresul ts in formation of an IDL that conta ins apo B100 and some remaining apo E. IDL, l ike HDL and chylomicron remnants , can be cleared by theapo E receptor on l iver (genetic defects in the apo E l igand or i ts receptor el ici t type II I hyperl ipidemia, in which IDL, chylomicron remnants ,and HDL are elevated). Further hydrolys is of triacylglycerol in IDL by LPL and transfer of the remaining apo E to HDL resul t in formation of LDLwith cholesteryl es ters from ei ther the l iver (endogenous) or chylomicron remnants (exogenous). VLDLs decrease in surface area as thei rtriacylglycerols are progress ively hydrolyzed unti l they are reduced to cholesteryl es ter-enriched LDL (Figure 16-8). In this way, VLDL functions todel iver fatty acids from endogenous ly synthes ized triacylglycerols to muscle and adipose ti ssue and endogenous ly synthes ized cholesterol tovarious ti ssues .

The primary function of LDL i s to provide cholesterol to periphera l ti s sues . LDL receptors , respons ible for this uptake, are associated withclathrin-coated pi ts on the plasma membrane of these cel l s and recognize apo B100 (Figure 16-8). LDL particles are then endocytosed into thevarious ti ssues including the l iver a long with the receptors . The receptors and clathrin mostly recycle back to the plasma membrane. Most ofthe LDL (70%) binds to receptors on l iver hepatocytes . The remainder of the LDL associates with receptors on periphera l cel l s . Lipoproteindisorders in which LDL receptors , or thei r capaci ty to bind the apo B100 l igand, are defective, resul t in an increased level of cholesterol in LDLremaining in ci rculation, caus ing hypercholesterolemia and atheroscleros is (see below). A s imi lar event occurs for clearance of IDL, a lsoknown as VLDL remnants , and for clearance of chylomicron remnants , a l though i t i s apo E on the surface of these particles that i s recognizedby a receptor primari ly found in l iver plasma membrane. In both instances , the number of receptors transcribed/trans lated and ava i lable onthe cel l s ’ surface i s di rectly related to the amount of cholesterol ins ide a cel l . By this mechanism, cel l s can di rectly regulate thei r intake ofcholesterol depending on current concentrations in the Golgi .

HDL

As discussed in Chapter 11, the l iver i s the only organ capable of dispos ing of s igni ficant quanti ties of cholesterol , ei ther via excretion intothe bi le or by metabol i sm to bi le acids , both of which are lost to some degree in the feces . HDL, synthes ized in l iver and secreted into plasmaas a discoida l particle (Figure 16-8), ass is ts in this function serving as a cholesterol scavenger and faci l i tating the transport of cholesterolfrom the periphery to the l iver for convers ion to bi le acids and eventual el imination. It i s this cholesterol -removing property that renders HDLthe des ignation of “good” cholesterol carrier.

Mature HDL particles conta in leci thin, cholesterol es ter, lecithin cholesterol acyl transferase (LCAT), apo A1, apo C, and apo E. Ci rculating HDLacquires cholesterol from periphera l cel l s in a process faci l i tated by cholesterol efflux regulatory protein (CERP), an ATP-binding, proteintransporter. CERP i s activated by apo A1 and fl ips unesteri fied cholesterol and leci thin to the outer layer of cel l membranes . CERP then del iversthe free cholesterol and leci thin as substrates for LCAT on HDL. Apo A1 a lso activates LCAT in the nascent HDL and functions as a l igand for acel l surface receptor that exis ts on periphera l cel l s . Cholesterol es ters , the product of LCAT cata lys is , move to the core of nascent HDL forsubsequent transport back to the l iver. The enti re process of LCAT extraction of cel l cholesterol and incorporation into HDL for l iver clearanceis ca l led “reverse cholesterol transport.”

Another major function of HDL i s to serve as a repos i tory for apo A1, apo C, and apo E. Transfer of apo C i s required for the metabol i sm ofchylomicrons and VLDL, and apo E i s crucia l for clearance of chylomicron remnants , IDLs and HDLs . Therefore, HDL contributes to bothexogenous and endogenous pathways of l ipid transport.

Familial hypercholesterolemia: Familial hypercholesterolemia, a l so known as hyperlipoproteinemia type IIa according to the Fredricksonclass i fi cation, i s a genetic disorder caused by defective LDL receptors or apo B. Affected patients have a decreased abi l i ty to remove LDL fromthe ci rculation, leading to very high levels of LDL and a resul ting high ri sk of heart attack and s troke. When only one copy of the LDL gene i saffected (seen in approximately 1 in 500 people), high LDL cause atherosclerotic patients by thei r 50s . The rarer homozygous s tate, whereboth copies of the gene are defective, (occuring in approximately 1 in 1,000,000 people) causes severe cardiovascular disease in infancy andchi ldhood, which can lead to early death. Beyond the early development of atherosclerotic plaques (and the associated ri sk of heartattacks , s trokes , and other cardiovascular problems), patients wi l l a l so often have xanthomas, yel lowish depos i ts of excess cholesterol andfat in the eyel ids (xanthelasma palpebra) or depos i ted in tendons (tendon xanthoma), especia l ly the Achi l les tendon. The cause of fami l ia lhyper-cholesterolemia i s normal ly divided into one of seven molecular abnormal i ties , five di rectly a ffecting the LDL receptor.

1. LDL receptor completely absent.

2. LDL receptor does not reach cel l surface because of improper transport from endoplasmic reticulum to Golgi apparatus/plasmamembrane.

3. LDL receptor does not bind to LDL ei ther due to mutation in the receptor or the essentia l apo B molecule. A known change of asparagineto glutamine at amino acid 3500 di rectly affects apo B binding to the LDL receptor.

4. LDL receptor, once bound to LDL, does not endocytose in clathrin-coated pi ts .

5. LDL receptor does not return to surface after del ivery of LDL ins ide of cel l .

Treatment for heterozygous patients i s usual ly the aggress ive use of medications and genetic counsel ing to cons ider the medica lconsequences for thei r offspring. Homozygous patients usual ly do not respond wel l to medication therapy a lone and may require LDLapheres is—the di rect removal of LDL from the blood via a system s imi lar to dia lys is . In severe cases , l i ver transplant may offerimprovement.

ATHEROSCLEROSISThe formation of vessel blockages , known commonly as plaques , occurs via a process known as atherosclerosis. Atheroscleros is , the mostcommon underlying cause of heart attacks and s trokes , i s a process that involves inflammation of blood vessel wal l s , particularly arteries ,fatty depos i ts that include l ipoproteins and cholesterol , and white blood cel l s , especia l ly macrophages . Chronic atheroscleros is leads tohardening of the plaque by ca lcium as wel l as thickening and s ti ffening (arteriosclerosis) of the affected arteria l wal l s .

The exact mechanism of the formation of a plaque i s s ti l l under investigation, but current opinion i s that the ini tia l s tep involves oxidation

of LDL, probably by the enzymatic action of lipoprotein-associated phospholipase A2. These oxidized LDL particles are more readi ly taken up bymacrophages that can invade endothel ia l cel l s in the wal l of an artery that are themselves damaged by oxidation (Figure 16-8). The injuryini tiates an immune response by macrophages (Chapter 15), which bind to the area via the protein vascular cell adhesion molecule-1 and va inlyattempt to remove the oxidized LDL molecules . Platelets are a lso recrui ted to the s i te of injury and attempt to cover the area. Theaccumulation of oxidized LDL and the monocytes/ macrophages attempting to ingest i t i s known as a fatty streak—continued inflammation andgrowth of the fatty s treak leads to an atheroma of the arteria l wal l .

As the immune response continues the monocytes/ macrophages die, releas ing inflammatory factors such as IL-1 and tumor necrosis factor-α.More white blood cel l s , including T-lymphocytes and mast cel l s , are recrui ted with continued inflammation of the arteria l wal l . The continuedinflammation a lso impacts smooth muscle cel l s (Chapter 12), both leading to increased numbers and movement toward the growing plaque,adding to i ts s i ze and compos i tion. In addition, smal l ca lcium crysta ls begin to form within the smooth muscle cel l s (microcalcification) of thearteria l wal l , which are adjacent to the atheroma. In an attempt to confine and cover the inflammation, a hard, fibrous capsule i s producedfrom continued ca lci fi cation and col lagen and elastin released from the death of adjacent smooth muscle cel l s .

When the plaque wal l ruptures , blood comes in contact with the thrombotic factors in the interior. The resul t can be a thrombus thatcompletely occludes the artery. This i s the most common cause of myocardia l infarction. Sometimes the thrombus i s only partia l ly orintermittently occlus ive. These processes are thought to underly the pathophys iology of unstable angina. Additional ly, part or a l l of theoverlying thrombus can break off to form an embolus, which can travel to and block smal ler arteries . Blockage of an artery can a lso causeirrevers ible di lation known as an aneurysm. Risk for development of atherosclerotic plaques depends on severa l factors , including genetic,envi ronmenta l , and chronic diseases , which wi l l not be discussed further here. This process i s summarized in Figure 16-9.

Treatment of atheroscleros is rel ies on lowering the associated ri sks , including the use of aspi rin to inhibi t platelet aggregation andstatins (see above and Chapter 7) to lower levels of LDL and other harmful l ipids/l ipoproteins . Omega-3 fatty acids , found in many deep seafish, may reduce the chance of death from heart attacks . Omega-3 fatty acids are bel ieved to act via the production of anti -inflammatoryeicosanoid synthes is (Chapter 3).

Cardiovascular Disease and Cholesterol/Lipoproteins: Lowering levels of cholesterol and particular l ipoproteins , namely LDL and triacylglycerols ,have been shown to often dramatica l ly reduce the ri sk for cardiovascular disease including heart attack and s troke. Statins , which reducetota l cholesterol and LDL (Chapter 7), are a mainstay of the prevention of cardiovascular disease. However, other medication classes may beused to lower cholesterol/l ipoproteins as wel l .

Fibrates, which include gemfibrozil, fenofibrate (see the figure below), and clofibrate, among others , activate the peroxisome proliferator-activated receptor alpha (PPAR-α). Activation of this receptor found in muscle, l i ver, and other ti s sues increases the β-oxidation of l ipids in thel iver (Chapter 7), increases LPL l ipolys is of triacylglycerol from chylomicrons and VLDL (see above), increases the concentration of HDL toass is t in the clearance of cholesterol from periphera l cel l s (see above), and decreases the secretion of triacylglycerol from the l iver into theblood s tream (Chapter 11). The vi tamin niacin (see Chapter 10) a lso helps reduce LDL and VLDL levels by blocking the catabol i sm of fats inadipose ti ssue. As a resul t, the tota l concentration of fatty acids i s decreased in the blood, leading to lowered secretion by the l iver of VLDLand cholesterol . Niacin may a lso increase HDL, thereby further increas ing the clearance of cholesterol . Newer medication l ike exetimibereduces cholesterol absorption at the intestina l vi l l i (Chapter 11). These agents may be used in combination with dietary measures andstatins and may influence ini tia l plaque formation (see above).

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

Figure 16-9. Overview of Poss ible Mechanism of Atheroscleros is Formation and Infarction. [Reproduced with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

BIOCHEMICAL MECHANISMS ASSOCIATED WITH HEART ATTACKA heart attack or myocardia l infarction (MI) occurs when blood flow and, therefore, oxygen and nutrient del ivery i s blocked to an area of theheart. It can be worsened by a marked reduction in number (anemia) or in the abi l i ty of red blood cel l s to carry these components (Chapter 14).Prolonged lack of oxygen leads to death of the cardiac muscle cel l s and, frequently, fata l dysrhythmias or heart fa i lure. During the course of aheart attack, severa l biochemica l events occur related to insufficient oxygen (hypoxia), insufficient blood flow (i schemia), lack of oxygen(anoxia/infarction and cel l death), as wel l as the resul ting pa in (angina).

An MI wi l l often be preceded by episodes of i schemia that resul t in the typica l squeezing chest pa in of angina pectoris . Complete blockageof blood vessels that feed the heart muscle produces a s tate of no oxygen (anoxia). The effect of lowered and/or absent oxygen i s multi -fold onbiochemica l pathways . Without oxygen, oxidative phosphorylation (Chapter 6) cannot occur and new ATP and NAD+ are not generated. As aresul t, the fi rs t s tep of glycolys is i s blocked, a l though glucose ava i labi l i ty wi l l a l so fa l l (hypoglycemia) wi thout blood flow/nutrient del ivery.NADP+ s tores wi l l rapidly disappear, reducing flux though the pentose phosphate pathway cease. Al though of no uti l i ty in this scenario,glycogen synthes is and s torage wi l l a l so s top because of the lack of glucose-6-phosphate molecules from glycolys is . Amino acid synthes is andbreakdown wi l l s top as wi l l any enzymes (Chapter 5) that rely on ATP or any other nucleotides derived from i t (Chapter 4), including the abi l i tyto generate more proteins and enzymes via transcription (Chapter 9). Motor proteins such as myos in, dynein, and kines in wi l l no longer haveATP to power the conformational changes required for thei r activi ty (Chapter 1). Cel l moti l i ty, including intracel lular transport processes that

rely on these motors , wi l l a l so end and membrane channels that rely on the Na +–K+-ATPase function (Chapter 8) and/or other energy-rel iantprocesses wi l l no longer work, caus ing the bui ldup of toxic waste products (carbon dioxide, etc.). The fa i lure of these membrane transporterswi l l a l so affect Ca 2+ gradients , a ffecting the ca lcium-dependent contraction of cardiac cel l s discussed above.

When the blockage i s suddenly rel ieved, reperfusion injury may occur, from the release of damaging, reactive oxygen species (e.g. superoxide,hydrogen peroxide, and hydroxyl radica ls ). In addition, the metabol i sm of ATP in i schemic conditions creates the molecule hypoxanthine, which,i tsel f, may be metabol i zed by xanthine oxidase to produce hydrogen peroxide. Both processes overwhelm the affected cel l s ’ normalantioxidant defenses , including vi tamins A, C, and E (Chapter 10), which a lso cannot be replaced because of blocked blood flow. Othermolecular processes dependent on guanos ine triphosphate such as the growth of microtubules (Chapter 1), G-protein s ignal ing (Chapter 8),and the binding of transfer RNA molecules during trans lation (Chapter 9) wi l l cease.

As i schemia continues and worsens to anoxia , an infarct, wi th i rrevers ible death of cel l s wi l l occur within 30 minutes . As the cel l s die, thei rmembranes wi l l lose their bi layer integri ty (partia l ly due to ca lcium activation of phosphol ipases as wel l as damage by free radica ls tophosphol ipids , membrane proteins , and glycosaminoglycans) essentia l to separate compartments [e.g., mitochondria and peroxisomes thatconta in molecules toxic to cel l s (Chapter 8)] and extracel lular and intracel lular compartments (Chapter 8) vi ta l for l i fe. As membranesbreakdown, the contents of the cardiac cel l s are released, including cardiac markers used to detect an MI. Simi lar processes occur in the bra induring s troke or to any ti ssue that i s deprived of efficient and constant ci rculation.

Cardiac Markers: Myocardia l infarction i s diagnosed by a combination of symptoms, ECG changes , and cardiac markers that leak into theblood s tream from dying heart cel l s . In the past various enzymes , including Lactate Dehydrogenase (LDH) and Creatine Kinase (CK) weremonitored but these proteins a lso ri se with damage to other organs . CK-MB, an i soform of CK, i s much more speci fic to the heart and i s s ti l lused in monitoring damage from MI. The troponin proteins , which have cardiac-speci fic i soforms, are now the most commonly used markersas part of a diagnostic workup of a suspected MI.

Death of cardiac cel l s wi l l a l so interrupt the normal propagation of depolarization that produces a normal ECG tracing. As cardiac cel l s diein particular areas of the heart, changes such as ST-segment elevation or depress ion and the development of Q-waves are diagnostic of boththe presence and location of a heart attack. MIs are often ini tia l ly categorized as an ST elevation MI (STEMI) or non-STEMI because of di fferenttreatment modal i ties . Heart attacks are a lso often described as transmural i f cel l death extends completely through a section of the heart wal l ,often as a resul t of a complete blockage of an artery for the heart.

Treatment of an MI and i ts associated angina from blockage of a heart blood vessel i s focused on lowering the heart’s demand for oxygenwhi le attempting to increase oxygen del ivery to those cel l s . Aspirin inhibi ts cyclooxygenase production of thromboxanes (e.g. Thromboxane A2),which inhibi ts platelet aggregation and further blocking of blood vessels (Figure 16-10).

As noted above, nitroglycerin, which generates NO, activates and di lates blood vessels by protein kinase G-induced dephosphorylation andinactivation of smooth muscle myos in molecules (Figure 16-5). Di lated blood vessels a l low increased oxygen del ivery in cases of narrowed butnot completely blocked heart arteries .

Prinzmetal’s Angina: One form of angina with a potentia l ly di rect biochemica l bas is i s Prinzmetal’s angina, caused by spasm of coronaryarteries that reduce blood flow rather than the blockage seen in routine atherosclerotic disease. Normal ly, acetylchol ine not only inducescontraction of the vessels but a lso causes heal thy endothel ia l cel l s to produce and release NO. The vasoconstriction effect of acetylchol ineis , therefore, overridden by the vasodi latory effect of NO. In patients with Prinzmeta l ’s angina, however, acetylcho-l ine-induced NOproduction i s deficient and vasoconstriction predominates . Diagnos is i s usual ly made by evidence of transmural i schemia on the ECG thatabruptly resolves spontaneous ly or with ni troglycerin. Treatment may include usual ly via ni troglycerin to provide external NO and CCBs thatblock ca lcium release and, therefore, acetylchol ine-mediated constriction.

β-Blockers, a l so described above, are a mainstay of this treatment as they block β1-receptors in the heart and β-2-receptors found in bloodvessel smooth muscle and decrease both heart rate and contracti le force lowering the work load and, therefore, oxygen demand. Thevasodi latation effects of CCBs and inhibi tion of angiotens in vasoconstriction (angiotensin-converting enzyme inhibitors), both described above,make these medications an additional part of i schemia/angina/MI treatment as wel l .

Varying mechanica l and medicina l techniques can a lso be used to reopen the blocked vessel , a l though the renewed flow of blood a lsocarries with i t toxic radica l molecules , inflammatory interleukin molecules , excess acid, and dis turbances of ca lcium transport that can resul tfurther to the heart and surrounding ti ssues as wel l as heart arrhythmias known col lectively as reperfus ion injury (see above).

Thrombolysis: Thrombolysis and embolysis, the breakdown of a thrombus or embolus by medica l means , are used as mainstay therapy for MI,s troke, and other acute medica l problems involving blockage of blood vessels . The treatment rel ies on plasminogen activators that ini tiatethe cascade of the body’s natura l clot-dissolving enzymes (Chapter 14) by promoting production of plasmin from i ts proenzyme (Chapter 5)plasminogen. Thrombolytics include streptokinase, urokinase, and arti fi cia l ly produced analogues of tissue plasminogen activator.

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

Figure 16-10. Action of Aspirin in Treatment of Myocardial Infarction. Aspi rin i rrevers ibly inhibi ts cyclooxygenase (COX), leading to a decrease inproduction of prostaglandins and thromboxanes from arachidonic acid. See text and chapter. [Adapted with permiss ion from Kibble JD andHalsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

REVIEW QUESTIONS1. In what ways does cardiac muscle di ffer from striated and smooth muscle?2. What are the three classes of ca lcium channel blockers , thei r primary effect(s ), and their medica l uses?3. What i s the function of the L-type channel in cardiac cel l s?4. What are the roles of very-low-dens i ty l ipoprotein and low-dens i ty l ipoprotein in the endogenous l ipoprotein pathway in the process ing

of cholesterol and triacylglycerols?5. What are the functions of apol ipoproteins A1, B100, C, and E in the process ing of l ipoproteins?6. Why i s high-dens i ty l ipoprotein termed the “good” cholesterol carrier?7. What are the sequences of events leading to an arteria l thrombus?8. What are the events associated with the onset of a myocardia l infarction and the associated metabol ic ramifications?9. What i s the advantage of measuring troponin I and troponin T as cardiac markers relative to tota l activi ty of creatine kinase?10. What are the various options in the treatment of a myocar-dia l infarction and the mechanism for each of these options?

CHAPTER 17THE RESPIRATORY SYSTEM

Editor: Howard J. Huang, MDIns tructor in Medicine, Divis ion of Pulmonary and

Cri tica l Care Medicine, Washington Univers i tySchool of Medicine, St. Louis , MO

Bas ic Anatomy and DevelopmentO2–CO2 Exchange in the Lung and Acid–Base Ba lance

Noninfective Diseases of the Respiratory SystemInfective Diseases of the Respiratory SystemReview Questions

OVERVIEWThe respiratory system faci l i tates the exchange of oxygen and carbon dioxide between the external envi ronment and the human body. Theairways (trachea and bronchi ), lungs , and a lveol i and pulmonary surfactant provide the biochemica l and biophys ica l bas is for this exchange.Acid–base ba lance in the body a lso rel ies on these ti ssues as wel l as on the carbonic anhydrase and other enzyme systems. Various diseasesof the respiratory system affect ei ther the s tructure or the biochemistry of the lungs ; medications and treatments for these diseases rely onknowing their biochemica l bas is .

BASIC ANATOMY AND DEVELOPMENTThe respiratory system, composed of the nose, nasa l cavi ty, larynx, trachea, bronchiole tubes , and lungs , i s derived from the endoderm,a l though the splanchnic mesoderm contributes to the viscera l pleura , carti lage, and connective ti ssue and a lso to the immediately associatedsmooth musculature and capi l laries (Figure 17-1). Other closely associated s tructures include the ribs and diaphragm and the centra lcardiovascular system, which provides pulmonary arteries and veins .

Figure 17-1. Overview of Lung Structure. The bas ic s tructure of lungs (A) includes the trachea and repeti tively branching bronchi/bronchiolesleading to a terminal bronchiole. Pulmonary arteries and veins provide ci rculation to this lung ti ssue. At the terminal bronchiole/ a lveol i (B),the exchange of oxygen and carbon dioxide between a lveol i and the surrounding vasculature i s conducted as discussed later in this chapter.

[Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

The respiratory system faci l i tates the transport of oxygen (O2) to the body’s ti s sues and enables el imination of gaseous waste products ofmetabol i sm. Muscles of the ribs , diaphragm, neck, shoulders , and abdomen generate a negative pressure gradient, which promotes the flowof external a i r into a system of branching, conducting a i rways and periphera l terminal bronchioles .

Exchange via pass ive di ffus ion through the cel l membranes of the type I epi thel ia l cel l s of the alveoli and the pulmonary capi l laries i srespons ible for the uptake and removal of O2 and carbon dioxide (CO2), respectively (Figure 17-2). Pulmonary arteries from the right ventricle ofthe heart del iver deoxygenated, CO2-laden blood through the branching blood vessels , to the capi l laries ; pulmonary veins return the freshlyoxygenated blood with decreased CO2 to the left auricle and ventricle for del ivery to the body’s ti s sues . Fina l ly, pos i tive pressure, a long withthe elastic wal l s of the a lveol i , springing back from ful l expans ion, expels the waste gas , including the CO2. Al though exhalation i s normal ly apass ive process , active expuls ion i s a lso poss ible, uti l i zing the same muscle groups noted above. Defects or injuries to any of the parts of therespiratory system or to those that affect the musculature and/or cardiovascular system can lead to respiratory disease.

Figure 17-2. Structure of the Alveolus. (A) The a lveolus a l lows the exchange of oxygen (O2) and carbon dioxide (CO2) between the a lveolar a i r andcapi l lary. (B) Magni fied view of the s tructure of the a lveolar/ pulmonary capi l lary interface showing the proximity of a lveolar a i r and thecapi l laries . This s tructure a l lows the efficient di ffus ion of O2 and CO2 through the s ingle-cel l layer of type I cel l s to/from erythrocytes in thecapi l laries and the a lveolus . The pulmonary inters ti tium and type II cel l s respons ible for secretion of surfactant, which helps to mainta in thefragi le s tructure of the a lveol i , are a lso shown. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology,1st edi tion, McGraw-Hi l l , 2009.]

The pass ive di ffus ion of O2 and CO2 rel ies on the close proximity, cel lular membranes , and interwoven connective ti ssues (col lagen andelastin fibers ; see Chapter 13) that a l low the a lveolar–capi l lary gas exchange. The overa l l large surface area of the approximately 300 mi l l ionalveol i in the average human body provides roughly 75 m2 of gas exchange area. The capi l lary network that surrounds these a lveol i coversabout 70% of this area. Gas exchange between the a lveol i and the pulmonary capi l lary i s driven by the higher “partia l pressure” in the a lveol iversus in the pulmonary capi l laries . This pressure gradient propels the smal l O2 and CO2 through the basement membrane and the l ipidbi layer of the a lveolar type I squamous epi thel ia l cel l (~0.2 μm thin) and the capi l laries one layer of endothel ia l cel l plasma membranes .

PULMONARY SURFACTANT AND THE DEVELOPING LUNG

Pulmonary surfactant, composed of lecithin and myelin, i s secreted on a continuous bas is by type II a lveolar cel l s and Clara cel l s beginning atapproximately week 20 of gestation. Pulmonary surfactant has both a l ipid (~90% of tota l ) and a protein (~10% of tota l ) component. About ha l fof the l ipids are dipalmitoylphosphatidylcholine (Chapter 4), the 16-carbon saturated l ipid with a charged amine group on i ts head group. Theremaining l ipids include phosphatidylglycerol, which modulates the fluidi ty of the surfactant as wel l as cholesterol and other l ipids . About ha l fof the proteins are apoproteins, which have both hydrophobic and hydrophi l i c portions ; the other ha l f i s composed of proteins normal ly found

in blood plasma. These l ipids and proteins a l l have the capabi l i ty of interacting with aqueous (hydrophi l i c) or nonaqueous (hydrophobic)envi ronments and i t i s this qual i ty that leads to thei r specia l i zed function in the lung (Figure 17-2). By di rectly interacting with a lveolar watervia thei r hydrophi l i c regions whi le the hydrophobic regions remain in the a i r, pulmonary surfactant creates a myel in mesh-work that l ines thealveol i—a s trong, intertwined l ipoprotein system that i s ana logous to the myel in sheath of nerve cel l s (Chapter 19).

This unique a lveolar l ining greatly reduces surface tens ion, a l lowing eas ier expans ion/stretching (known as pulmonary compliance) andcol lapse of a lveol i during respiration and the resul ting changes in pressure. This reduction in surface tens ion decreases the work ofrespiration and the tota l amount of pressure that must be generated for efficient and effective inspi ration and expiration. The pulmonarysurfactant a lso helps a l l the lung a lveol i to expand (inspi ration) and shrink (expiration) at the same rate, thereby reducing the chance forisolated overexpans ion and the tota l col lapse (atelectas is ) of the a lveolar sacs .

Pulmonary Surfactant and Premature Birth: The respiratory system develops only partia l ly during pregnancy and becomes ful ly functional onlyafter bi rth. One medica l consequence i s seen in premature infants in whom pulmonary surfactant has only s tarted to form. Speci fica l ly,premature babies born before 32 weeks of gestation must be treated aggress ively before bi rth to help augment surfactant or i t may resul tin Infant Respiratory Distress Syndrome (IRDS). This condition, a lso known as Hyal ine Membrane Disease because of i ts findings onpathologica l examination, resul ts in respi ratory dis tress of the newborn infant a long with accessory muscle use, grunting, nostri l flaring,and cyanos is and, i f not appropriately treated, cessation of breathing and death. Multiple compl ications can a lso resul t from IRDS, makingi t the leading cause of death of infants up to 1 month old in the developed world. The most success ful treatment i s frequent s teroidinjections to the mother, which prompts surfactant production whi le delaying bi rth for as long as poss ible.

O2–CO2 EXCHANGE IN THE LUNG AND ACID–BASE BALANCE

A major function of blood and red blood cel l s i s the transport of O2 to cel l s and CO2 from cel l s from/to the lung, respectively. As a resul t,diseases involving this system wi l l often resul t in poor oxygenation (hypoxia) and/or increased CO2 levels (hypercapnia) of the blood and

tissues . Approximately 60% of CO2 i s carried back to the lungs dissolved in the blood plasma as bicarbonate molecules (HCO3–). The binding of

O2 and exchange with CO2 i s discussed in deta i l in Chapter 14.The impact of O2–CO2 exchange extends beyond s imple oxygenation of ti s sues and el imination of waste CO2. Levels of CO2, as regulated by

the lung, kidneys (Chapter 18), blood (Chapter 14), and even the pancreas (Chapter 11), are respons ible for mainta ining proper and acceptablelevels of hydrogen ions (H+) and, therefore, the body’s acid–base ba lance (Figure 17-3). Ranges of pH outs ide the normal body with a range ofless than 7.35 (acidosis) or more than 7.45 (alkalosis), i f uncompensated, can quickly lead to death. This ba lance i s cri ti ca l in the centra l nervoussystem where ti ssues and cel l s are even more sens i tive to pH variations .

Figure 17-3. Regulation of Acid–Base Balance by the Lung. El imination of excess carbon dioxide (CO2) can occur by increased respiratory rate

(hyperpnea), which removes bicarbonate (HCO3–) and hydrogen ions (H+) from other ti s sues (e.g., red blood cel l s ). See the text for further

deta i l s . [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Carbonic anhydrase (Figure 17-4), found in severa l ti s sues including the lungs , kidneys , gastrointestina l tract, sa l iva , bra in, and red bloodcel l s , i s essentia l for the rapid convers ion of CO2 to HCO3

– (then carried in the plasma) and for the s lower reverse reaction of HCO3– to H2CO3

(carbonic acid) to CO2 (Figure 17-3) in the lungs or kidneys . About 75% of the carbon in the body i s in the form of H2CO3 or HCO3–. In the absence

of carbonic anhydrase, an equi l ibrium of the components of the CO2 to HCO3– interconvers ion (Figure 17-4) i s es tabl i shed. Carbonic anhydrase

al lows the body to bypass the carbonic acid s tep. In the lungs , carbonic anhydrase a lso faci l i tates HCO3– to CO2 via H2CO3.

Figure 17-4. Interconversion of Carbon Dioxide (CO2) to Bicarbonate. CO2 i s revers ibly converted to bicarbonate ion (HCO3–) via the action of the

enzyme carbonic anhydrase. Carbonic acid (H2CO3) i s a bypassed intermediate in both reaction di rections . Water molecule (H2O), essentia l to

the process , and hydrogen ions (H+) are a lso shown.

Deficiencies in the management of CO2/bicarbonate in the lungs can lead to the medica l conditions of respiratory acidosis and respiratoryalkalosis. Both have acute and chronic forms. Acute respiratory acidos is occurs when there i s a short-term decrease in respiratory function(hypoventi lation), leading to a decrease in the amount of CO2 exhaled, and because CO2 i s a lways being produced by the body, an increase in

HCO3– per the reaction below (Figure 17-4). Chronic respi ratory problems such as those caused by chronic obstructive pulmonary disease

(COPD), inters ti tia l lung diseases (see below), mechanica l abnormal i ties (e.g., diaphragm dysfunction, deformities of the chest), respi ratorymuscle fatigue (e.g., chronic lung disease, extended asthma attack), or other disorders with longer term hypoventi lation, increase CO2 in the

blood, and resul t in higher concentrations of H+ (acidos is ). Al ternatively, acute or chronic increases in exhalation of CO2 wi th lowering of H+

concentrations (a lka los is ) can resul t from lung (pneumonia and fever with increased rate of breathing), psychiatric (s tress and anxiety),and/or neurologica l (s troke, meningi ti s ) diseases , pregnancy, l iver disease, overuse of aspi rin and/or caffeine, recent increases in a l ti tude,and even overly aggress ive mechanica l venti lation of patients . Various mechanisms attempt to compensate for any respiratory acidos is ora lka los is . Cel l s are able to ei ther take up or release bicarbonate as part of a buffering system that can respond on a time sca le of minutes .Red blood cel l s have a speci fic membrane transport protein for the movement of bicarbonate ions (Chapter 14). The kidneys are a lso able toincrease or decrease bicarbonate excretion or reabsorption in the proximal tubules over a period of a few days (Chapter 18). Acid–basebalance i s discussed in more deta i l in Chapters 14 and 18.

NONINFECTIVE DISEASES OF THE RESPIRATORY SYSTEMNoninfective diseases of the respiratory system are grouped into obstructive and restrictive, a l though patients can suffer from one or both.Obstructive lung diseases, as the name impl ies , include medica l conditions where a i rflow is impaired by some manner. Class ic obstructivediseases of the respiratory system include emphysema, chronic bronchitis, bronchiectasis, and asthma. Because emphysema usual ly has aninflammatory component, chronic bronchi ti s i s often present at the same time. The two can be diagnosed separately or together as COPD.Restrictive lung diseases have loss (i .e., restriction) of complete lung expans ion. This may be because of fibros is or scarring damage to the lungtissue, mechanica l problems such as severe scol ios is , chest wal l deformities , and/or increased abdominal pressure (e.g., hernia , etc.).

OBSTRUCTIVE DISEASES—EMPHYSEMA

Emphysema resul ts from the destruction of the elastin network that provides the phys ica l support for the lung’s bronchioles and acini (clustersof a lveol i ), which di rectly impacts the pulmonary compl iance discussed above. The destruction decreases the surface area that i s cri ti ca l forO2–CO2 exchange (see above) forcing patients to breathe faster (hyperventi lation), to help compensate for lowered O2 levels in the blood.Patients with emphysema a lso have di ffi cul ty exhal ing because of the partia l or tota l col lapse of the a lveol i , a lveolar s tructures , andbronchioles during exhalation. As a resul t, they are sometimes referred to as “pink puffers” because they must exhale s lowly often throughpursed l ips .

Emphysema is divided into panacinar and centroacinar types because of i ts location but, more importantly, because of the biochemica l bas isof the disease. A thi rd type of emphysema, congenital lobar, seen in some newborns , resul ts from the overgrowth of a lung or pulmonary lobeand the resul ting compress ion and bronchia l narrowing seen on other parts of the respiratory system, including the pulmonary vasculature.This type of emphysema wi l l not be discussed any further here.

Panacinar emphysema impacts bronchioles and a lveol i located in the lower lungs and resul ts from a deficiency of the enzyme α-1-antitrypsin.As the name impl ies , this deficiency causes pathology throughout the acinar s tructure to include severa l a lveol i and their associatedbronchiole(s ). The long-s tanding hypothes is of α-1-antri tryps in’s role i s based on i ts relationship with neutrophil elastase known as the “Pro-tease-antiprotease Theory.” Neutrophi l elas tase i s respons ible for the destruction of respi ratory bacteria (e.g., Escherichia coli, Salmonella,Shigella, and Yersinia) by degrading a protein in thei r outer bacteria l membrane. Unfortunately, neutrophi l elas tase a lso destroys collagen IV(Chapter 13) respons ible for the supportive archi tecture of bronchioles and a lveol i . α-1-Anti tryps in i s a di rect inhibi tor of neutrophi l elas taseand, therefore, l imits the damage to the respiratory s tructures . A genetic deficiency of α-1-anti tryps in resul ts in unopposed neutrophi lelastase and, in smokers , emphysema. As a resul t of the genetic cause of this type of emphysema, i t i s suspected in younger patientspresenting with s igni ficant obstructive lung disease. However, α-1-anti tryps in deficiency only accounts for a smal l percentage (~2%) ofemphysema cases especia l ly in older smokers who have s topped smoking for severa l years . A poss ible prominent disease role for otherenzymes (e.g., matrix metalloproteases) in the lung i s currently being explored.

The second major type of emphysema is centroacinar, a ffecting cel l s mainly at the end of the bronchioles (i .e., the center of the acinus) withless impact on the more external a lveolar s tructures . Cigarette smoke, speci fica l ly, the effects of nicotine on neutrophi l s and the neutrophi lelastase enzyme, has been found to be a major cause of centroacinar emphysema in susceptible patients . Nicotine serves as a majorattractant for neutrophi l s via an increased production of nuclear factor-kappa b (NF-kβ), which increases production of tumor necrosis factor (TNF)and interleukin (IL)-8. IL-8 activates secondary s ignal ing pathways by the Gq-protein pathway (phosphol ipase C, diacylglycerol and IP3, proteinkinase C, and ca lcium ion release). IL-8 a lso activates the mitogen-activated protein kinase (MAPK), whose Gα protein pathway leads to increasedprotein tyrosine kinase (PTK) activi ty. PTK phosphorylations promote neutrophi l chemotaxis and cel l adhes ion for the accumulation ofneutrophi l s as wel l as the activation of neutrophi l elas tase, leading to destruction of the lung archi tecture. Nicotine a lso inhibi ts the activi tyof antiproteases (e.g., α-1-anti tryps in) di rectly as wel l as indi rectly by generating free-radica l , reactive oxygen species (e.g., H2O2/ O2

–) addingto the destructive cascade. Thus , the same imbalance between proteases and antiproteases resul ts this time from the toxic effects of nicotineand smoking. Other factors can s igni ficantly impact the development of disease in these patients , including poorly understood contributionsof inflammatory factors (Chapter 15) and genetic variation, which affect disease susceptibi l i ty.

OBSTRUCTIVE DISEASES—BRONCHITIS

Bronchitis, inflammation of the bronchia l mucus membranes , can be divided into acute and chronic forms. Al l share an inflammatory response(Chapter 15), which leads to this disease. Acute bronchi ti s i s usual ly caused by a temporary i rri tant such as an infective agent [vi rus (~90% ofa l l cases ) or bacteria] or a temporary envi ronmenta l i rri tant. Cough with poss ible production of excess ive mucus i s the major symptom thatresolves as soon as the agent i s el iminated. Chronic bronchi ti s , however, i s a long-s tanding disorder that i s uniquely diagnosed not bylaboratory samples or testing but, rather, by the presence of a cough productive of sputum that las ts for three months or longer per year for atleast 2 years . Symptoms include shortness of breath and/or wheezing, especia l ly a fter exertion, because of obstruction of a i r flow and a i rexchange from a i rway inflammation, edema, and mucus hypersecretion. Chronic bronchi ti s can a lso lead to spasms of the bronchi , whichfurther reduce lung function. Cigarette smoking and/or other envi ronmenta l or occupation i rri tants are the predominant cause of this medica lcondition. Unl ike emphysema, destruction of lung parenchyma is not present. However, emphysema and chronic bronchi ti s frequently co-exis t.

OBSTRUCTIVE DISEASES—ASTHMA

Asthma, a chronic inflammatory condition of the bronchi , has ga ined increas ing notoriety because of i ts growing preva lence in a l l societies ,both developed and undeveloped. Asthma leads to an intermittent variable and partly revers ible constriction of the bronchi smooth musclesknown as bronchospasm and a i rway obstruction with breathing di ffi cul ties , including the characteris tic as thmatic wheeze of an asthmaexacerbation. In very severe cases , obstruction becomes so severe and a i rflow so l imited that wheezes become inaudible. Pathologica l growthand enlargement of smooth muscles and mucus-producing cel l s as wel l as pro-inflammatory cytokines, and elevated immunoglobulin (Ig) E (seebelow and Chapter 15) are a lso ha l lmarks of as thma. Chronic, poorly control led, or severe asthma can lead to remodel ing of the lung,including depos i tion of fibrotic ti s sue that leads to i rrevers ible constriction of the bronchi . Severe asthma attacks cause thousands of deathsin a year. Unl ike COPD and bronchi ti s , the inflammation i s largely revers ible and affects only the bronchi and not the a lveol i as in

emphysema.A genetic component to asthma has been noted for severa l years with over 100 genes having been associated with varying degrees and with

varying populations to asthma. However, an envi ronmenta l component i s a l so essentia l for onset and progress ion of the disease. Theseenvironmenta l factors can include cigarette smoking (di rect and pass ive smoke), a i r pol lution, grass/mold/plants (especia l ly whensporulating), pet dander, cockroach droppings , cold temperatures , various vi ra l infections (e.g., chi ldhood respiratory vi ra l infections such asRSV), s tress , phys ica l exertion, decreased exposure to di rt, and/or overuse of antibiotics in chi ldhood, medicines including aspi rin andacetaminophen, and a myriad of other variables . The presence of other immunologica l diseases , including eczema, a l lergic rhini ti s (hayfever), and a l lergies , i s a l so associated with the preva lence of as thma. Repeti tive and long-term exposure not only to chemica ls but a lso tosubstances such as flour can a lso lead to an occupational form of asthma because of chronic inflammation and resul ting bronchospasms.

Regardless of the precipi tating factor(s ), as thma is associated with an inflammatory disease resul ting in the overproduction of IgE, whichmay block β2-receptors on bronchi smooth muscle cel l s . This “β-adrenergic theory of as thma,” fi rs t proposed in 1968, has led to an improvedunderstanding of the biochemica l mechanism behind asthma and a l lergies in genera l as wel l as the development of speci fic medicines tocounteract these effects . This theory proposes the fol lowing pathway to a hypersens i tivi ty of the cel l s l ining the bronchi and asthmaticdisease.

Genetic and other variables make cel l s in the bronchi “hypersens i tive” to particular envi ronmenta l triggers . Envi ronmenta l triggers areingested by antigen-presenting cells (e.g., macrophages , B-cel l s , and dendri tic cel l s ), which interact with immature, helper T cel l s (TH0) as part ofa normal antigen-mediated immune response (Chapter 15). Normal ly, no inflammatory response would resul t. However, in patients with apredi lection to asthma, genetic or otherwise, a B-cel l /helper T cel l (Th2) response i s el i ci ted with the production of antibodies to the trigger(Chapter 15). Repeated exposure to this trigger resul ts in further activation of the immune system via a type I hypersensitivity response (Chapter15). This type I response produces IgE from plasma cel l s . The IgE interacts with mast cel l s and basophi l s caus ing them to secrete a variety ofmolecules , noted with their associated functions in Table 17-1.

Table 17-1. Asthmatic Type I Hypersens i tivi ty Response

The sum resul t of the various molecules i s di lation of blood vessels in the lung and smooth muscle contraction. In addition, chronic a i rwayinflammation with excess mucus production (because of increased prol i feration and growth of mucus producing cel l s in the bronchi ) leads toremodel ing of the bronchi , including thickening of the wal l s and a resul tant a i rway narrowing. Fina l ly, parasympathetic nerves (Chapter 19),which inner-vate the bronchia l wal l s , are themselves s timulated by the triggers , leading to release of acetylchol ine. Acetylcholine binds to i tsreceptor and activates Gq-proteins , which activate phosphol ipase-C-producing diacylglycerol (activates protein kinase C) and IP3, which leadsto ca lcium release (activates protein kinase C and ca lmodul in-dependent kinase). The resul t i s an additional s igna l for bronchia l smoothmuscle contraction and bronchoconstriction. Al l effects exacerbate breathing problems, including bronchospasm and asthma exacerbations ,and, in a vicious ci rcle, increase and perpetuate the inflammatory response unless broken by appropriate treatment.

BIOCHEMICAL BASIS OF ASTHMA MEDICATIONS

The increased understanding of the biochemica l processes that lead to the inflammation of as thma and the bronchospasm of acute asthmaexacerbations has led to a number of medications des igned to speci fica l ly counteract these mechanisms. They include the fol lowing: Short-Acting β2-Agonists [Albuterol (Salbutamol), Leval-buterol, and Terbutaline]: Counteract the inhibi tory effect on the β2-adrenergic receptor

resul ting in relaxation of bronchia l smooth muscle and di lation of bronchia l passages (Figure 17-5). Effect las ts for 4–6 hrs .

Figure 17-5. Actions of Bronchodilators. A mainstay of as thma treatment i s via bronchodi lators , which include short- and long-acting β-agonis tsthat activate adenyl cyclase (AC) and by muscarinic antagonis ts that block acetylchol ine-enhanced bronchoconstriction. See figures and text forfurther deta i l s . ATP, adenos ine triphosphate; cAMP, cycl ic adenos ine monophosphate. [Adapted with permiss ion from Katzung BG, et a l .: Bas icand Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009. Long-Acting β2-Agonists (Salmeterol, Formoterol, and Bambuterol): Same mechanism (Figure 17-5) as short-acting, only effect las ts for

approximately 12 hrs . Anticholinergics/Muscarinic Antagonists (Ipratropium Bromide): Blocks the action of acetylchol ine (Figure 17-5), leading to relaxation of bronchia l

smooth muscle and di lation of bronchia l passages . These medications are more frequently used for COPD patients . Leukotriene Antagonists (Montelukast, Zafirlukast, and Zileuton): Montelukast and Zafi rlukast block the binding of leukotrienes to thei r bronchia l

cel l receptors . Zi leuton inhibi ts the enzyme 5-l ipoxygenase and, therefore, the synthes is of leukotrienes B4, C4, D4, and E4 (Chapter 15). Al lleukotriene antagonis ts resul t in the blockage of leukotriene-associated bronchoconstriction and any activation of inflammatory pathways(Figure 17-6, Chapter 15).

Figure 17-6. Actions of Leukotriene Antagonists and Corticosteroids. Leukotriene antagonis ts block l ipoxygenase (LOX) or leukotriene receptors ,thereby l imiting thei r inflammatory effects . Corticosteroids promote the inhibi tion of phosphol ipase A2 and, therefore, the arachidonic acidpathway. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009. Corticosteroids (Various Oral and Inhaled Forms): Bind to the glucocorticoid receptor found in the nucleus leading to homodimerization and

binding to speci fic DNA sequences , which increases the express ion of anti -inflammatory proteins . Some of these proteins block theexpress ion of pro-inflammatory molecules in the cytosol . Corticosteroids have emerged as an essentia l component for the treatment of anypatient with mi ld, intermittent asthma or worse.

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009. Monoclonal Antibodies (Mepolizumab and Omalizumab): Mepol izumab blocks the activi ty of IL-5 (eos inophi l i c activation) and omal izumab blocks

the activi ty of IgE.

RESTRICTIVE DISEASES—ACUTE RESPIRATORY DISEASE SYNDROME

Acute respiratory distress syndrome (ARDS), a l though fi rs t described in 1967, ga ined much notoriety in recent years because of the high rate offata l i ty (over 30%). The disease can resul t from a variety of insul ts , including trauma, aspi ration of acidic gastric contents , pancreati ti s ,pneumonia , drug overdose, and/or seps is , and i s characterized by inflammation of the lung ti ssue. Cl inica l ly, i t i s defined as impairedoxygenation with bi latera l pulmonary infi l trates on chest X-ray in the absence of left heart fa i lure. Phys iologica l ly, i t i s associated withdecreased pulmonary compl iance and increased a i rway pressures . Just as with the other obstructive diseases , ARDS i s associated with severeimpairment of O2 and CO2 di ffus ion.

The biochemica l bas is of the disease a lso shares the ha l lmarks of an ini tia l release of cytokines leading to increased vascular permeabilityand inflammation wi th neutrophi l s , monocytes , and macrophages present. The inflammation resul ts in damage to the a lveol i , includingswel l ing, which increases the space between a lveolar membrane and the surrounding capi l laries , worsening O2–CO2 exchange and caus ingrespiratory acidos is , shortness of breath, increased breathing rate, severe decrease in oxygenation, and often respiratory fa i lure requiringmechanica l venti latory support. However, the mechanica l breathing ass is tance, i tsel f, may a lso contribute to the continuing inflammatoryresponse and must, therefore, be used with care, often at low levels of pressure and tida l volume uti l i zing pos i tive end-expiratory pressure tohelp reinflate and keep open affected a lveol i . Further inflammation leads to depos i tion of fibrous (hyaline membrane) materia l . A notedreduction in pulmonary surfactant production, due to dys function of the type II a lveolar cel l s , may resul t in the complete col lapse of a lveol iand may a lso impact pulmonary compl iance and work of breathing. Corticosteroids at high doses are often used to help l imit inflammation.The use of arti fi cia l pulmonary surfactants i s a l so sometimes cons idered. Unfortunately, nothing has been shown to clearly lower morta l i tyother than lung-protective venti lation and supportive care.

RESTRICTIVE DISEASES—OCCUPATIONAL EXPOSURES

A number of restrictive lung diseases resul t from the continued exposure to i rri tants , often as a resul t of an occupation or other constantenvironmenta l condition. The genera l term pneumoconiosis i s used for any restrictive lung disease caused by the chronic exposure andinhalation of particles smal l enough to bypass the defense mechanism of the upper a i rway. Examples of severa l pneumoconios is diseasesand their causative agent(s ) are l i s ted in Table 17-2.

Table 17-2. Examples of Pneumoconios is Diseases

In a l l cases , the occupational exposure to the agents in the dust leads to a chronic inflammatory process often resul ting from thestimulation of lung fibroblasts to depos i t fibrous connective ti ssue (Chapter 13) leading to permanent scarring. This scarring dis torts theairways and thickens the wal l s of the smal ler bronchioles and a lveol i , leading to restrictive disease and reducing pulmonary compl iance andlung function. As a resul t, O2–CO2 exchange can be markedly reduced.

RESTRICTIVE DISEASES—INTERSTITIAL LUNG DISEASES

A subset of restrictive lung diseases affects the inters ti tium (the ti ssue and areas around the a lveol i , including the a lveolar and capi l larywal l s and the involved connective ti ssue, blood vessels , and lymphatic ti s sue) and are known as interstitial lung diseases (ILDs). ILD includes awide variety of conditions , such as desquamative inters ti tia l pneumonia , idiopathic pulmonary fibros is , lymphoid inters ti tia l pneumonia ,nonspeci fic inters ti tia l pneumonia , and respiratory bronchiol i ti s -associated inters ti tia l lung disease (Table 17-3) and involves aninflammatory response that often leads to fibros is and scarring of the lung inters ti tium. Hypersens i tivi ty pneumoniti s (not l i s ted in Table 17-3), which can appear l ike lung ti ssue fibros is when advanced, can a lso be included as an ILD. The damage to the lung ti ssue reduces O2–CO2

exchange, which can lead to hypoxia and the need for supplementa l O2. Treatment, including corticosteroids , i s usual ly a imed at decreas ingthe inflammation.

Table 17-3. Examples of Inters ti tia l Lung Diseases

Interstitial Lung Disease Secondary to Medical Treatments: Certa in ILDs resul t from particular medica l treatments as a known but unwanted s ideeffect. These treatments include radiation and chemotherapy (e.g., bleomycin), resul ting from ei ther constant radiation exposure of the lungsor the toxic effects of cancer medications . Other medications , including amiodarone and methotrexate, are known offenders . The mechanismof ILD by these treatments i s poorly understood, a l though the roles of IL-18 (produced by macrophages with s timulation of natura l ki l lercel l s and T lymphocytes ) and IL-1β (produced by macrophages and involved in severa l aspects of activation of an inflammatory response)have been impl icated in some.

Cystic Fibrosis (CF): Al though hundreds of variations of CF are known, the prototypica l type i s caused by an autosomal recess ive mutation,caus ing the deletion of a cri ti ca l phenyla lanine at amino acid 508 of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR i s amembrane-bound, adenos ine triphosphate-driven, chloride ion transport channel essentia l in the production of lung mucus as wel l assweat and pancreatic digestive solution. The mutation leads to a nonfunctional transporter, which resul ts in a thickened mucus in the lungwhich further blocks narrow bronchioles leading to chronic inflammation and remodel ing of the lung ti ssue and, often, chronic s inus i ti s .Pancreatic secretions are a lso affected, leading to accumulation of digestive enzymes in the pancreas and intestina l blockages by thickfeces . This same effect can a lso lead to inferti l i ty, especia l ly of men, due to thickened ejaculatory fluid. The lack of chloride ion transporta lso inhibi ts the production of NaCl in sweat, leading to a common, noninvas ive test of tasting a baby’s sweat i f CF i s suspected. Breathingproblems and constant lung infections usual ly lead to bronchiectas is and obstructive defects . Li fe expectancy i s dependent on theparticular mutation and can range from the late teens to early 20s up to the late 30s or early 40s .

INFECTIVE DISEASES OF THE RESPIRATORY SYSTEM

Infections of the respiratory system are caused by a wide variety of bacteria and vi ruses and, as such, wi l l not be ful ly covered here. However,certa in infections uti l i ze biochemica l mechanisms that offer ins ight into thei r cl inica l effects as wel l as treatments .

Bordetella pertussis (the cause of whooping cough) produces and releases an enzymatica l ly active protein toxin respons ible for the i l lness .Released in i ts inactive form, pertuss is toxin binds to receptors on a cel l membrane and i s transported via the Golgi apparatus to theendoplasmic reticulum. Upon activation, the toxin adds adenos ine diphosphate (ADP) molecules to the α-subunits of G proteins, blocking theirnormal binding to G-protein-associated receptors (Chapter 8). Gi proteins are among those affected and, being inhibi ted by the ADPribosylation, are unable to s top adenyl cyclase production of cycl ic adenos ine monophosphate (cAMP). The resul ting a l tered s ignal ing leadsto the cl inica l mani festations of whooping cough.

Pertuss is toxin’s inhibi tion of Gi protein activi ty a lso affects other organ systems. Macrophage phagocytic functions are dis rupted, restrictingtheir abi l i ty for bacteria l ki l l ing. In patients with pertuss is , B cel l s and T cel l s that leave the lymphatics show an inabi l i ty to return. Theseeffects may expla in the high frequency of secondary infections that accompany pertuss is . Pertuss is toxin a lso inhibi ts his tamine activation ofphosphol ipase C and adenylyl cyclase affecting gastric, cardiac, smooth muscle cel l s , and immune cel l s . Fina l ly, the block of cAMP productionin pancreatic β-cel l s by pertuss is toxin coupled with the actions of epinephrine can lead to overproduction of insul in and hypoglycemia.

Aspergi l los is fungal infections represent a spectrum of diseases ranging from benign colonization of the a i rways to invas ive infections ,especia l ly in patients with chronic lung diseases or immune compromise. Infection by Aspergillus ini tiates both type I and type IIhypersens i tivi ty responses leading to IgE and IgG production, respectively. Further immune reactions ini tiate type II I hypersens i tivi tyresponses with depos i tion of immune complexes in the lung membranes , activation of helper T (Th2) cel l s , which recrui t IL-4 and IL-5, andrecrui tment of eos inophi l s and neutrophi l s , the latter by IL-8 secretion. This immune response resul ts in bronchoconstriction and increasedvascular permeabi l i ty via mechanisms s imi lar to asthma (see Table 17-1). Continued or repeated infections resul t in the release of enzymesby immune cel l s , which degrade and scar the lung ti ssue resul ting in fibros is and the breakdown of the lung archi tecture.

Tuberculos is , the disease cause by severa l Mycobacterium species , depends on activation of macrophages , T and B lymphocytes , andfibroblasts . Enci rclement and i solation by lymphocytes and interferon-γ secretion to activate phagocytic macrophages and cytotoxic T cel l s leadto the formation of a granuloma—a compact col lection of immune cel l s (e.g., macrophages and his tiocytes ) and necrotic cel l s , which i solatesthe bacteria l infection. Granulomas are a lso seen in the infective diseases of leprosy, his toplasmos is , cryptococcos is , coccidiomycos is ,blastomycos is , and cat scratch disease (Bartonella infection) as wel l as in severa l noninfectious conditions such as sarcoidos is , Crohn’sdisease, Wegner’s granulomatos is , Churg–Strauss syndrome, and rheumatoid arthri ti s .

REVIEW QUESTIONS1. What are the mechanisms of inhalation and exhalation?2. What i s the function of a lveol i?3. What i s the role of pulmonary surfactant?4. What i s compl iance?5. What are the obstructive and restrictive lung diseases and their associated consequences?6. What are the defects/mechanisms, and consequences associated with the fol lowing diseases affecting lungs : emphysema, bronchi ti s ,

as thma, acute respiratory dis tress syndrome, pneumoconios is , and inters ti tia l lung diseases?7. What are the symptoms of lung infections associated with pertuss is , aspergi l los is , and tuberculos is?8. What are the mechanisms of action of as thma medications and their appl ication(s ) to other diseases?9. What i s the role of oxygen/carbon dioxide exchange and the carbonic anhydrase reaction in acid–base ba lance associated with the lung?

CHAPTER 18THE URINARY SYSTEM

Editor: Armando J. Lorenzo, MD, MSc, FRCSC, FAAPStaff Pediatric Urologis t, The Hospi ta l for Sick Chi ldren,

Univers i ty of Toronto, Toronto, Ontario, Canada

Bas ic Anatomy and Phys iologyRenal Fi l trationRenin–Angiotens in–Aldosterone System (RAAS)Acid–Base Ba lanceSynthetic FunctionsReview Questions

OVERVIEWThe urinary system, composed of kidneys , ureters , bladder, and urethra , i s respons ible for the excretion of waste products in urine andregulation of important ions and molecules in the body. The biochemica l components of the basa l lamina and varying cel l types found in therenal corpuscle create the fi l tering mechanism that ini tiates this process . Ionic channels and pumps found in the nephron a l low the fina lcompos i tion of the urine to be modi fied according to the needs of the body. This i s regulated by various hormones that help regulate sodium,potass ium, chloride, ca lcium, magnes ium, ammonia/acid–base ba lance, tota l body water, blood pressure, and other molecules and ions . Therenin–angiotens in–aldosterone hormonal system as wel l as vasopress in and atria l naturetic peptide are keys in this process . Fina l ly, thekidneys are essentia l for red blood cel l s production via erythropoietin synthes is and a lso add an essentia l biochemica l s tep in the activationof vi tamin D.

BASIC ANATOMY AND PHYSIOLOGYThe urinary system, embryologica l ly derived a long with the reproductive system from the intermediate mesoderm, cons is ts of the kidneys andassociated blood vessels , ureters , bladder, and urethra . The kidney (Figure 18-1) conta ins hundreds of thousands to mi l l ions of nephrons andassociated tubules and col lecting ducts where blood i s fi l tered by the renal corpuscles for waste products ; fluid ba lance (and, secondari ly,blood pressure) i s adjusted; electrolytes , glucose, amino acids , and other molecules are ba lanced to appropriate levels ; acid–base levels aremainta ined; and the process of urine production i s completed. Additional ly, the enzyme renin, the hormone erythropoietin, and the fina lactive form of vi tamin D are produced by the kidney.

Figure 18-1. Basic Anatomy of the Kidney. The kidney conta ins the renal artery and vein, which transport blood to and from the pelvis and into aca lyx and medul la where fi l tration occurs . Urine produced by this process exi ts via the ureters . [Reproduced with permiss ion from Kibble JDand Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

RENAL FILTRATION

THE RENAL CORPUSCLE

The s tructure of the renal corpuscle (Figure 18-2A) a l lows the kidneys to perform i ts ini tia l fi l tering function. The overa l l s tructure i s composedof a glomerulus, which receives blood via a smal l col lection of capi l laries bringing blood to (afferent) and away from (efferent) the renalcorpuscle, and the surrounding Bowman’s capsule a long with the nephron. The glomerulus forms the fi rs t part of the fi l tration apparatus withfenestrations (“openings” from the Latin for window) formed between the cel l membranes of i ts endothel ia l cel l s . The fenestrations arerelatively large, though, and only fi l ter materia l the same s ize or smal ler than blood cel l s (Figure 18-2A–B). Next i s a three-layered s tructureca l led the basal lamina (a l so ca l led the basement membrane), which i s approximately s ix times thicker than other basement membranesfound in the body (Figure 18-2C). The biochemica l components of the basa l lamina form an efficient and highly selective, s ieve-l ike fi l ter.Negatively charged glycosaminoglycans, including predominately heparin (Chapter 2), comprise two of the layers , the lamina rara interna and thelamina rare externa, creating a selective, electrostatic barrier to other negatively charged molecules . The middle layer, the lamina densa, i scomposed of type IV collagen, di ffering from other col lagen types because of i ts lack of regular glycine res idues (Chapter 1). This a l lows i t toform flat, kinked sheets . Also found in the lamina densa i s laminin, a glycoprotein (Chapter 2), which forms multiple l inks to other molecules

because of i ts unique cross -shape. The kinked, fi lamentous type IV collagen molecules , l inked by laminin, form a network that fi l ters a l lmolecules with a molecular weight less than 66,000 Da.

Type IV Collagen and Basal Lamina Disorders: Al though a multi tude of kidney diseases exis t, many affect various consti tuents of the renalcorpuscle. Two such diseases are Goodpasture’s and Alport’s syndromes, both of which are bel ieved to involve the type IV collagen, whichmakes up the basal lamina.

In Goodpasture’s syndrome, a l so known as antiglomerular basement membrane disease, the immune system erroneous ly attacks the basa llamina of the glomerul i and a lveol i in the lung, leading to inflammation of the glomerul i (rapidly progressive glomerular nephritis) wi th acutekidney fa i lure (sometimes with increased protein in the urine), ri se in blood pressure, edema as wel l as cough, breathlessness because ofbleeding within the lung, and the requirement for supplementa l oxygen. Smoking and prior lung damage increase symptom severi ty andprogress ion. The molecule that appears to be attacked in both anatomica l locations i s type IV col lagen. The ini tiating cause of the sel f-attack i s unknown, a l though exposure to certa in chemica l solvents , herbicides , and/or vi ra l infections has been impl icated.

In Alport’s syndrome, genetic mutations in type IV col lagen lead to inflammatory destruction of the glomerul i (glomerulonephritis) wi thblood in the urine and progress ive kidney fa i lure as wel l as decreas ing hearing (progress ive destruction of the basa l lamina in the innerear affecting ha i r cel l s at other s tructures ) and vis ion [lens dis tortion (anterior lenticonus), cataracts, and rarely lens rupture as wel l as “flecks”around the macula , which cause retina l damage and progress ive loss of s ight]. Severa l di fferent mutations inheri ted in X-l inked, autosomalrecess ive, or autosomal dominant manners can cause Alport’s syndrome.

Abnormal i ties in col lagen and the kidney matrix are a lso seen in the autosomal dominant form of polycystic kidney disease (PCKD) inwhich multiple cysts form in the kidneys , leading to eventual damage of the ti ssue and renal insufficiency/fa i lure. Patients with PCKD a lsoexhibi t s imi lar col lagen-related abnormal i ties in the bra in, blood vessels , heart va lves , and abdominal/pelvic wal l s , i .e., hernias .

The next part of the fi l ter, surrounding the glomerulus , i s Bowman’s capsule with an outer layer of epi thel ia l cel l s but a lso a layer ofspecia l i zed epi thel ia l cel l s ca l led podocytes. These podocytes have “foot processes ,” which wrap around and fuse with the glomerulus andinterdigi tate with nearby podocytes (Figure 18-2B). These foot processes are a lso l ined by two specia l proteins ca l led podocin and nephrin anda negatively charged glycoprotein ca l led glycocalyx, which help to create and s tabi l i ze s tructures ca l led slit diaphragms. Defects in podocin areknown as one cause of minimal change disease, a condition associated with loss of the podocyte foot processes with resul ting loss of proteininto the urine (proteinuria) and swel l ing of the body (edema). The s l i t diaphragms, l ike the fenestrated endothel ia l cel l s , l imit passage ofnegatively charged and/or medium-s ized molecules such as serum a lbumin. Mesangial cells, modi fied smooth muscle cel l s (Chapter 12) in theglomerulus (Figure 18-2A), modulate the flow of the blood from the afferent capi l laries and, in addition, produce protein and proteoglycancomponents of the extracel lular matrix of the renal corpuscle, fatty acid-derived prostaglandins (Chapter 3), and protein/glycoproteincytokines . Mesangia l cel l s a l so phagocytica l ly remove molecules trapped by the fi l tration barrier—a molecular fi l ter cleaner. Fina l ly,juxtaglomerular (JAG) cells, a second type of specia l i zed smooth muscle cel l s of the renal corpuscle that synthes ize, s tore, and secrete theenzyme renin, important in the control of blood pressure (Figure 18-2A and see text below). The unique features of a l l these componentscreate a meshwork of cel l s , proteins , glycoproteins , and other biochemica l molecules , which selectively a l low the passage of water, ions , andsmal l molecules (e.g., creatinine and urea) but prevent the passage of large and/or negatively charged molecules .

Figure 18-2. A–C. Renal Corpuscle. (A) Cel lular components of the renal corpuscle are shown. Blood enters the corpuscle via the afferent capi l lary(black arrow) and i s fi l tered through the glomerulus (blue arrows). Resul ting fi l trate proceeds into the proximal tubule to be el iminated asurine. Blood returns to the body via the efferent capi l lary. Additional components of the renal corpuscle include mesangia l cel l s (not shown)and juxtaglomerular cel l s . The associated dis ta l tubule i s shown. (B) Expanded view shows deta i l of how blood i s fi l tered through thefenestrated endothel ia l cel l layer ins ide the capi l lary lumen and through the glomerular basement membrane and podocyte foot processeslayers . (C) The glomerular basement membrane i s composed of three layers , including a lamina rara interna, lamina densa, and lamina rara

externa (not shown, see text). The afferent capi l lary (not shown) del ivers blood to the endothel ia l layer, with resul ting fi l trate emergingthrough to Bowman’s space and subsequent col lection by the proximal tubule. The speci fic role of each layer and i ts components i s describedin the text. [Adapted with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

Because of the many components that form the complex fi l tering mechanism of the glomerulus , many disorders exis t, which affect thestructure and, therefore, the function of the glomerulus . The dys function of the s tructura l and functional components of the glomerularmeshwork noted above resul ts in the spi l lage of proteins (e.g., a lbumin) and red blood cel l s into the urine, termed nephritic syndrome, or largeamounts (>3.5 g/ day) of protein, termed nephrotic syndrome. Both are cl inica l indicators of a renal problem. Glomerular disorder diseases arenormal ly divided into prol i ferative—involving the increased growth or number of glomerular cel l s or matrix materia l—and nonprol i ferative—no increased cel lulari ty or matrix. These varying diseases and the biochemica l effect on glomerular s tructure are l i s ted in Table 18-1.

Table 18-1. Summary of Glomerular Disorders

The fina l components of the urinary system, the ureters , bladder, and urethra , are l ined by a trans i tional epi thel ium with severa l layers ofcel l s that a l low the bladder to s tretch whi le s ti l l mainta ining bladder wal l integri ty. A thick and elastic lamina propria layer, composed of amesh network of col lagen, elastic fibers , and proteoglycans (Chapters 1, 2, and 13), holds and attaches the epi thel ia l cel l layers together toprovide additional flexibi l i ty and s trength to the fluid-proof but s tretchable s tructure. The two to three smooth muscle layers beneath theepithel ium increase movement of urine from the kidneys to the bladder and, ul timately, for excretion. When urination occurs , an internalurethral sphincter, composed of smooth muscle (Chapter 12), i s control led by the autonomic nervous system (Chapter 19), whereas an externalsphincter, composed of skeleta l muscle and innervated by the pudendal nerve, offers voluntary control .

Urinary Tract Infections (UTIs) and Cranberry Juice: Bacteria l UTIs of the bladder (cystitis) or kidney (pyelonephritis) a ffect mi l l ions of patients .Al though antibiotics are often needed for treatment, the use of cranberry juice may a lso help. Cranberry juice, speci fica l ly, conta ins theplant flavenoid molecule A type proanthocynidin, which di rectly inhibi ts attachment of p-type fimbriae of Escherichia coli, the cause of up to 90%of UTIs , to the urinary tract wal l . As a resul t of the use of cranberry juice, bacteria are swept away by urinary flow, accelerating recovery fromor even preventing a UTI.

Diuretics and the Nephron: Diuretics are any substance, including medications , which increase the loss of water via excretion in urine. Diuretic

medications are fundamenta l to many treatments of high blood pressure and heart fa i lure, and work via speci fic effects on the channelsand pumps of the nephron.

Loop diuretics, named so because their action affects the loop of Henle, include furosemide, bumetanide, torasemide, and ethacrynic acid(see figure). These medications actively compete (Chapter 5) for the Cl –-binding s i te, blocking Na+–K+–2Cl– transport and reducing the Na + andK+ ion concentration in the renal medul la . The lower ion concentration and, therefore, osmotic pressure gradient decreases the amount ofwater reabsorbed from the urine in the col lecting duct, lowers the tota l fluid volume of the blood plasma, and, consequently, lowers bloodpressure. Magnes ium ion (Mg2+) and ca lcium ion (Ca 2+) reabsorption i s a lso decreased, adding to this effect. Because of this mechanism, aposs ible s ide effect of loop diuretics i s low K+ (hypokalemia), low Mg2+ (hypomagnesemia), low Ca 2+ (hypocalcemia), and low Cl –

(hypochloremia). Because of the effect on ca lcium excretion, loop diuretics are a lso used in cl inica l s i tuations associated with elevatedserum levels of ca lcium (hyperca lcemia). Nevertheless , elevated levels of urinary ca lcium may lead to formation of kidney s tones , aphenomenon sometimes seen in premature babies who are exposed to high doses early in l i fe. Nonsteroidal anti-inflammatory moleculesblock the action of loop diuretics by decreas ing glomerular fi l tration of the blood and passage of the diuretics to the loop of Henle.

Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.Thiazide diuretics, including hydrochlorothiazide, chlorothiazide, metolazone, indapamide, and the parent molecule benzothiadiazine,

work by blocking the Na+–Cl– cotrans-porter in the distal tubule. Blockage of the membrane channel in this part of the nephron decreases thereabsorption of these two ions into the inters ti tium and the blood s tream, decreas ing water influx, tota l blood volume, and blood pressure.The resul ting increased Na + concentration and water content a lso activate an a ldosterone-regulated (see below) K+ secretion channel inthe col lecting ducts , leading to loss of K+. As a resul t, thiazide diuretics can lead to cl inica l ly s igni ficant low K+ levels in the blood. Thiazidediuretics a lso decrease calcium excretion and, therefore, levels in the urine, lowering the chance of ca lcium-derived kidney s tone formationand other diseases involving high levels of serum ca lcium. The effects of thiazide diuretics are mimicked by the diseases of Bartter andGitelman syndromes wherein rare genetic defects lead to dys function of the above-mentioned cotransporters in the ascending part of theloop of Henle or the dis ta l convoluted tubule, respectively.

NEPHRON

The production of urine by the kidneys involves a complex, wel l -regulated series of membrane-bound protein pumps and channels , whichselectively develop gradients of ions and molecules to determine the fina l content of urine. The body has mechanisms to sense the makeupof the fi l trate via osmotic measurement (e.g., macula densa) and hormonal response, as i t progresses from the renal corpuscle to the ureters .

Fi l tering of the blood by the renal corpuscle removes waste products , but this process i s not perfect by any means . The complex task ofreturning particular molecules to the ci rculatory system whi le progress ively concentrating the resul ting fluid that wi l l become urine i s the jobof severa l protein channels (active and pass ive) and pumps (Chapter 8) found in the membranes of the nephron (Figure 18-3).

Figure 18-3. Overview of Nephron and Urine Production. I l lus tration of components of the nephron and summary of functions found in eachsegment, including proximal convoluted tubule, proximal s tra ight tubule, thin descending l imb, loop of Henle, thin and thick ascending l imbs ,dis ta l convoluted tubule, and col lecting duct. Molecules leaving (reabsorption) and entering (secretion) as wel l as medications and/orhormones affecting each segment (numbered) are indicated. See text for further deta i l s . ADH, antidiuretic hormone; PTH, parathyroid hormone.[Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

The nephron i s the tubular s tructure, which continues from the renal corpuscle, leading to the col lecting system and subsequently ureters ,bladder, and urethra . It i s surrounded by inters ti tia l ti s sue (the connective ti ssue of the kidney found outs ide the tubule and surrounding thenephrons), which conta ins “peri tubular” capi l laries . Water and selected molecules are transferred across the nephron members by thesechannels and pumps and returned to the body via these capi l laries , a process ca l led reabsorption (the ini tia l absorption i s cons idered to havetaken place in the intestines ). Water and molecules moving from the inters ti tium into the nephron for el imination in urine undergo a processtermed secretion. The tubule system beyond the glomerulus i s divided functional ly into four dis tinct sections (Figure 18-3): (1) the proximalconvoluted tubule, (2) the loop of Henle (descending and ascending parts ), (3) the distal convoluted tubule (proximal and dis ta l parts ), and (4) thecollecting duct. Each section i s characterized by speci fic channels/ pumps and osmotic conditions required for i ts particular function in urineproduction. A summary of the actions on certa in molecules by the various segments of the nephron i s given in Figure 18-3 and Table 18-2.

Table 18-2. Summary of Nephron Actions on Various Molecules

The col lective functions of a l l the segments of the nephron, in tota l , a l low the kidney to not only regulate water but a lso the conservationor el imination of electrolytes ; the proper maintenance of acid–base ba lance; the excretion of waste products ; and the preservation ofimportant proteins , carbohydrates , and even medications .

INULIN/CREATININE CLEARANCE

The measurement of the glomerular filtration rate (GFR) (volume of fluid fi l tered from the afferent arteriole into the renal corpuscle per uni ttime) a l lows cl inicians to determine the heal th of the kidneys and to quanti fy any degree of kidney or renal fa i lure. GFR i s most accuratelymeasured by injection of inulin into the bloodstream, a polysaccharide molecule from plants , which i s nei ther reabsorbed nor secreted by thekidney. A more practica l method of estimating GFR, though, i s to measure blood creatinine, a molecule derived in muscle from the breakdownof creatine phosphate, a rapidly ava i lable source of adenos ine triphosphate (ATP) energy for muscle and bra in. Creatinine i s exclus ively excretedby the kidneys , completely fi l tered at the renal corpuscle, and only smal l amounts are secreted into the peri tubular capi l laries . The natura loccurrence of s teady levels of creatinine in the blood and urine offers an easy way to establ i sh GFR and, therefore, kidney function. Varyingmathematica l formulas a l low for correction of known variations depending on the patient’s muscle mass , age, gender, race, and/or s i ze. Thenormal range of GFR i s (100–130) ml/min/1.73 m2 but this varies in chi ldren and older adults . A GFR of over 60 ml/min/1.73 m2 i s usual lysufficient for normal heal th, a l though high blood pressure, diabetes , and other chronic diseases can decrease kidney function and resul t inchronic kidney disease (CKD). CKD is enumerated in s ix s tages , depending on the patient’s GFR, as noted below (Table 18-3).

Table 18-3. Stages of Chronic Kidney Disease and GFR

RENIN–ANGIOTENSIN–ALDOSTERONE SYSTEM (RAAS)The RAAS i s a complex, regulatory pathway involving the kidney, l iver, lung, adrenal glands , pi tui tary gland, and selected arterioles found inthe kidney. It includes precursor and activated peptides as s igna ls among these various organs , the enzymes that activate the peptides , andregulation by two major hormones of the renal and cardiovascular systems (Figure 18-4). The JAG apparatus i s located between the afferentarteriole and the renal corpuscle and dis ta l convoluted tubule of the same nephron. This specia l i zed col lection of cel l s i s able to sense andregulate blood flow volume into and out of the renal corpuscle/glomerulus by the production of the s ignal ing peptide hormone/enzyme reninand by two, s imple biochemica l mechanisms of the macula densa, involving Na + and the gaseous molecule nitric oxide (NO).

Figure 18-4. Renin–Angiotensin–Aldosterone System. Overview of effects of renin, angiotens in I I and a ldosterone on organs control l ing volumestatus and blood pressure. aa , amino acid. See text for further deta i l s . [Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review ofMedica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

RENIN AND BLOOD PRESSURE

Renin i s produced by specia l i zed cel l s (granular cel l s of the JAG apparatus) in response to low blood pressure sensed in the JAG and bydecreased NaCl levels detected by macula densa cel l s (see below). Blood pressure in the JAG is detected by specia l i zed nerve cel l s , which areactivated (Chapter 19) by changes in the arteriole blood pressure, leading to a loca l i zed nerve s ignal and secretion of the hormone renin. Aswi l l be discussed further below, sympathetic nerve activi ty (Chapter 19) a lso leads to increased renin secretion. Secreted renin enzymatica l lyconverts the precursor protein angiotens inogen, produced in the l iver, into angiotens in I , which, when converted to angiotens in I I (see below),leads to increased blood pressure and, therefore, the pressure of perfus ion in the renal corpuscles .

Renin Inhibitors: Recently developed inhibi tors of renin have introduced a novel class of medications to help treat high blood pressure. Twosuch medications , aliskiren and remikiren, bind di rectly to renin and competi tively inhibi t binding of angiotensinogen and, therefore,production of angiotensin I. The efficacy and safety of these new drugs i s s ti l l being determined but may offer a novel method of patient care.

MACULA DENSA AND BLOOD FLOW/OSMOLARITY

With high blood flow through the arterioles of the renal corpuscle, increased Na + are fi l tered into the glomerulus . Cel l s found in a part of theJAG, termed the macula densa, conta in a Na+–Cl– cotransporter channel, which transports Na + into these cel l s . A l imited number of Na+–K+-ATPasepumps a ttempt to transport the Na + back out of the cel l but, i f Na + levels are high (i .e., osmolari ty i s high), they are soon overwhelmed. The

internal Na + concentration increases and creates an osmotic gradient, which brings water molecules into the macula densa cel l s . These cel l s ,swol len with Na + and water, conta in a “stretch-activated” channel, which a l lows ATP to escape fol lowed by convers ion to s imple adenos ine.These adenos ine molecules are bound by membrane-bound proteinaceous receptors on the adjacent afferent and efferent arterioles of theglomerulus , leading to constriction of the afferent arteriole and di lation of the efferent arteriole. The resul t i s decreased blood flow throughand fi l tration by the renal corpuscle. As a resul t, less Na + get reabsorbed and the loca l concentration decreases .

Al ternatively, low flow through the arterioles leads to a lower Na + concentration in the macula densa cel l s . The lower Na + concentration(i .e., low osmolari ty) activates the enzyme nitric oxide synthetase (NOS) in these cel l s . NOS cata lyzes the reaction, which converts the amino acidarginine and an oxygen molecule to ci trul l ine and NO, uti l i zing reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. NOthen activates speci fic Gs proteins , which produce cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). cGMPactivates a protein kinase G, which, in turn, leads to inactivation of myos in molecules via phosphorylation of thei r regulatory l ight cha in.Inactivated myos in molecules in the blood vessel wal l s leads to vessel relaxation and di lation. cAMP leads to the increased release of renin.

Erectile Dysfunction (ED), NO, cGMP, and Phosphodiesterase Inhibitors: Understanding the role of NO and cGMP in the relaxation of arteriole wal lsmooth muscle has led to the development of phosphodiesterase inhibitor medications , which have revolutionized the treatment of ED. As inthe kidney, NO-mediated production of cGMP causes di lation of arteries in the corpus cavernosum, increas ing blood flow and leading toerection. Various factors including age, chronic diseases such as high blood pressure and diabetes , as wel l as s ide effects of certa in drugsdecrease the blood flow and lead to ED. Speci fic phosphodiesterase inhibi tors [e.g., s i ldenafi l (Viagra), vardenafi l (Levi tra), and tadalafi l(Cia l i s )], which have s tructures mimicking the cGMP molecule, competi tively block the enzyme cGMP-specific phosphodiesterase type 5, whichbreaks down cGMP (see figure below). By inhibi ting this enzyme, the cGMP s ignal remains , promoting vasodi lation and leading to betterand susta ined erection. The mechanism of cGMP relaxation of arteriole wal l s and the actions of the phosphodiesterase inhibi tors are a lsoused for certa in types of pulmonary hypertension and pulmonary edema due to altitude sickness, in which pulmonary artery di lation lowerspressure in the lungs reducing disease damage to lung ti ssue.

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

Both constant monitoring of blood pressure (renin) and blood flow/osmolari ty (macula densa) through the arterioles and, therefore, therenal corpuscles and nephrons , provide feedback to portions of the nephron (to a l ter electrolyte and water reabsorption and secretion) and

the rest of the body. The remainder of the RAAS i s respons ible for these actions .

ANGIOTENSINOGEN/ANGIOTENSIN I AND II

Angiotensinogen i s a 452-amino-acid-long protein, which i s produced on a constant bas is by the l iver (Figure 18-5). Angiotensin I, compris ing thefi rs t 10 amino acids of angiotens inogen, i s produced by cleavage of a leucine–val ine bond by renin. The 10-amino-acid angiotens in I i scompletely inactive but i s subsequently converted to the eight-amino-acid peptide hormone, angiotensin II. This reaction i s cata lyzed byangiotensin-converting enzyme (ACE), an enzyme found throughout the body but especia l ly concentrated in the lung’s capi l lary beds . Angiotens inII has severa l activi ties throughout the body, including on the kidney, adrenal glands , heart and blood vessels , sympathetic nervous system,and hypothalamus. Al l of these effects are propagated via Gq or Gi protein-coupled angiotens in receptors , which activate phospholipase C,

leading to phosphorylation via protein kinase C (PKC) and Ca 2+ release (Chapter 8). Both PKC and an influx of ca lcium can lead to myos in l ight-chain phosphorylation and, therefore, contracti le activi ty. Activation of the Gi-coupled receptor a lso inhibi ts adenyl cyclase and, therefore, theproduction of cAMP, which inhibi ts smooth muscle relaxation (which a lso promotes contracti le activi ty). Fina l ly angiotens in I I activates certa intyrosine kinases, leading to other effects in the body. These effects are i l lus trated in Figure 18-4 and l i s ted in Table 18-4.

Table 18-4. Angiotens in I I Effects on the Body

Figure 18-5. Conversion of Angiotensinogen. Angiotens inogen i s converted to angiotens in I by the action of renin from the kidney by the removal ofmore than 400 amino acids by proteolytic cleavage (s i te indicated by arrow). Angiotens in I i s subsequently converted to angiotens in I I by theaction of angiotens in-converting enzyme (ACE), found mainly in the lung, by the removal of an additional two amino acids (cleavage s i teindicated by arrow). The molecule i s further processed as shown, a l though the majori ty of functions in the body are via angiotens in I I assummarized in Table 18-4. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

The sum effect of angiotens in I I i s the increase in body blood pressure with muting of many of the body’s compensating mechanisms.Angiotens in I I can a lso be converted to a seven-amino-acid angiotens in I I I wi th decreased effect on blood pressure but a s imi lar effect onaldosterone secretion and a s ix-amino-acid angiotens in IV, which has even less activi ty.

ACE Inhibitors: The marked effect of angiotens in I I on blood pressure, including increased pressure and, therefore, potentia l vessel damagein the kidney, led to the development of medications ca l led ACE inhibitors. This drug class , a l l of which end in the suffix “-pri l ,” speci fica l lyblock the enzyme, which converts inactive angiotens in I into i ts active angiotens in I I counterpart. The effectiveness in lowering bloodpressure and providing protection to the kidney aga inst damage has led to wide use of ACE Inhibi tors in high blood pressure, diabetes ,kidney disease with protein loss in the urine (proteinuria), a fter heart attack, congestive heart fa i lure, and kidney diseases . Of note, ACEInhibi tors may a lso affect the kinin–kallikrein system by increas ing the production of bradykinin, a nine-amino-acid peptide involved in pa in,inflammation, and blood pressure control . Bradykinin may be respons ible for some of the ACE inhibi tors ’ adverse effects (such as coughand the potentia l ly fata l s ide effect of angioedema). These s ide effects can be avoided by use of the related angiotensin receptor blockerclass of medications , whose names a l l end in the suffix “-sartan,” which competi tively block the effects of angiotens in I I at i ts G-proteinreceptors .

Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.

ALDOSTERONE

Aldosterone, the fina l player of the RAAS, i s the minera locorticoid s teroid hormone (Chapter 3), which acts to increase Na + and waterreabsorption and, as a resul t, K+ secretion. The overa l l effect i s an increase in blood volume and, therefore, blood pressure. Aldosteroneproduction/excretion i s mainly related to the concentration of K+ in the serum, probably measured by carotid artery sensors . Sympathetic nerveactivi ty can a lso influence a ldosterone production but on a much lesser sca le. Aldosterone’s activi ty i s predominately seen in the dis ta lconvoluted tubule and col lecting duct.

Aldosterone and Disease—Conn Syndrome and Addison’s Disease: Increased amounts of a ldosterone lead to primary aldosteronism (Connsyndrome), whereas decreased levels lead to adrenal insufficiency (Addison’s disease).

Conn syndrome resul ts from overproduction of aldosterone, usual ly caused by uncontrol led growth (hyperplas ia) of the adrenal gland(s ) oran a ldosterone-secreting tumor. The excess ive a ldosterone leads to an increase in sodium and water reabsorption with increasedsecretion/ excretion of potass ium that a lso produces excess hydrogen ion (H+) secretion (metabol ic a lka los is ). The sum effect i s high bloodpressure, which can only be treated by removal of the hyperplastic adrenal gland or tumor and/or use of the potass ium-sparing diureticsspi ronolac-tone or eplerenone (see below).

Addison’s disease, a l ternatively, resul ts from underproduction of a ldosterone from ei ther genetic or disease/ trauma causes or frominhibi tion of a ldosterone production by the adrenal glands . Effects on the nephron lead to increased loss of sodium and water and

increased potass ium and H+ (metabol ic acidos is ) levels in the blood. This leads to low blood pressure, which may be severe enough tocause patients to fa int, especia l ly when s tanding up. Severe symptoms may be seen during an “Addisonian crisis” in which loss ofconsciousness and death may resul t.

Aldosterone speci fica l ly increases the reabsorption of Na + and water out of and the secretion of K+ back into the tubule by binding tospeci fic membrane-bound receptors and increas ing the membrane permeabi l i ty of cel l s l ining the tubule. In addition, a ldosterone increasesthe number of and s timulates the ATP-dependent phosphorylation of Na+–K+ pumps, leading to a conformational change of the pump, whichexposes Na + to the intersti tium and decreases Na + binding. As a resul t, increased Na + are reabsorbed a long with Cl – (to mainta in electrica lneutra l i ty) and water (to mainta in osmotic ba lance). The resul t i s an increase in Na + and water in the intersti tium, leaving them to be takenup by intersti tia l capi l laries back to the blood s tream. As a resul t of the inactivated Na +–K+ pumps but secondary to secretion via increasedmembrane permeabi l i ty, K+ accumulate in the tubules and, subsequently, in the urine. Fa l l ing K+ concentrations , subsequently, decreasealdosterone secretion. Aldosterone secretion i s addi tional ly a ffected by conditions in which blood volume and/or electrolyte concentrationsare changed such as s igni ficant blood loss , pregnancy, burns , shock, and prolonged phys ica l exercise.

Pseudohyperaldosteronism and Licorice: Pseudohyperaldosteronism, in which a ldosterone levels are normal , can a lso occur as a resul t of diet.Excess ive l i corice in the diet, conta ining the sweetener molecule glycyrrhizin, inhibi ts 11-β-hydroxysteroid dehydrogenase, which reduces theconvers ion of excess cortisol to inactive cortisone in a ldosterone-selective epi thel ia l cel l s . Inhibi tion of this enzyme a l lows the extra corti solto produce a ldosterone-l ike effects , including high Na + and blood pressure as wel l as low K+ levels in the blood. Tissue res is tance to theeffects of a ldosterone (poss ibly because of decreased action on the receptors ) and/or Liddle’s syndrome (a genetic defect leading tooveractivi ty of reabsorbing sodium channels ) can a lso lead to pseudohypera ldosteronism. Both can be treated us ing potass ium-sparingdiuretics (see below).

Potassium-Sparing Diuretics and Aldosterone Regulation: Spi ronolactone and eplerenone are members of a group of diuretics known as“potassium-sparing.” These two medications , used for high blood pressure and heart fa i lure, mimic the s teroid s tructure of aldosterone (seefigure below) and function as a competi tive inhibi tor for a ldosterone receptors in the col lecting duct. Thus , the effects of a ldosterone onmembrane permeabi l i ty and number/activi ty of Na +–K+ pumps are not seen, decreas ing reabsorption of Na + and water but decreas ing (i .e.,sparing) the secretion and loss of K+.

Aldosterone a lso influences pH by s timulating the secretion of H+. Fina l ly, a ldosterone influences reabsorption of water in the kidneys and,therefore, blood pressure, by increas ing the release of the peptide hormone vasopress in (a lso known as antidiuretic hormone, ADH) from thepitui tary gland. Vasopress in’s actions wi l l be discussed in greater deta i l below.

VASOPRESSIN

Although not formal ly part of the RAAS, vasopressin, a l so known as ADH, i s intimately involved in s imi lar process and, in fact, completes theprocess of determining the fina l water content of urine and, therefore, fluid ba lance in the body (Table 18-4). Vasopress in, a peptide hormoneproduced as a prohormone in the hypothalamus, i s essentia l for enhanced reabsorption of water by increas ing the presence of kidney-speci fic, membrane-bound water channels ca l led aquaporin-2. Vasopress in ra ises the number of these channels by enhancing the geneexpress ion for the protein and increas ing i ts del ivery to the appropriate plasma membrane. These functions occur via speci fic vasopress inreceptors coupled to a Gs protein (Chapter 8) found on cel l s of the dis ta l convoluted tubule and col lecting ducts . Binding of vasopress in to thereceptor activates an adenyl cyclase, which converts ATP to cAMP. The resul ting s ignal ing pathway activates protein kinase A, whichphosphorylates aquaporin-2 protein, speci fica l ly on serine 262 whi le in the Golgi apparatus , promoting del ivery of the ves icles to andmembrane fus ion with col lecting duct membranes .

Vasopressin, Aquaporin-2, and Water Balance: The proper ba lance of water and, therefore, plasma volume is essentia l for the properfunctioning of the body and di rectly influences blood pressure and the heart. Aquaporin-2 plays a major role in the regulation of this fluidstatus and a l terations or mutations of this protein lead quickly to disease s tates . Nephrogenic (kidney-associated) diabetes insipidus, acondition of poor fluid ba lance regulation and notable for large output of very di lute urine, i s closely associated with mutations of theaquaporin-2 channel . Changes in the express ion of the aquaporin-2 gene are l inked to the unwanted water retention seen in congestiveheart fa i lure, l i thium toxici ty, and during pregnancy. Neurogenic (brain-associated) or “central” diabetes insipidus occurs when vasopressin i s notsynthes ized because of hypothalamus or pi tui tary gland fa i lure. Common causes include head trauma, tumors , or unknown reasons . Fina l ly,ethanol intake inhibi ts the action of vasopress in and, therefore, aquaporin-2, leading to decreased water reabsorption and associatedthirs t/mi ld dehydration.

Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.

Therefore, the cooperative actions of a ldosterone, which mainly influences electrolytes , and vasopress in (ADH), which influences water,control the fina l water content of the urine, including how di lute i t i s . In fact, in times of extreme dehydration, the col lecting duct a lone canreabsorb approximately one quarter of the water in the fi l trate.

ATRIAL NATRIURETIC PEPTIDE (ANP)

ANP (Figure 18-6) i s another important peptide hormone that controls body water as wel l as Na + and K+ levels . ANP i s released in times of bodyfluid overload and/or high blood pressure from muscle cel l s of the heart. ANP binds to speci fic receptors , which activate a guanyl cyclase tomake cGMP. The effects of ANP on the kidney include the fol lowing:

Figure 18-6. Natriuretic Peptides. Atria l natriuretic peptide (ANP) and bra in natriuretic peptide (BNP) amino acid sequences and s tructures arei l lus trated. See text for further information. [Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11thedition, McGraw-Hi l l , 2009.]

Increased glomerular fi l tration by• di lation of the afferent arteriole,• constriction of the efferent arteriole,• relaxation of the mesangia l cel l s .

Increased reabsorption of NaCl by• increased blood flow through the vasa recta ,• increased osmotic drive for further water and NaCl excretion. cGMP-driven phosphorylation/deactivation of sodium channels (located on the dis ta l convoluted tubule and col lecting duct). Inhibi tion of renin and a ldosterone production.

The sum effect of ANP’s actions i s increased excretion of water, sodium, and potass ium by the kidneys to help compensate for excess fluidand high blood pressure. Because of these qual i ties , ANP and brain natriuretic peptide (Figure 18-6), which, despi te the latter’s name are bothsecreted by heart cel l s , have shown uti l i ty in fol lowing the progress ion and resolution of congestive heart fa i lure.

ACID–BASE BALANCEThe maintenance of the proper acid–base balance between pH 7.35 and 7.45 i s essentia l to mainta in the human body. Imbalances , includingacidos is and a lka los is , not only adversely affect multiple protein and enzymatic functions but, as a resul t, can a lso lead rapidly to death.Various mechanisms found in the lungs , kidneys , and blood s tream constantly adjust this equi l ibrium as per the needs of the body. Otherbuffers , including ammonia (NH3) (see below), proteins , and phosphate a lso contribute to this important functional and protective

mechanism by combining with H+ to remove them from solution.The kidney plays an essentia l role in regulating the body’s acid–base ba lance mechanism and rel ies on the selective and regulated

reabsorption and secretion of H+ and bicarbonate ions (HCO3–), two primary molecules of the bicarbonate buffering system, involving the

reaction . This i s done by two types of cel l s , which secrete or reabsorb H+ or HCO3– via

H+-ATPase proton pumps, and H+–K+ and Cl ––HCO3– channels . Acid–base regulation i s a lso found in the col lecting duct.

Increased acidi ty in the body augments reabsorption of bicarbonate from the proximal convoluted tubule and loop of Henle, and col lectingduct cel l s whi le a lso caus ing increased secretion of hydrogen from the col lecting duct. Increased base causes the kidney to decrease H+

secretion and causes less reabsorption/more secretion of bicarbonate from the nephron. Table 18-2 provides speci fics regarding the speci ficmechanisms involved with these two ions .

NH3 AND ACID–BASE BALANCE

Another acid–base ba lance mechanism (Figure 18-7) inherent in the kidney’s functions i s the handl ing of ammonia (NH3) as part of a secondarybuffering system. Al though the primary management of ammonia levels i s via the urea cycle in the l iver (Chapter 11), any necessary el iminationof urea and/or excess , toxic ammonium ions (NH4

+) occurs in the kidney’s nephrons .

Figure 18-7. Overview of Ammonia (NH3) Balance in the Body.

In particular, NH3 can freely cross the membranes of the nephron and, a fter combining with a H+ to form NH4+, be excreted in the urine

(Figure 18-8A) with a net acid loss . Fina l ly, NH3 i s produced by the enzymatic degradation of the amino acid glutamine, converted fi rs t toglutamate and then to α-ketoglutarate, which continues through the Krebs ’ cycle (Figure 18-8B). If a base excess exis ts , the convers ion ofglutamine and, therefore, the production of NH3 i s inhibi ted to help the body re-establ i sh the proper acid–base ba lance.

Figure 18-8. A–B. The Ammonia (NH3) Buffering System. (A) Interconvers ion of NH3 to ammonium (NH4+), which captures one H+. This reaction

occurs freely in the human body, depending on concentrations of each respective molecule. (B) Convers ion of glutamine to glutamate, via theenzyme glutaminase, with production of one NH3 molecule (upper) and glutamate to α-ketoglutarate via the enzyme glutamatedehydrogenase with production of a second NH3 and NADH (lower).

Ingested sources of NH3 such as protein and amino acids are processed via the gastrointestina l system and el iminated in feces ortransferred through the porta l ci rculation to the l iver or to the body via systemic ci rculation. In the l iver, NH3 i s converted to urea and returnedto the systemic ci rculation where i t can be el iminated as urine by the kidneys or sent back to the gastrointestina l system. The ba lance of NH3

depends on varying contributions of l iver, kidney, and intestina l metabol i sm and/or el imination. [Reproduced with permiss ion from BarrettKE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Acid–base ba lance can a lso have a marked effect on the efficacy of medications by influencing how they are el iminated from the body.Many medications are excreted via the kidneys but, once in the nephron, they can be ei ther reabsorbed and returned to the ci rculation (Figure18-3) or secreted/ el iminated in the urine. Medications secreted in the proximal tubule are indicated below a long with predominate form(acid, base, or nei ther) at secretion (Table 18-5). The form of a medication can influence secretion, as acidic medications are secreted morewhen the urine i s bas ic and vice versa . In cri ti ca l care and other cl inica l settings , changing urine pH, ei ther as the resul t of an i l lness orselective medica l intervention, can affect the ul timate el imination of acidic and bas ic drugs from the blood s tream. In very speci ficci rcumstances , this abi l i ty can be uti l i zed to a id in patient care.

Table 18-5. Medications Secreted in Proximal Tubule

Hyperammonemia and NH3 Toxicity: High levels of free NH3 in the body cause harm to varying parts of the body. Most prominent are theeffects on the bra in and nervous system, which subsequently lead to other cl inica l mani festations . Menta l retardation, seizures , andunusual and uncontrol led muscle movements as wel l as breathing i rregulari ties and poor control of body temperature are common. Comaand death may resul t without treatment.

There are multiple enzyme deficiencies , most due to autosomal recess ive mutations , which resul t in excess ammonium in the body.These various disease s tates are separated into primary—involving enzymes from the urea cycle (see Figure 5.9a) and secondary—involvingenzymes that are not part of the urea cycle. Examples are l i s ted below.

SYNTHETIC FUNCTIONS

SYNTHESIS OF ERYTHROPOIETIN

Erythropoietin, a l so known as hematopoietin, i s a major glycoprotein cytokine and hormone that regulates the production of red blood cel l s(Figure 18-9). Erythropoietin i s produced by endothel ia l cel l s that are part of the kidney’s peri tubular capi l laries . The amount of erythropoietinproduced i s bel ieved to be regulated by a transcription factor that becomes hydroxylated (OH–) and then gets degraded when oxygen i sabundant. When oxygen levels are low, the transcription factor binds to erythropoietin receptors on red blood cel l membranes and bonemarrow cel l s and activates an associated Janus kinase 2 resul ting in phosphorylation of tyros ine res idues (Chapter 8). Activation byerythropoietin protects the red blood cel l s from normal ly programmed cel l death (apoptos is ) and s timulates the production of new red bloodcel l s from their speci fic precursor cel l s .

Figure 18-9. Regulation of Red Blood Cell Production by Erythropoietin. If the abi l i ty of blood to carry oxygen decreases because of a fa l l in numbersof red blood cel l s (e.g., normal cel l death, pathologica l destruction of red blood cel l s , bleeding, etc.), the kidney senses lower pO2 levels andincreases the levels of erythropoietin (EPO). EPO then s ignals the bone marrow to increase production of red blood cel l s . See text for furtherdeta i l s . [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Renal disease leads to a drop in erythropoietin production and, therefore, a low red blood cel l count or anemia. Arti fi cia l ly producederythropoietin can be used to increase these patients ’ red blood cel l s count as wel l as those suffering from certa in chronic diseases such asheart fa i lure, undergoing chemotherapy or radiation therapy for cancer, and/or cri ti ca l ly i l l and in need of the additional oxygen carryingcapaci ty. Erythropoietin has a lso been shown to be active on bra in cel l s and has been useful in the treatment of schizophrenia and relateddiseases . It has a lso been abused by athletes who uti l i ze this cytokine to increase red blood cel l counts and, therefore, oxygencarrying/performance capaci ties .

ROLE IN VITAMIN D SYNTHESIS

The kidney a lso plays a vi ta l role in the synthes is of active vitamin D and, secondari ly, the regulation of Ca 2+ concentration in the body. VitaminD3 i s ini tia l ly hydroxylated in the l iver to produce 25(OH)D3 and s tored there unti l required (Chapter 3, Figure 3-10). A second hydroxyl group i sadded when parathyroid hormone (PTH) activates 1-α-hydroxylase in the proximal convoluted tubule of the kidney to produce the active form1,25(OH)2D3 or calcitriol. Vi tamin D and the vi tamin D receptor found mainly in the dis ta l convoluted tubule a lso act to increase Ca 2+ and

phosphate ion (PO43–) reabsorption by increas ing the number of these ion channels . PTH a lso increases reabsorption of Ca 2+ and Mg2+ in the

proximal portion of the dis ta l convoluted tubule and the ascending l imb of the loop of Henle in oppos i tion to the action of the hormonecalcitonin (for more deta i l , see Chapter 13).

Kidney Stones: Kidney stones (nephrolithiasis) can form because of a number of di fferent biochemica l means often secondary to a diseasestate. Types of kidney s tones and potentia l causes are l i s ted below:

REVIEW QUESTIONS1. What are the roles of the renal corpuscle, glomerulus , nephron, tubules , and col lecting systems?2. How do reabsorption, secretion, and excretion di ffer?3. What are the functions of renin, angiotens in I and II , angiotens in-converting enzyme, a ldosterone, vasopress in, and atria l natriuretic

peptide and how does each relate to the renin–angiotens in system?4. What i s the function of erythropoietin?5. How do the components of the glomerulus act together to create a molecular fi l tering mechanism?6. What receptors are activated and by what s igna l ing pathways to increase or decrease renal reabsorption or secretion of molecules?

7. What i s the role of the renin–angiotens in system in fluid and electrolyte homeostas is and what i s the impact of abnormal i ties in triggeringsecondary hypertens ion?

8. What i s the role of the kidney in the synthes is of erythropoietin and the active form of vi tamin D?9. What are the bas ic concepts , reactions , and role of the kidney in the renin–angiotens in–aldosterone system?

CHAPTER 19THE NERVOUS SYSTEM

Editor: Kathryn Beck-Yoo, MDDepartment of Anesthes iology, Swedish Medica l Center at

Bal lard, Seattle, Seattle, Washington, USA

Components of the Nervous SystemNerve Impulse ConductionAutonomic Nervous SystemNeurotransmittersBiochemistry of Vis ionAnesthes iaReview Questions

OVERVIEWThe nervous system provides a network of coordinated s ignal ing for bodi ly functions . Somatic nerves promote skeleta l muscle activi ty and,therefore, gross movements . Autonomic nerves provide s ignals for internal organs , including regulation of thei r function. These s ignals aretransmitted by nerves , which rely on membrane-bound protein pumps for conduction of the impulse. Specia l i zed l ipids form myel in sheaths ,which a id in the fast and efficient transmiss ion of these s ignals . Neurotransmitters , often smal l peptides , a l low continuation of the nerves ignal between neurons and from neurons to target ti s sues . The breakdown of this network of s igna l ing and regulation leads to severa lneurologica l diseases .

COMPONENTS OF THE NERVOUS SYSTEMThe nervous system is anatomica l ly and functional ly composed of two main parts : the central nervous system (CNS) and the peripheral nervoussystem (PNS) (Figure 19-1). The CNS cons is ts of the bra in and spina l cord and the PNS i s composed of a l l nerves outs ide of the CNS, including a l lspina l and crania l nerves . In addition, there are further class i fi cations for the nerves of the PNS. Fi rs t, nerves are dis tinguished by thedirection of nerve propagation. Afferent (sensory) neurons conduct action potentia ls toward the CNS. Efferent (motor) neurons transmit impulsesaway from the CNS to effectors such as muscles or glands . Second, nerves of the PNS can be further divided into the somatic (skeletal muscle)and autonomic (organs, glands, and smooth muscle) nervous systems. The autonomic nervous system (ANS) i s divided into the sympathetic (SNS) andparasym-pathetic nervous system (PSNS). Genera l ly, both interact with the same effectors , but often paradoxica l ly. The SNS i s involved in theactivi ties associated with the fight-or-fl ight response, helping the body to cope with s tress . The PSNS promotes activi ties that support the bodywhi le at rest, including digestion.

Figure 19-1. Overview of the Nervous System. Schematic diagram comparing some anatomic and neurotransmitter features of autonomic andsomatic motor nerves . Only the primary transmitter substances are shown; parasympathetic gangl ia are not indicated. See text for moredeta i l s . Ach, acetylchol ine; D, dopamine; Epi , epinephrine; M, muscarinic receptors ; N, nicotinic receptors ; NE, norepinephrine. [Reproduced

with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Neurons are the fundamenta l components of the nervous system. The main components of a neuron include the soma (or cel l body),dendri tes , axon, and the axon terminals (Figure 19-2). The cel l body, or soma, i s the centra l part of the neuron that conta ins the nucleus andother cel l organel les . The dendrites are the branching s tructures of the neuron that receives s timul i or messages via chemoreceptors . The axoni s a s lender, cable-l ike extens ion of a neuron that carries nerve impulses away from the soma toward the axon terminal . The axon terminalsare the ha i r-l ike terminals of the axon where neurotransmitters are released from the synaptic knobs . Neurons communicate with oneanother via synaptic transmiss ion, where the axon terminal of one neuron comes into close contact with the dendri tes of another neuron.Neurons can communicate chemica l ly, via neurotransmitters , or electrica l ly with electrica l ly conductive junctions between the cel l s .

The myelin sheath i s a phosphol ipid membrane that surrounds and protects the axons of some nerves (Figure 19-2). The primaryphosphol ipid of the membrane i s ga lactocerebro-s ide, a sphingol ipid (Chapters 3 and 8). Such phosphol ipids in nerve membranes s trengthenthe sheath. Myel in i s produced by Schwann cel l in the PNS and by ol igodendrocytes in the CNS. The neurofibrillar nodes (a l so known as nodes ofRanvier) are the gaps in the myel in sheath, where the action potentia l occurs during conduction a long the axon.

Figure 19-2. General Structure of a Nerve Cell. The genera l s tructure of a nerve cel l includes the cel l body with a nucleus and nucleolus , multipledendri tes , and an axon. Further deta i l s are provided in the text. The ini tia l segment, immediately after the axon hi l lock, conta ins no myel insheath but does conta in a very high dens i ty of vol tage-gated Na + channels for continued propagation of the nerve impulse. In the myel inatedsection, the impulse i s conducted from node to node (see below) unti l reaching the terminal boutons where neurotransmitter molecules arestored for release and nerve s ignal propagation (see below). [Reproduced with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Textand Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

NERVE IMPULSE CONDUCTIONSuccess ful conduction of nerve impulses rel ies on three elements : an electrica l ly insulated membrane, energy-driven pumps to establ i sh andmainta in ion gradients , and ionic selective channels .

Demyelinating Disorders: Demyel inating disorders refer to any condition that dis rupts myelin in ei ther the CNS or PNS resul ting in aninterruption of normal nerve transmiss ion. The process of demyel ination can be because of an i schemic, metabol ic, genetic, or infectiousinsul t. However, because a demyel inating disorder can fol low an infection or an inoculation, i t has been hypothes ized that an autoimmunemechanism is to blame. In some disorders , remyel i -nation can occur with an associated degree of neura l recovery. However, many times ,neurologic defici ts are permanent. Because myel in i s formed in the CNS by ol igodendrocytes and in the PNS by Schwann cel l s ,demyel inating disorders can target di fferent locations and have a variable presentation as i l lus trated below.

NEURON AT REST

Neurons mainta in a resting membrane potentia l of –70 mV by adenos ine triphosphate (ATP)-mediated active transport and pass ive di ffus ionof ions . The Na+–K+-ATPase pump of the cel l membrane transfers three Na + out of the cel l whi le transferring two K+ intracel lularly (Chapter 8 andFigure 19-3). The s teady-s tate condition resul ts in an extracel lular Na + concentration of 150 mmol/L and an intracel lular Na + concentration of5–10 mmol/L. The extracel lular K+ concentration i s 3–4 mmol/L and intracel lular i s 120–135 mmol/L. This gives ri se to the negative s tate withinthe cel l , and i t creates a concentration gradient that wi l l favor the extracel lular di ffus ion of potass ium and the intracel lular di ffus ion ofsodium upon depolarization.

Figure 19-3. The Na+–K+-ATPase Pump. The pump and associated sodium (Na +)- and potass ium (K+)-gated channels are shown. These gatedchannels a l low the s low restoration of Na + and K+ to thei r respective oppos i te s ide (see Figure 19-4 and associated text). ADP, adenos inediphosphate; ATP, adenos ine triphosphate. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

Figure 19-4. A–B. The Nerve Impulse. (A) The resting membrane i s mainta ined at a membrane potentia l of –70 mV. The onset of an actionpotentia l opens the gated Na + channels (see Figure 19-3 and Figures below upper graph), leading to the efflux of Na +. Once the threshold leveli s reached at –55 mV (dashed red l ine), the depolarization of the membrane caused by Na + i s rapid unti l a maximum membrane potentia l of

+30–35 mV is reached. At this maximum vol tage, the gated Na + channels close and the gated K+ channels open (see Figure 19-3 and Figuresbelow upper graph) and an influx of K+ leads to a drop in the membrane potentia l . The Na +–K+-ATPase pump (Figure 19-3) a lso contributes torestoration of the resting membrane potentia l (not shown). (B) I l lus tration of relative membrane permeabi l i ty of Na + (PNa+) and K+ (PK+) duringpropagation of the nerve impulse. [Reproduced with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion,McGraw-Hi l l , 2010.]

NERVE IMPULSE

Severa l s timul i can activate a neuron leading to the generation of a nerve impulse (Figure 19-4). These include chemica l , mechanica l , orelectrica l exci tation. Impulse propagation i s accompanied by smal l membrane depolarizations . When the depolarization exceeds thethreshold level of -55 mV, activated sodium channels cause a flood of Na + ions into the cel l which drives further depolarization. This causes arelative excess of cations in the cel l , which eventual ly leads to a membrane potentia l of +35 mV. If the ini tia l s timulus i s less than –55 mV,few Na + channels are opened and the threshold level i s never reached. As a resul t, nerve conduction i s an a l l -or-none response.

Niemann–Pick C Disease: Niemann–Pick C Disease i s an autosomal recess ive, l i fe-threatening lysosomal s torage disease, s triking mainly inmid-to-late chi ldhood but a lso in infants and adults . A majori ty of cases involve a mutation on the NPC1 gene on chromosome 18 resul tingin lysosomal cholesterol and glycosphingol ipid accumulation in neurons and gl ia l cel l s in the CNS. The l ipid bui ld-up resul ts in a dis tortionof the neuron mainly by erroneous formation of dendri tes , which subsequently a l ters neurotransmiss ion. The cl inica l presentation i s highlyvariable but usual ly presents with the onset of movement and learning disabi l i ties and early impairment of vertica l eye movement (vertica lsupranuclear gaze pa lsy). In addition, patients can experience loss of muscle tone (dystonia), seizures , disabl ing joint problems, problemsswal lowing (dysphagia), and, in adults , varying cognitive impairment as wel l as psychos is and/or depress ion. Most patients die due tocompl ications of this disease before the age of 20 years .

As the neuron’s action potentia l depolarizes , vol tage-gated ca lcium channels cause an influx of ca lcium ions (Ca 2+). This Ca 2+ influx triggersthe fus ion of s torage ves icles with the terminal bud membrane, releas ing a neurotransmitter into the synaptic cleft (Figure 19-5). Receptors onthe adjoining neuron bind with the neurotransmitter caus ing an a l teration in the receptor’s configuration. These neurotransmitter-gated ionchannels act as an internal pore a l lowing Na + to flow down their electrochemica l gradient into the cel l . As the intracel lular concentration ofNa + ri ses , the charge within the cel l becomes pos i tive caus ing a depolarization of the membrane. The resul t i s a propagating electrica l s igna lknown as an action potential. This process repeats unti l the target ti s sue has been reached.

Figure 19-5. The Synaptic Cleft. After the nerve impulse reaches and depolarizes the presynaptic membrane, the influx of Ca 2+ resul ts in thefus ion of presynaptic ves icles to the surface of the membrane. The neurotransmitter released into the cleft binds to the receptor s i te on thepostsynaptic membrane, which wi l l propagate the action potentia l onto the adjacent neuron. [Adapted with permiss ion from Kibble JD andHalsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Alzheimer’s Disease (AD): AD i s a progress ive and fata l degenerative disease of the bra in and the leading cause of dementia . Alzheimer’s i sdue to the accumulation of amyloid plaques between neurons and neurofibrillary tangles (NFTs) wi thin the neurons . The exact mechanism ofhow the plaques and tangles affect bra in function i s not ful ly understood. Patients vary in both amount and ratio of plaques versus tangles .However, patients who have primari ly only amyloid plaques deteriorate s lower than those with primari ly tangles .

Amyloid plaques cons is t of β-amyloid protein, which i s a protein fragment of amyloid precursor protein (APP). Normal ly, these proteinfragments are degraded and el iminated via one of two pathways . In the case of AD, APP i s cleaved by β-secretase and γ-secretase to formamyloid-β-derived di ffus ible l igands . The accumulation of the amyloid forms hard, dense plaques . These plaques phys ica l ly a l ter thearchi tecture of the surrounding ti ssue, and therefore a l ter normal neura l functioning. In addition, amyloid depos i ts cause mitochondria ldys function leading to premature, scheduled cel l death (apoptos is ).

The second major finding in AD i s NFT. These tangles are made of s ix i soforms of tau proteins, highly soluble microtubule-associatedproteins found normal ly in heal thy bra ins . In a heal thy person, phosphorylation of tau protein destabi l i zes axonal microtubules , a l lowingnormal neuron function. In AD, the process i s a l tered when hyperphosphorylation of tau resul ts in the formation of NFTs . Al l s ix tau i soformsare present in the hyper-phosphorylated s tate. As they are a l tered, they become insoluble aggregates within the neuron leading toinstabi l i ty in the neura l tubes . This impacts normal nerve conduction within the bra in.

Myel in sheaths enable action potentia ls to travel fas ter than in unmyel inated axons and at decreased energy expenditure (Figure 19-6). Theunsheathed nodes of Ranvier conta in a high dens i ty of vol tage-gated ion channels . The action potentia l i s conducted a long the axon via

conduction jumping from one node of Ranvier to the next.

Figure 19-6. Effect of the Myelin Sheath on Nerve Impulse Propagation. Pos i tive charges from the membrane ahead of and behind the actionpotentia l flow in the area of negative charge as the potentia l travels down the axon (see di rection of propagation). Unmyel inated nervespropagate the nerve impulse by s low, continuous conduction. Nerves with a myel in sheath are able to conduct the nerve impulse much quickervia sa l tatory conduction from one node of Ranvier to the next. [Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica lPhys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

REPOLARIZATION

Repolarization i s dependent upon severa l factors . The sodium channels are inactivated and cannot reopen unti l membrane polarization i sreset to the resting membrane potentia l of -70mV. Inactivi ty of the sodium channels causes a consequent drop in sodium permeabi l i ty and anincrease in potass ium conductance. During this time, the membrane i s unable to repeat exci tation (refractory period). Eventual ly, the sodium–potass ium pump re-establ i shes the origina l basel ine gradients and the cel l i s returned to i ts resting potentia l .

AUTONOMIC NERVOUS SYSTEMThe PNS i s divided into the somatic nervous system and ANS. The somatic nervous system includes voluntary movements of skeleta l muscles aswel l as touch sensation, vi s ion, and hearing. The ANS coordinates and mainta ins a s teady s tate among internal organs , such as regulation ofcardiac muscle, smooth muscle, and glands . It i s cons idered to be an involuntary system. The ANS i s further subdivided into the sympatheticand parasym-pathetic nervous systems, the activi ty of which i s integrated by the hypothalamus. An autonomic nerve pathway from the CNS to aviscera l effector cons is ts of two major neurons that synapse in a gangl ion outs ide of the CNS: the preganglionic and postganglionic neurons . Thisdi ffers from the somatic nervous system, wherein a s ingle motor neuron travels from the CNS to the innervated ti ssue.

SYMPATHETIC NERVOUS SYSTEM

This system is dominant in a s tress s tate or the “fight-or-fl ight” response. It serves to mobi l i ze energy s tores in times of need. Activation ofthe SNS resul ts in tachycardia (increased heart rate), di lation of large blood vessels in skeleta l muscles , vasoconstriction of skin and viscera lblood vessels , di lation of bronchioles , mobi l i zation of fatty acids from triglycerides in adipose ti ssue, and mobi l i zation of l iver glycogen toglucose. The latter changes are des igned to supply energy to the body and to provide muscle ti s sue with di rect energy via anaerobic glycolys is .In addition, digestive secretions and intestina l peris ta ls i s decrease. The SNS’s pregangl ionic neurons originate in the thoracolumbar spina lcord (from T1 to L2). The sympathetic gangl ia are located in two chains just outs ide of the spina l column, in the paravertebra l gangl ia . Thepregangl ionic, unl ike the postgangl ionic, neurons are myel inated.

PARASYMPATHETIC NERVOUS SYSTEM

The role of the parasympathetic nervous system is to conserve and restore energy or, in oppos i tion to the sympathetic system, to “rest anddigest.” It i s the primary functioning divis ion in relaxed s i tuations to promote the normal functioning of severa l organ systems. The resul t inthe body i s decreased heart rate (bradycardia), bronchiolar constriction, pupi l constriction, and increased peris ta ls i s and secretions . Thepregangl ionic neurons originate in severa l of the crania l nerve nuclei (CN 3, 7, 9, and 10) and sacra l segments (S2, S3, and S4) of the spina lcord, giving ri se to the term craniosacra l divis ion. In contrast to the SNS, the gangl ia are located close to the target organs , and the post-gangl ionic neurons are relatively short. The effects of both the sympathetic and parasympathetic systems on the body are summarized inTable 19-1.

Table 19-1. Summary of the Autonomic Nervous System

Guillain–Barré Syndrome (GBS, Acute Idiopathic Polyneuropathy): GBS i s an acute inflammatory disease leading to demyel ination affecting thePNS. It i s the most commonly acquired inflammatory neuropathy. GBS i s a medica l emergency, but a majori ty of patients ful ly recover over aperiod of months . As many as one-thi rd of patients wi l l exhibi t res idual weakness 3 years after the event, necess i tating retra ining, orthoticappl iances , and rehabi l i tations . Fata l i ties occur in more than 2% of a l l patients .

GBS i s typica l ly characterized by a progress ive symmetric muscle weakness , loss of sensation, para lys is , and decreased reflexes(hyporeflexia) s tarting in the lower extremities and progress ing rapidly to the arms and face. As the symptoms progress , the respiratorymuscles can a lso be affected necess i tating mechanica l venti lation. In addition, the ANS can be dis rupted resul ting in severe tachycardia ,arrhythmias , and hypo- or hypertens ion. The diagnos is of GBS i s a cl inica l one.

Al though the precise mechanism of GBS i s unknown, there i s probably an immunologic bas is to the disorder resul ting in damage to themyel in sheath of the PNS. GBS sometimes fol lows an infective i l lness , inoculation, or surgica l procedure. The most common antecedentinfection i s the bacteria Campylobacter jejuni. Other known pathogens include enteric vi ruses , cytomegalovi rus , Epstein–Barr vi rus , andMycomplasma. Lymphocytic infi l tration and macrophage-mediated demyel ination of periphera l nerves i s the underlying pathology, andsymptoms genera l ly resolve with remyel ination. Symptoms usual ly appear 1–3 weeks after the antecedent event.

NEUROTRANSMITTERSNeurotransmitters are molecules (often amino acids/smal l peptides and/or thei r derivatives ) which carry and often ampl i fy a s igna l from onenerve to another nerve or to another cel l type. Class ic examples of neurotransmitters include glutamine, γ-aminobutyric acid (GABA),acetylchol ine (Ach), dopamine, norepinephrine (NE), and serotonin among many others . Neurotransmitters can affect thei r target nerve or cel lei ther via exci tation or inhibi tion as i s summarized in Table 19-2. Description of individual neurotransmitters and their actions fol low.

Table 19-2. Summary of Neurotransmitters

DOPAMINE

Dopamine i s a monoamine and i s synthes ized from the amino acid tyros ine in a series of enzymatic reactions that fi rs t produces L-DOPA, thendopamine, then NE, and fina l ly epinephrine (Figure 19-7A). Dopamine i s ei ther exci tatory or inhibi tory depending upon which of i ts receptorsubtypes i s activated. In the bra in, dopamine functions in the role of behavior, cogni tion, voluntary movement, s leep, mood, and learning. Theaction of dopamine i s mostly in the substantia nigra , arcuate nucleus of the hypothalamus, and the ventra l tegmenta l area of the midbra in.Dopamine i s a lso very important in the reward system. Dopamine i s inactivated by reuptake via the dopamine transporter fol lowed byenzymatic breakdown via the combined effects of monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) (Figure 19-7B, upper).

Figure 19-7. A–B. Synthesis and Degradation of Dopamine, Epinephrine, and Norepinephrine. (A) The formation of the catecholamines , dopamine,epinephrine, and norepinephrine i s shown, each separated by a s ingular enzymatic s tep. (B) Breakdown of the catecholamines , including(upper) dopamine to homovani l l i c acid (HVA), and (lower) epinephrine and norepinephrine to vani l lylmandel ic acid (VMA). Both HVA and VMAare subsequently el iminated by the kidneys . COMT, catechol -O-methyl transferase; MAO, monoamine oxidase. [Adapted with permiss ion fromKibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Via the dopamine receptors (D1–5), dopamine increases the actions of the di rect pathway within the basa l gangl ia . Receptors D1 (regulationof growth/development of neurons , behaviora l response, and D2-receptor s igna l ing) and D5 (bel ieved to play a role in control of bloodpressure) activate a Gs protein to activate adenyl cyclase and cycl ic adenos ine monophosphate (cAMP) s ignal ing (Chapter 8). Receptors D2

(probable role in regulation of muscle tone; association with schizophrenia), D3 [bel ieved to play a role in emotions (e.g., depress ion) andcognitive thought; a lso may play a role in drug addiction and schizophrenia], and D4 (poss ible role in “thri l l seeking” behavior as wel l as anassociation with schizophrenia) activate a Gi protein to inhibi t cAMP production (Chapter 8). Normal ly, dopamine promotes smooth,coordinated muscle movement within the body. Insufficient dopamine biosynthes is in the dopaminergic neurons can cause Parkinson’s disease(see below); receptor types D3 and D4 are poss ibly impl icated. In the fronta l lobes , dopamine controls the flow of information from otherareas of the bra in. Dopamine disorders in this region of the bra in can cause a decl ine in neurocognitive functions , especia l ly memory,attention, and problem-solving.

NE/EPINEPHRINE

NE i s synthes ized from tyros ine (Figure 19-7A) in the cytoplasm and packaged into ves icles of postgangl ionic fibers . It i s released viaexocytos is . NE i s terminated by reuptake into the postgangl ionic nerve ending, di ffus ion from receptor s i tes , or metabol i sm by the enzymesmonoamine oxidase (MAO), and catechol-O-methyltransferase (COMT) (Figure 19-7B, lower). NE i s uti l i zed in sympathetic postgangl ionic neurons .Epinephrine, sometimes referred to as adrenaline, i s the fina l product of the tyros ine pathway in adrenal medul la chromaffin cel l s that a lsoproduces dopamine and NE. Epinephrine has a wide variety of effects on severa l organ systems as wel l as metabol ic functions , which arereviewed in Chapter 10. Epinephrine fol lows a s imi lar degradation pathway as NE (Figure 19-7B, lower). Both NE and epinephrine act ashormones and neurotransmitters via adrenergic receptors .

Adrenergic receptors are subdivided into α and β types as i l lus trated below. Both NE and epinephrine bind to a l l adrenergic receptor typesbut with di ffering affini ty.

1. α1-receptors are postsynaptic adrenoreceptors located in smooth muscle, eye, lung, blood vessels , gut, and the geni tourinary system.Stimulation of these receptors activates a Gq protein resul ting in increased phosphol ipase activi ty that releases diacylglycerol and inos i toltri sphosphate with the latter promoting increased ca lcium (Chapter 8). These s ignals lead to exci tation, which i s exhibi ted by di lation of thepupi l s (mydrias is ), bronchodi lation, constriction of blood vessels , increased s trength of heart contraction (pos i tive inotropy), and decreasedheart rate (negative chronotropy). NE binds this receptor better than epinephrine.

2. α2-receptors are located chiefly on the presynaptic nerve terminals . Gi protein activation inhibi ts adenyl cyclase activi ty/cAMP production and

decreases the entry of Ca 2+ into the neuronal terminal . This reduces the amount of released NE. In addition, i t produces vasoconstrictionand reduces sympathetic outflow in the CNS. Epinephrine binds these receptors better than NE.

3. β1-receptor s timulation activates a Gs protein, which increases adenyl cyclase activi ty, ATP convers ion to cAMP, and protein kinasephosphorylation of a variety of proteins . This action resul ts in pos i tive chronotropy (heart rate increase), dromotropy [conduction of theimpulse through the heart’s AV node (Chapter 16)], and inotropy (force of heart contraction). Whether NE and epinephrine bind equal ly to thisreceptor or epinephrine binding i s preferred over NE, remains controvers ia l .

4. β2-receptors are mostly postsynaptic adrenoceptors located in smooth muscle and glands . They not only activate adenyl cyclase via a Gs

protein but a lso inhibi t the same enzyme by a Gi protein. The di rectly oppos i te effects are bel ieved to a l low di fferent functions in speci ficcel l and ti ssue locations . The β2 s timulation relaxes smooth muscle resul ting in vasodi lation and bronchodi lation as wel l as release ofinsul in and the induction of gluconeogenesis. Epinephrine binds β2-receptors much more s trongly than NE.

5. β3-receptors are mostly found in fat/adipose ti ssue. They a lso activate adenyl cyclase via a Gs protein. The β3-receptors induce the breakdownof fats/l ipids (lipolysis) and a lso help to regulate heat production (thermogenesis), especia l ly in skeleta l muscle. NE binds this receptor betterthan epinephrine.

Figure 19-8. Synthesis of Serotonin. The formation of serotonin (5-hydroxytrypatmine) from tryptophan i s i l lus trated. [Adapted with permiss ionfrom Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

SEROTONIN

Serotonin (5-HT) i s a l so a monoamine neurotransmitter involved in s leep, eating, arousa l , dreaming, and the regulation of mood, temperature,and pa in transmiss ion. Serotonin i s synthes ized from the amino acid tryptophan by a short metabol ic pathway (Figure 19-8) cons is ting of twoenzymes: tryptophan hydroxylase and amino acid decarboxylase. At least seven serotonin or 5-HT receptors have been characterized uti l i zing Gs,

Gq, or Gi proteins that activate an intracel lular second messenger cascade; one receptor functions as a Na +–K+ channel leading to membranedepolarization. Serotonin i s terminated via uptake at the synapse on the presynaptic neuron. Various agents can inhibi t 5-HT reuptake,including MDMA (ecstasy), amphetamine, cocaine, dextromethorphan, tricyclic antidepressants (TCAs), and selective serotonin reuptake inhibitors (SSRIs).

ACETYLCHOLINE (Ach)

Ach i s the neurotransmitter uti l i zed in somatic efferent neurons , sympathetic pregangl ionic neurons (including the adrenal medul la andsweat glands), and the enti re parasympathetic nervous system. Ach i s synthes ized in the nerve terminal by choline acetyltransferase, whichcata lyzes the reaction between acetyl coenzyme A and choline (Figure 19-9). In the synapse, Ach i s degraded by acetylcholinesterase. Cholinergicreceptors refer to those that bind to Ach. They are further subdivided into nicotinic and muscarinic. Nicotinic receptors are located in the somaticsystem on the motor end plates of skeleta l muscle cel l s , a l l postganglionic neurons, and the adrenal medulla. When Ach binds to a nicotinicreceptor, the resul t i s exci tatory. Muscarinic receptors activate end-organ effector cel l s in bronchia l smooth muscle, sa l ivary glands , and thes inoatria l node. These effects are reviewed in Table 19-3.

Figure 19-9. Synthesis of Acetylcholine. The formation of acetylcho-l ine from acetyl -CoA and chol ine i s i l lus trated. CoA, coenzyme A.

REGULATION OF CATECHOLAMINES

Table 19-3. Summary of Effects of Acetylchol ine on Nicotinic and Muscarinic Receptors

The group of neurotransmitters ca l led catecholamines include dopamine, NE, and epinephrine and are secreted by the nervous system inei ther an acute or chronic response to s tress . Acutely, the neura l s igna l i s del ivered from the hypothalamus and bra in s tem to the adrenalmedul la via release of Ach from pregangl ionic neurons (Figure 19-10). Neura l s timulation of the adrenal medul la causes depolarization of theaxonal membrane and thus promotes the influx of ca lcium. Ca lcium, in turn, s timulates the release of catecholamines from the neurosecretorygranules. These s torage granules fuse with the plasma membrane, leading to the exocytotic release of NE (minor component) and epinephrine(major component). Nerve s timulation a lso promotes a chronic response resul ting in the synthes is of catecholamines . Prolonged s tress andsympathetic nerve activi ty resul ts in an induction of phenylethanolamine N-methyltransferase (PNMT) by glucocorticoids as a means of adapting tophys iologic s tress (Figure 19-10). Cortisol i s transported from the adrenal cortex to the medul la via the intra-adrenal porta l system. This offersan advantage because the corti sol concentration in this system is 100-fold enhanced relative to the arteria l blood. This response para l lelssynthes is and release of insul in in response to Ach.

Figure 19-10. Regulation of the Release of Catecholamines and Synthesis of Norepinephrine/Epinephrine in the Adrenal Medulla Chromaffi Cell. Immediaterelease of norepinephrine/epinephrine resul ts from neuronal s igna ls via the neurotransmitter acetylchol ine and a resul ting influx of ca lciumions . This ca lcium pulse leads to the release of the neurotransmitters via exocytos is . Longer term release in response to s tress or otherfactors rel ies on hormones from the hypothalamus and the resul ting induction of PNMT. See text for deta i l s . ACTH, adrenocorticotropichormone; CRH, corticotropin-releas ing hormone; DD, dopa decarboxylase; DH, dopamine hydroxylase; DPN, dopamine; Epi , epinephrine; NE,norepinephrine; PNMT, phenylethanolamine N-methyl transferase; TH, tyros ine hydroxylase. [Adapted with permiss ion from Murray RA, et a l .:Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Myasthenia Gravis (MG)–Lambert–Easton Syndrome: MG i s a disorder characterized by fluctuating weakness and easy fatigabi l i ty of commonlyused skeleta l muscles . This disorder produces symptoms such as double vis ion (diplopia), eyel id drooping (ptos is ), and problemswal lowing (dysphagia). MG typica l ly s trikes not only young women in thei r 30s but a lso men in thei r 60–70s . MG can occur in associationwith tumors of the thymus as wel l as other autoimmune disorders (e.g., lupus , rheumatoid arthri ti s ). MG is due to the autoimmunedestruction or inactivation of postsynaptic Ach receptors at the neuromuscular junction, functional ly reducing the number of Ach receptors .MG can be relaps ing–remitting in nature, but usual ly i s s lowly progress ive that can lead to severe respiratory weakness and compl ications .Infections , s tress , surgery, menses , and pregnancy can often worsen symptoms temporari ly. Treatment i s with antichol inesterase drugs ,s teroids , plasma exchange (plasmapheres is ), and surgica l removal of the thymus (thymectomy). Antichol inesterase drugs (such aspyridostigmine) treat the symptoms of MG by increas ing the amount of ava i lable Ach in the neuromuscular junction via inhibi tion ofacetylchol inesterase.

In contrast, Lambert–Eaton Syndrome (or Myasthenic syndrome) features proximal muscle weakness caused by a presynaptic defect ofneuromuscular transmiss ion. Antibodies to ca lcium channels on the nerve terminal cause the dramatic decrease of Ach release at themotor end-plate. Unl ike MG, s trength improves with susta ined contraction. Treatment i s usual ly provided by s teroid therapy andplasmapheres is . Antichol inesterase therapy gives only a modest improvement.

Parkinson’s Disease (PD): PD i s a bra in disorder caused by the destruction of dopamine-producing neurons in the substantia nigra. This leads toa chemica l imbalance between dopamine and Ach in the corpus s triatum. The four cardina l symptoms of PD are tremor (often at rest), rigidi ty,s lowness of movement (bradykinesia), and postura l ins tabi l i ty and reflect the loss of dopamine’s impact on smooth muscle movement. AsPD progresses , patients begin to have di ffi cul ty in walking (s low, shuffl ing walk) and muffled speech, depress ion, and di ffi cul ty inswal lowing (dysphagia). Making an accurate diagnos is of PD can be di ffi cul t given that many other neurodegenerative disorders mani festwith parkinsonian-type symptoms. No blood test exis ts that can confi rm or refute a diagnos is of PD, but severa l are used in conjunction withmagnetic resonance images (MRIs ) more to rule out other poss ible disorders . The diagnos is i s , therefore, based on his tory, a thoroughphys ica l examination and exclus ion of other causes .

There i s no cure for PD. Treatment i s di rected at reducing the chemica l imbalance by blocking Ach and increas ing dopamine with levodopaand carbidopa. Levodopa i s the precursor to dopamine but has severa l neuromuscular/ movement s ide effects (dyskines ias ) i tsel f.Carbidopa inhibi ts the enzyme that breaks down levodopa to dopamine a l lowing a reduction of the amount of levodopa that needs to betaken and, therefore, the number of s ide effects from levodopa. Al though levodopa can dramatica l ly improve symptoms, especia l lybradykines ia , i t does not ha l t the progress ion of the disorder. Other drugs used for PD include bromocriptine, which mimics the role ofdopamine in the bra in. An antivi ra l drug, amantadine, a l so appears to reduce symptoms. Whereas levodopa primari ly rel ieves bradykines ia ,antichol inergic drugs help to control tremor and rigidi ty by decreas ing the ava i lable amount of Ach. Drug-res is tant patients can benefi t froma therapy ca l led deep brain stimulation (DBS), which uses electrodes implanted into the bra in and connected to a smal l electrica l deviceca l led a pulse generator that can be external ly programmed. DBS can reduce the need for levodopa and related drugs , which in turndecreases the l ikel ihood of dyskines ias that are a common s ide effect of levodopa.

GLYCINE, GLUTAMATE, AND GABA

Glycine (Figure 19-11) i s found widely dis tributed in the spina l cord, bra instem, and retina and serves an inhibi tory function. The receptor i s agated channel (Chapter 8), composed of five protein subunits , which a l lows an influx of chloride ions . This influx acts as an inhibitorypostsynaptic potential and lessens the abi l i ty for the occurrence of future postsynaptic or motorneuron action potentia ls . The poison strychninei s a competi tive inhibi tor of glycine receptor activi ty. Caffeine i s another glycine receptor inhibi tor.

Figure 19-11. Glycine Structure.

Glutamic acid (Figure 19-12A) i s the most abundant exci tatory neurotransmitter in the CNS and the most common neurotransmitter in thebra in. It i s a lways exci tatory via glutaminergic receptor opening of nonselective ion channels . Glycine serves an important coagonis t for thereceptor. Glutamic acid s timulation i s terminated by a membrane transport system that i s used for reabsorbing glutamate and aspar-tateacross the presynaptic membrane. Glutamic and aspartic acid re-enter the cel l as sodium enters the cel l and potass ium exi ts . Thus , glutamicacid/aspartic acid entry i s indi rectly powered by the ATP-driven sodium pump. Both neurotransmitters work together to control manyprocesses , including the bra in’s overa l l level of exci tation. Many of the drugs of abuse affect ei ther glutamic acid or GABA or both to exerttranqui l i zing or s timulating effects on the bra in.

Figure 19-12. A. Structure of Glutamic Acid (Glutamate and Synthesis of GABA). The formation of GABA (γ-aminobutyric acid) from glutamic acid i si l lus trated. B. GABA Receptors. The functional aspects of GABAA- and GABAB-type receptors are i l lus trated. See text for addi tional deta i l s . GABA,γ-aminobutyric acid; AC, adenyl cyclase. [(Left channel ) Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11thedition, McGraw-Hi l l , 2009.] [(Remainder part) Adapted with permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rdedition, McGraw-Hi l l , 2010.]

GABA (Figure 19-12A) i s an amino acid neurotransmitter and i s the most widespread inhibi tory neurotransmitter in the bra in. It i s mosthighly concentrated in the substantia nigra , hypothalamus, and the hippocampus . GABA reduces the exci tabi l i ty of neurons by hyperpolarizingthem. There are two types of GABA receptors (Figure 19-12B). GABAA receptor activation opens chloride channels , and GABAB receptors act via asecond messenger to ei ther open potass ium channels or to close ca lcium channels . GABA is synthes ized from glutamic acid and i s inactivatedby active transport into the astrocyte gl ia l cel l s near the synapses .

Huntington’s Chorea (HD): HD i s an autosomal dominant, progress ive degenerative disorder caused by a genetica l ly programmed destructionof neurons in the basa l gangl ia as wel l as the cortex. It usual ly mani fests i tsel f in middle age with progress ive deterioration unti l death10–30 years later. As i ts name impl ies , HD i s characterized as an abnormal involuntary movement disorder (chorea) caused by overactivi ty ofdopamine. These movements are brief, i rregular, nonrepeating contractions that appear to move from one muscle group to the next and i t i soften accompanied by wri thing movements . HD can a lso cause dementia , clums iness , inabi l i ty to swal low (dysphagia), and personal i tychanges . Biochemica l ly, there i s an underactivi ty of GABA and Ach as wel l as a relative overactivi ty of dopamine. HD has no cure, and theprogress ion of the disease cannot be ha l ted. Therefore, a l l therapies are di rected purely at the symptoms, including the use ofphenothiazines to block dopamine receptors and control abnormal movements and the drug reserpine to deplete monoamines .

Eating Disorders: The satiety center of the bra in i s a col lection of neurons in the latera l aspect of the ventromedia l hypothalamus.Stimulation of this area increases appeti te and destruction of this center causes suppress ion of food intake. The media l area of theventromedia l hypothalamus demonstrates a regulatory role over the aforementioned latera l aspect. Destruction of this area has beenfound to cause gross overeating (hyperphagia) with resul tant obes i ty in animal models . The protein leptin targets the hypothalamus andcauses decreased food intake by activating melanocyte-stimulating hormone, a satiety factor, whi le suppress ing the hunger s igna l from AGRP(Agouti -related peptide). Additional hormones such as glucagon and cholecystokinin a l so inhibi t appeti te. The role of these centers in eatingdisorders such as bul imia and anorexia i s incompletely understood. There i s some evidence to suggest that neurotransmitter imbalancesexis t within the hypothalamus of patients who suffer from anorexia nervosa. The pathology behind such eating disorders i s multi factoria l ,including genetic, hormonal abnormal i ties with decreased levels of serotonin, and psychosocia l .

NEUROPEPTIDES

Neuropeptides are cha ins of l inked amino acids produced in the bra in. They are made from larger polypeptides that are cleaved. Theendogenous opiods (endorphins) or enkephalins produce analges ia and euphoria . Their receptors are catagorized as del ta (δ), kappa (κ), mu (μ),or nociceptin-type receptors and function via a variety of G proteins . Substance P i s found in the synaptic ves icle of unmyel inated C fibers , whichenhance the transmiss ion of pa in s igna ls . The substance P receptor i s a Gq protein, which activates phosphol ipase C and inos i tol

triphosphate/Ca 2+ production.

BIOCHEMISTRY OF VISIONNutri tional vitamin A (retinol esters ) i s converted in the retina to 11-cis-retinal, an i somer of a l l -trans-retina l forms by retinal isomerase. 11-cis-retina l then covalently attaches to a lys ine res idue on the visua l protein opsin. The resul ting rhodopsin molecule becomes a G-protein-coupledreceptor with the “l igand” for activation being l ight. A deficiency of vi tamin A in the retina wi l l , therefore, decrease the sens i tivi ty of rods ,resul ting in night bl indness .

Normal ly, cycl ic guanos ine monophosphate (cGMP) keeps inward Na +–Ca 2+ channels open to mainta in membrane depolarization (Figure 19-

13). When l ight s tikes the retina, photoexci ted rhodops in binds to a multi subunit membrane protein ca l led transducin, which in turn activates acGMP phosphodiesterase by caus ing dissociation of inhibi tory subunits . Stimulation of the cGMP phosphodiesterase leads to the exchange of abound guanos ine diphosphate for guanos ine tri -phosphate (GTP). This process releases the α subunit of transducin with bound GTP,reminiscent of the mechanism by which cAMP activates protein kinase A. The phosphodiesterase cata lyzes the destruction of cGMP (cGMP →GMP) near the membrane of the rod cel l . When cGMP concentrations fa l l , inward Na +–Ca 2+ channels close, thereby lowering both intracel lularNa + and Ca 2+ concentrations . The fa l l in Na + el i ci ts hyperpolarization caus ing the rod cel l to release less glutamate neuro-transmitter. Thereduced amount of this inhibi tory neurotransmitter generates an electrica l impulse to the occipi ta l lobe of the bra in that triggers theperception of l ight.

Figure 19-13. Biochemistry of Vision. Photoreceptors of the eye are normal ly depolarized because of the influence of cGMP on Na +–Ca 2+ channels .When l ight s trikes the receptor, transducin activates a cGMP phosphodiesterase, which decreases cGMP concentration, leading to closure ofthe channels . In this hyperpolarized envi ronment, the neurotransmitter glutamate i s released to provide an impulse to a l low the perceptionof l ight. cGMP, cycl ic guanos ine monophosphate. [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica lPhys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Because of i ts dynamic nature and the need to rapidly perceive ever-changing intens i ties of l ight, bui l t-in “cessation” and “recovery” facetsof the cycle exis t. When the l ight activation s ignal i s terminated (“cessation”), the active rhodops in molecule i s phosphorylated by rhodopsinkinase. Arrestin, a soluble protein, then binds the phosphorylated rhodops in to inactivate i t. Binding to arrestin a lso s lows rhodops in’s abi l i tyto propagate another l ight s igna l . The “recovery” phase, during which the cycle returns to a s tate that i s preparatory for the next flash of l ight,involves replenishment of the cGMP. Production of cGMP is triggered by the low intracellular Ca2+ that was induced by the decl ine in theconcentration of cGMP (see above). The low Ca 2+ leads to cGMP production from GTP via guanyl cyclase, which i s s timulated by low intracel lularca lcium via a “recoverin” mediator protein. Further breakdown of cGMP is accompl ished through di rect inhibi tion of phosphodiesterase.

ANESTHESIAGenera l anesthesia i s an a l tered phys iologic s tate characterized by a temporary loss of consciousness , rel ief of pa in (analges ia), amnes ia , andvariable levels of muscle relaxation. Despi te the loss of awareness , the vi ta l phys iologic functions are unaffected. Broadly speaking, genera lanesthetics can be categorized as ei ther inhalation (breathed-in) or intravenous (by vein).

Inhalation anesthetics (e.g., ether, chloroform, ha lothane, and sevoflurane) do not appear to have a s ingle s i te of action. However, knownareas of the bra in that are affected include the reticular activating system, cerebra l cortex (unconsciousness and amnes ia), cuneate nucleus ,the ol factory cortex, and the hippocampus . Al l inhalation anesthetics work the same at the molecular level , but thei r exact mechanism ofaction remains only partia l ly understood. The hydrophobic s i tes on a neuron’s membrane bind to the l ipophi l i c anesthetic, expanding thephosphol ipid bi layer and a l tering membrane function. Another postulated theory i s that anesthetics decrease membrane conductance. Manyanesthetics a lso enhance GABA inhibi tion of the CNS. Action at the bra instem level depresses the withdrawal from pain (most notably at thedorsa l horn interneurons). In addi tion, inhalation anesthetics depress exci tatory transmiss ion in the spina l cord. Despi te not understandingthe exact mechanism, the end resul t i s the inhibi tion of synaptic function.

Intravenous anesthetics (e.g., barbi turates , benzodiazepines , propofol , and opiates ) depress the reticular activating system, located in thebra instem control l ing consciousness . It works by depress ing transmiss ion of exci tatory neurotransmitters such as Ach and enhancinginhibi tory neurotransmitters such as GABA.

REVIEW QUESTIONS1. What are the centra l nervous system and periphera l nervous system, and what roles do they perform in the human body?2. What are the bas ic parts and the function(s ) of unmyel inated and myel inated neurons?3. What are the main s teps in nerve impulse conduction, including the role of membrane-bound channels?4. What are the bas ic roles of the sympathetic and parasympathetic nervous systems?5. What are the major neurotransmitters and what are thei r major functions?6. How is the production and release of catecholamine neuro-transmitters regulated acutely and chronica l ly?7. What i s the biochemica l process behind vis ion?

CHAPTER 20THE REPRODUCTIVE SYSTEM

Editor: Catrina Bubier, MDWomen’s Heal th Care Associates , Englewood,

Colorado, USA

Bas ic Anatomy and DevelopmentFemale Reproductive SystemThe Menstrua l CycleFerti l i zationBreast Development and LactationMale Reproductive SystemReview Questions

OVERVIEWThe female and male reproductive systems develop selectively as a resul t of speci fic hormonal s igna ls (sex-determining region on the Ychromosome, anti -Mul lerian hormone, testosterone/5α-dihydrotestosterone, and estrogens) that lead to further development or regress ion ofembryologica l s tructures . Additional hormones (gonadotropin-releas ing hormone, fol l i cle-s timulating hormone, luteinizing hormone,progesterone, and human chorionic gonadotropin) influence further development and subsequent adult functions , including the menstrua lcycle, ferti l i zation and pregnancy, lactation, and oogenes is/ spermatogenes is . These hormones work via s igna l ing proteins , including severa lvariations of G proteins , to selectively activate or inhibi t these developmenta l and functional events .

BASIC ANATOMY AND DEVELOPMENTThe reproductive system is derived from the intermediate mesoderm and includes the reproductive organs of both males and females derivedfrom the Wolffian ducts (male), Mullerian ducts (female), and the gonads (male and female), including the testes and ovaries . The influence ofhormones and biochemica l s igna ls in the formation and activi ty of the reproductive system is vastly important and, when these s ignals goawry, disease ensues .

During the ini tia l s tages of gestation, a l l humans begin with both Wolffian and Mul lerian ducts and the development of male and femaleembryos i s indis tinguishable. At about gestational day 56, further growth and development into male or female sexual organs i s dependenton the effects of the sex-determining region of the Y chromosome (sry). In the genetic male, the s ry product binds to deoxyribonucleic acid (DNA)and dis torts i t dramatica l ly out of shape. This a l ters the properties of the DNA and l ikely a l ters the express ion of a number of genes . One ofthese genes produces anti-Mullerian hormone (AMH), a dimeric, glycoprotein hormone a lso known as Mullerian inhibiting factor (MIF). AMH isproduced by Sertol i cel l s in the testes and s ignals , via i ts receptor, a member of the transforming growth factor (TGF)-β I and II receptor fami ly.Binding of AMH to TGF-βtype II receptor a l lows i t to bind to the type I receptor. The type I receptor i s then able to phosphorylate serine and/orthreonine amino acids to activate transcription factors in the nucleus , which regulate gene express ion. The presence of AMH leads to ful ldevelopment of the Wolffian ducts and male s tructures ; only a few remnants of the Mul lerian ducts survive in males .

Figure 20-1. Summary of Hormonal Involvement in Sexual Differentiation. DHT, dihydrotestosterone; MIF, Mul lerian inhibi ting factor; s ry, sex-determining region of Y chromosome; T, testosterone; 5αR, 5α-reductase-2. [Adapted with permiss ion from Kibble JD and Halsey CR: The BigPicture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

In males , the Leydig cells a l so appear and testosterone synthes is begins , leading to male sexual characteris tics , including testi s formation.Testosterone’s effect on the semini ferous tubules (see below) i s a l so cri ti ca l for modulating s ignal ing and gene express ion and, therefore,male development (Figure 20-1). In the absence of s ry, embryos spontaneous ly develop into phenotypic females . With no s ry product, AMH andtestosterone are not produced and the Wolffian ducts regress with only a few remnants remaining. The mechanism of AMH suppress ion ofMul lerian duct formation i s unknown. Further development of the Mul lerian ducts creates the female sexual organs and s tructures . In bothsexes , the Wolffian duct i s respons ible for development of the bladder trigone.

Androgen Insensitivity Syndrome (AIS): AIS, a l so previous ly known as “testicular feminization,” resul ts from a mutation on the X chromosome,which yields a defective androgen receptor. Because this i s an autosomal recess ive condition, a l l patients are genetic males and, therefore,produce s ry, AMH, and testosterone/5α-dihydrotestosterone (DHT) normal ly. However, the defective androgen receptors do not a l low thenormal androgenic functions to be expressed, leading to fa i lure of Wolffian duct development and complete feminization of the externalgeni ta l ia and bl ind ended vagina. These patients are, therefore, phenotypica l ly female with a male karyotype. A s imi lar medica l condition,17α-hydroxylase deficiency, resul ts in the inabi l i ty to form any androgens (Chapter 3) and, therefore, estrogens as wel l . The condition, a lsoreferred to as “sexual infantilism,” leads to underdevelopment of the gonads (hypogonadism).

FEMALE REPRODUCTIVE SYSTEMAs noted above, the development and function of the female reproductive system is under the influence of severa l hormone s ignals .Fol lowing development, primary hormones in the female system are follicle-stimulating hormone (FSH), luteinizing hormone (LH), and the hormonethat regulates thei r express ion—gonadotropin-releasing hormone (GnRH). These hormones are a lso essentia l in the male reproductive system(see below). GnRH production and release begins at puberty and remains active during the male and female reproductive years .

GnRH

GnRH i s a smal l peptide produced in the hypothalamus by specia l i zed nerve cel l s ; as such, GnRH is ca l led a neurohormone, a class ofhormones that include thyrotropin-releas ing hormone, oxytocin (see below), antidiuretic hormone (Chapter 18), and corticotropin-releas inghormone. Release of GnRH resul ts in activation of a speci fic GnRH receptor, located in the gonadotropes of the pi tui tary gland. This receptor i sa membrane-bound G-protein-coupled s timulator of phosphol ipase C, which resul ts in ca lcium release and protein kinase C activation viaconvers ion of plasma membrane phosphatidyl inos i tol into inos i tol triphosphate and diacylglycerol (Chapter 8). These s ignals resul t inproduction and release of FSH and LH, as wi l l be described below. Regulation of this important s igna l i s multi fold. GnRH is degraded withinminutes so i t i s constantly produced in pulses , and the s i ze and frequency of these pulses i s important in s igna l ing. These GnRH pulses areconstant in males but vary in females , depending on the menstrua l cycle. Interestingly, the frequency of the GnRH pulses resul t in di fferentexpress ion of FSH (low frequency) and LH (high frequency). As a resul t FSH and LH are variably expressed during the female menstrua l period(Figure 20-2). Levels of testosterone, estrogen, and prolactin (increased during pregnancy) as wel l as increased concentration of FSH and LHcreate a negative feedback loop, which can decrease GnRH pulses .

Figure 20-2. Variable Expression of Reproductive Hormones During the Menstrual Cycle (Follicle-Stimulating Hormone, Luteinizing Hormone, Estradiol, andProgesterone). Periods of the menstrua l cycle, including menstruation, fol l i cular phase, ovulation, and lutea l phase are indicated. [Adapted

with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

FSH

FSH i s a dimeric glycoprotein hormone produced and secreted by the anterior pi tui tary gland. FSH has an α- and β-subunit, wi th the α-subunitbeing identica l in amino acid sequence to the α-subunit of LH, thyroid-s timulating hormone (TSH), and human chorionic gonadotropin (hCG)(see below). The β-subunit binds to and activates the FSH receptor.

The FSH receptor i s bel ieved to exis t in two conformational s tates (Chapter 1)—one active and the other inactive. Binding of FSH to i tsreceptor locks the receptor’s conformation into the active form, which activates a Gs-coupled protein caus ing release of an αs-subunit. This αs-subunit then activates adenyl cyclase to increase the cycl ic adenos ine monophosphate (cAMP) s ignal and protein kinase A activi ty (Chapter 8).Phosphorylated s ignal ing proteins bind to cAMP response elements on DNA, leading to activation of particular genes . FSH can a lso activatethe fami ly of extracellular signal-related kinases. This fami ly of receptor-activated kinase, a lso known as mitogen-activated protein (MAP)kinases , respond to a variety of s igna l molecules , including FSH, which regulate meios is and mitos is of selected cel l s . Al though manymechanisms may be in play, the activation of tyros ine kinases via the ras GTPase protein i s cons idered important in the subsequent activationof transcription factors .

FSH performs many functions in the development of female and male and i s a lso essentia l for the proper timing and phas ing of themenstrua l cycle. FSH, as i ts name impl ies , s timulates granulosa cel l s in the fol l i cles in the ovary to ini tiate egg growth and development(oogenesis). FSH speci fica l ly blocks programmed death or atresia of early developing egg fol l i cles . As one egg fol l i cle becomes dominant,reaching about 10 μm in diameter, i t begins to secrete estradiol, which negatively feedbacks on GnRH and, therefore, FSH production. The blockof atres ia in a l l fol l i cles except the dominant one leads to the surviva l of only one egg.

The activi ty of FSH i s regulated by severa l means . FSH has a biologica l ha l f-l i fe of only about 3–4 hrs . Unless GnRH pulses continue topromote FSH synthes is and secretion, i ts biochemica l effects wi l l begin to diminish. Increased estrogen wi l l a l so decrease FSH synthes is viai ts effect on GnRH. Activin (Figure 20-3, right), a protein heterodimer (composed of two di fferent subunits ) or homodimer (composed of twoidentica l subunits ), serves to increase FSH production and secretion via a mechanism extremely s imi lar to the FSH receptor, leading to proteinkinase A activi ty and upregulation of gene express ion. However, the same smal l antra l fol l i cles that FSH s timulates a lso produce the proteininhibin (Figure 20-3, left), a protein heterodimer with one subunit identica l to the activin subunit and one di fferent. Inhibin decreases FSHsynthes is and secretion and, therefore, works in a s imi lar manner to the dominant fol l i cle’s estrogen production to decrease FSH andincrease non-dominant fol l i cle atres ia .

As the lutea l phase ends , a smal l ri se in FSH can be detected that helps to s igna l the s tart of the next menstrua l cycle. In addition, estrogenand progesterone levels fa l l and decrease their negative effect on GnRH, a l lowing FSH levels to ri se with an ini tia l peak at day 3 (Figure 20-2).In a di rectly oppos i te s igna l ing mechanism, estrogen increases GnRH and, therefore, FSH secretion at the time of ovulation (see below).Fina l ly, as a woman approaches menopause, the number of ini tia l fol l i cles recrui ted to egg production decreases and, as a resul t, the level ofinhibin i s a l so lessened. This leads to an overa l l ri se in serum FSH levels that i s one s ign of perimenopause/menopause.

Figure 20-3. Inhibins and Activins. I l lus tration of s tructures of inhibins (left), heterodimers of three separate proteins (represented as threedi fferent colors ), and activins (right), homodimers or heterodimers of the green and purple proteins . [Adapted with permiss ion from Barrett KE,et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

LH

LH i s a dimeric glycoprotein, composed of an α- and β- subunit, produced by the anterior pi tui tary gland. The α-subunit i s identica l in aminoacid sequence to the α-subunit of FSH, TSH, and hCG (see below). The β-subunit i s s imi lar but not identica l to that of hCG and i s respons iblefor binding to the LH receptor. LH i s not produced unti l puberty and then i s generated in response to GnRH s ignals , regulated according toreproductive need. Like FSH, LH levels a lso ri se in females after menopause.

Like the FSH receptor, the LH receptor i s bel ieved to exis t in two conformational s tates (Chapter 1)—one active and the other inactive.Binding of LH to i ts receptor locks the receptor’s conformation into the active form, which activates a Gs-coupled protein caus ing release of anα-s-subunit. This αs-subunit then activates adenyl cyclase to produce the cAMP s ignal and increased protein kinase A activi ty (Chapter 8).Phosphorylated s ignal ing proteins bind to cAMP response elements on DNA, leading to activation of particular genes . LH has a biologica l ha l f-l i fe of about 20 min, so gene regulation via i ts receptor may s top unless additional LH or other s igna l ing molecules continue the nuclears ignal .

In females , LH provides the hormone s ignal for release of an egg from the ovary. Increas ing estrogen, which resul ts from FSH s timulation ofovarian granulosa cells, increases the presence of LH receptors . In addition, estrogen activates the pi tui tary to increase LH secretion. Both ofthese functions act to produce an LH “surge” over a 24–48 hr period (Figure 20-2). Unl ike FSH, activin, inhibin, and the sex hormones do notaffect LH production at the DNA level . The LH surge produces ovulation and a lso s ignals the corpus luteum, a temporary s tructure created fromthe remnant ovarian fol l i cle after egg release. The activated corpus luteum produces the hormone progesterone, which, in turn, s igna ls theuterine wal l to prepare for egg implantation i f ferti l i zation should occur. The presence of LH a lso s tarts and, in the case of ferti l i zation andimplantation of the egg, mainta ins the luteal phase (Figure 20-2) for 8 weeks . LH promotes androgen and estrogen production via s timulation oftheca l cel l s in the ovary. The LH surge a lso ini tiates the continuation of meios is in the oocyte, the completion of which occurs after the spermenters the ovum. Fina l ly, LH i s important to luteinize the granulosa cel l s to produce progesterone for maintenance of the lutea l phase.

Ovulation Prediction by LH Measurement: The 24–48 hr LH surge that leads to ovulation and, therefore, predicts an optimal time for conceptionhas led to the development of “ovulation predictor ki ts” to ass is t couples wanting chi ldren. These ki ts have anti -LH antibodies , which bindto LH excreted in the urine. Binding of the LH and a pos i tive color change via a chemica l reaction indicates when the level i s above thenormal 1–20IU/L—levels indicating maximum ferti l i ty times . Digi ta l measurements of LH level are a lso ava i lable.

ESTROGENS

Estrogen i s the inclus ive term for a group of s teroids that primari ly impact the female reproductive system. Estrogens are mainly synthes izedwhen FSH and LH s timulate theca l and granulosa cel l s in the ovarian fol l i cles , the corpus luteum after release of a dominant egg, and, in the

case of pregnancy, the placenta. The l iver, adrenal glands , adipose ti ssue, and breast ti s sue can a lso produce s teroida l es trogen. The highestlevel of estrogen occurs immediately prior to ovulation.

As a class of s teroid hormones , a l l es trogens function by cross ing the cel l membrane and activating estrogen receptors (ERs). Unbound ERsare mainly found in the cytosol but, upon binding of an estrogen molecule, the receptors move into the nucleus , form dimers , and bind tospeci fic sequences of the DNA molecule known as hormone responsive elements. The bound ER–DNA activates particular proteins that s tarttranscription of DNA to resul t in synthes is of speci fic proteins . ERs can a lso be found in the nucleus where they serve to regulate thetranscription of other proteins and may a lso associate with plasma membrane G proteins , which activate tyros ine kinases . Three major formsof estrogen molecules occur in humans (Figure 20-4 and Chapter 3).

Figure 20-4. The Three Major Classes of Estrogen Steroid Hormones in Humans. [Adapted with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica lPharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Estrone (E1), produced by the enzyme aromatase from androstenedione in adipose cel l s , i s found predominately in menopausa l women aswel l as in men. 17β-Estradiol (E2) i s formed from testosterone by aromatase and i s the main estrogen in nonpregnant, ferti le females .Aromatase in the granulosa cel l s of the ovaries i s activated by FSH. Estradiol can a lso be produced in smal ler amounts by aromataseconvers ion in the l iver and fat cel l s (Chapter 3). Estriol (E3) i s the most abundant estrogen during pregnancy and i s formed in the placenta andfeta l adrenal glands and l iver as i l lus trated in Figure 20-5. Other nonsteroida l molecules , found in nature and arti fi cia l ly produced, canexhibi t es trogen activi ty.

Figure 20-5. Steroid Production by Mother, Placenta, and Fetus During Pregnancy. Overview of production of s teroids from cholesterol duringpregnancy, showing various sources for the production of s teroids in the placenta and feta l compartment (e.g., feta l adrenal glands and l iver)as wel l as transport of maternal , placenta l , and feta l molecules . See text for further discuss ion. DHEA, dehydroepiandrosterone; DHEAS,dehydroepiandrosterone sul fate; LDL, low-dens i ty l ipoprotein. [Reproduced with permiss ion from Kibble JD and Halsey CR: The Big Picture:Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Estrogens have a wide-ranging set of functions in both men and women and only the major functions wi l l be covered here. In females ,estrogens are essentia l for the ini tia l growth of the columnar epi thel ia l cel l s of the endometrium. From a developmenta l s tandpoint,estrogen helps to ini tiate and propagate the development of secondary sexual characteris tics to include breast formation, growth of theuterus and vagina, growth of pubic and underarm hair, onset of adul t body changes (e.g., hip widening and deceleration of teenage growth),and the onset and regulation of menstruation (see below). Es trogen a lso influences a number of biochemica l and metabol ic processes toinclude bone reabsorption; increased clotting of blood via modulation of clotting factors I I , VII , IX, and X as wel l as plasminogen andanti thrombin II I (Chapter 14); increases in high-dens i ty l ipoprotein and triglycerides with decreases in LDL and fat depos i ts (Chapter 7); and

al terations in fluid ba lance and hormone levels . The role of estrogens in the female menstrua l cycle wi l l be examined in ful ler deta i l below.

PROGESTERONE

Progesterone i s a s teroid hormone from the hormone class ca l led progestogens, important in menstruation, pregnancy, and embryodevelopment. Progesterone i s made from pregnenolone, derived from cholesterol , in the corpus luteum after ovulation. If ferti l i zation occurs ,the placenta becomes the primary source by week 8, uti l i zing ci rculating cholesterol from maternal blood. Progesterone i s a lso produced inthe adrenal glands . Levels of progesterone are low in the fol l i cular/ prol i ferative phase and higher in the lutea l/secretory phase (Figure 20-2and below) because of the contribution of the corpus luteum.

Progesterone, l ike other s teroid hormones , passes through the plasma membrane and binds to an intracel lular progesterone receptor.Also, l ike other s teroid hormones , binding of progesterone leads to receptor dimerization, entrance into the nucleus , and activation ofselected genes via DNA binding. Transcriptional activation by the progesterone receptor i s normal ly inhibi ted by the receptor’s carboxy-terminal end. Binding of the s teroid hormone estrogen to the progesterone receptor causes a conformation change that releases thisinhibi tion and i s , therefore, essentia l for progesterone/progesterone receptor functions . Es trogen a lso acts to increase the tota l number ofprogesterone receptors , thereby ampl i fying progesterone effects . However, two di fferent i soforms of the progesterone receptor exis t: type Aand type B, the latter of which has an amino-terminal , transcriptional activation domain. The two receptor types appear to have s imi lar butunique activi ties that are s ti l l being investigated. Interestingly, progesterone a lso binds very s trongly to the receptor for a ldosterone,competi tively inhibi ting the minera lcorticoid’s function. Increased levels of progesterone, therefore, block a ldosterone’s effects (Chapter 18)and lead to increased loss of sodium and water by the kidneys . A sudden decrease in progesterone subsequently causes sodium and waterretention.

Progesterone Antagonists: The actions of estrogen and progesterone on progesterone receptor activi ty have led to the development of a classof medications ca l led selective progesterone receptor modulators (SPRMs). SPRMs affect the two i soforms of the progesterone receptordi fferently, depending on binding s trength and activation/inhibi tion activi ty. SPRMs may, therefore, a lso offer the option of selectivereceptor-type effects . Examples include treatment of uterine leiomyoma, endometriosis, and hormone-responsive breast cancers. One SPRM, RU-486, i s a l ready in selected use as a medica l abortant.

Progesterone’s main role i s to prepare the endometria l l ining of the uterus for poss ible implantation of a ferti l i zed egg and maintenanceof the uterine l ining to support the fetus during pregnancy. Progesterone’s influence on the endometrium is di fferent from estrogen, though,as i t turns the endometrium from a prol i ferative to a secretory phase, including the marked growth of spi ra l arteries (Figure 20-6). In this role,progesterone i s often used to support in vi tro ferti l i zation treatments and/or i rregular uterine bleeding. During pregnancy, progesterone a lsoinhibi ts lactation (see below) and smooth muscle contraction; decreased progesterone levels are one potentia l trigger for labor and a lsoini tiate mi lk production. As a resul t, recent l i terature suggests giving weekly intramuscular injections of progesterone to help avoid pretermlabor in at-ri sk mothers . If pregnancy does not occur, there i s a regress ion of the corpus luteum and, therefore, decreased production ofprogesterone, which ini tiates menstruation. Apart from the reproductive system, progesterone a lso impacts nerve function via binding to aserine/ threonine kinase ca l led glycogen synthase kinase 3. Progesterone’s protective effect on myel in sheaths (Chapter 19) of nerves has ra isedi ts potentia l to be used in patients suffering from multiple scleros is . Progesterone may a lso serve as a protective factor aga inst endometria lcancer by oppos ing the effects of estrogen.

Figure 20-6. Human Menstrual Cycle. Changing levels of hormones and their influence on fol l i cle/egg development; corpus luteum functions ; anddegeneration, growth, and thickening of the endometrium during the two phases of the cycle. The figure assumes an average 28-day cycle andstarts at the onset of menstruation. [Adapted with permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion,McGraw-Hi l l , 2009.]

hCG

hCG i s a heterodimeric (two di fferent subunits ) glycoprotein hormone produced by newly ferti l i zed embryos and, subsequently by the placenta.hCG has an α-subunit that i s identica l to the α-subunit of FSH, LH, and TSH and a unique β-subunit that binds to the luteinizinghormone/choriogonadotropin receptor (LHCGR). LHCGR is a Gs-coupled protein receptor found predominately in the ovary and testes , which spansthe membrane. LHCGR a lso binds with LH and, as a resul t, hCG and LH share severa l functions . Just l ike the LH receptor (see above), uponbinding of hCG or LH, the receptor undergoes a conformational change to i ts active s tate, leading to cAMP production, activation of proteinkinase A, and increases in express ion of selected genes . Like LH, hCG a lso mainta ins the corpus luteum and causes increas ing production ofprogesterone (see above).

Pregnancy Testing and hCG: Because hCG is produced by the newly ferti l i zed egg, the abi l i ty to measure i ts levels in blood and even urine hasled to the advent of eas i ly performed pregnancy tests. Most of these tests uti l i ze a monoclonal antibody to the β-subunit of hCG, thereby

avoiding interference from the s imi lar FSH and LH. Urine tests do not provide actua l numerica l levels of hCG and are, therefore, referred toas a qual i tative pregnancy test. Blood serum tests uti l i ze detection methods that can determine actua l β hCG levels ; therefore, this test i sreferred to as a quanti tative test. Quanti tative measurement of hCG is a lso important in the diagnos is and treatment of a variety of ovarian-, testicular-, and/or placenta l -derived cancers (e.g., germinomas, chorio-carcinoma, hydatidi form mole, and teratoma/dermoid cyst) as wel las ectopic pregnancy and miscarriage. Levels of hCG are a lso one of four components of a cl inica l quad screen test, used in early pregnancy todetermine the ri sk of Down syndrome, Edward’s syndrome, Patau’s syndrome, and neura l tube defects in the fetus .

THE MENSTRUAL CYCLEThe female menstrua l cycle i s control led by a series of hormones that ini tiate and regulate growth of the endometria l l ining, development ofan egg, release and poss ible implantation of the egg, and, i f pregnancy does not ensue, complete purging of the uterine l ining to a l low arepeat of the same process (Figure 20-6).

MENSTRUATION (DAYS 1–4)

The menstrua l cycle’s s tart i s cons idered at the onset of menstruation (day 1), the evacuation of the endometria l l ining in the nonpregnantfemale, which may rely, in part, on a smal l surge of FSH and then decl ine in the progesterone level (see above). Subsequently, increas ingestrogen s tops loss of the endometria l l ining and begins and ini tiates new thickening of the endometrium on approximately day 4.

FOLLICULAR/PROLIFERATIVE PHASE (DAYS 5–13)

Day 5 i s the approximate s tart of the follicular/proliferative phase because of the growth and development of the uterine endometria l l ining andof the ovarian fol l i cles . Speci fica l ly, a fter days 1–7 of the menstrua l cycle, activin as wel l as low levels of estrogen (estradiol ) andprogesterone a l low GnRH pulses to secrete increas ing amounts of FSH, which, then, ini tiates development of severa l ovarian fol l i cles . As adominate follicle emerges , es tradiol i s increas ingly synthes ized, which modulates FSH via inhibi tion of GnRH. Inhibin, produced by the samefol l i cles , a l so decreases FSH activi ty. Es tradiol a l so inhibi ts synthes is and secretion of LH unti l approximately day 12. At this point, a cri ti ca llevel of estradiol i s reached and promotion of LH production s tarts , leading to the LH surge. The mechanism behind the di rectly oppos i teeffects of estradiol on LH i s not completely understood but one poss ibi l i ty i s the di fferentia l activation of ei ther of the two ERs . ERα i s knownto oppose LH secretion, whereas ERβ increases LH levels and inhibi ts α-receptor activi ty. How preferentia l activation of the α- or β-receptorsoccurs i s unknown, but di fferentia l express ion, di fferent estradiol binding, or some other mechanism may expla in this observation.Regardless of the mechanism, the increas ing LH level leads to the breakdown of the fol l i cular wal l and release of the egg, known as ovulationon approximately day 14.

THE LUTEAL/SECRETORY PHASE (DAYS 15–28)

The luteal/secretory phase i s named so because of the activi ty centers on the corpus luteum, the remnant of the ovarian fol l i cle, and i tssecretion of progesterone. If the egg i s not ferti l i zed within about 24 hrs , i t dies and i s reabsorbed by the body. Meanwhi le, the remainingovarian fol l i cle converts into a corpus luteum and begins to produce progesterone in increas ing amounts (Figure 20-6). Increased levels of FSHand LH a lso help to develop the corpus luteum. Progesterone acts on the endometrium for poss ible ferti l i zed egg implanation. If the egg doesnot implant, hCG is not secreted and the corpus luteum dis integrates , caus ing markedly lower levels of progesterone, which ini tiates theshedding of the endometria l l ining as menstruation. Es trogen and progesterone synthes ized by the growing corpus luteum inhibi t GnRH and,therefore, FSH and LH production, leading to a rapid fa l l from ovulation levels . The fa l l of FSH and LH levels adds to the atrophy of the corpusluteum and a subsequent decrease in progesterone synthes is , aga in leading to menstruation. In the case of ferti l i zation and pregnancy (seebelow), the new embryo begins to produce hCG, which acts l ike LH in preserving the corpus luteum during the fi rs t 6–8 weeks of pregnancyunti l placenta l synthes is can take over.

FERTILIZATIONThe process of ferti l i zation, the entrance of a sperm cel l or, in rare cases , sperm cel l s into an egg, rel ies on severa l fundamenta l biochemica lprocesses , including cel l moti l i ty and plasma membrane fus ion, involving changing fluidi ty and s tructure of the sperm and egg membranes(Chapter 8, Figure 20-7).

Figure 20-7. Mechanism of Fertilization. Steps of ferti l i zation are indicated, including the acrosomal reaction (see text). [Adapted with permiss ionfrom Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

The sperm ta i l i s a class ic flagel lum (Chapter 12), producing whipping or lashing movements via adenos ine triphosphate hydrolys is thatpropels the sperm through the female reproductive organs toward the egg. As discussed in Chapters 1 and 12, this process rel ies on thestructura l proteins tubul in–microtubules and their interaction with the motor protein, dynein. Once a sperm reaches the egg, i t binds to theouter layer and then attempts to penetrate the hard zona pellucida. Upon reaching the corona radiata layer of the egg, the acrosome, a cap-l ikestructure at the tip of each sperm, releases the enzyme hyaluronidase, which breaks down hyaluronic acid conta ined in the presenting egglayer. Next, the enzyme acrosin digests the zona pel lucida and promotes changes in the s tructure and fluidi ty of the membranes of the spermtip and the egg to cause them to fuse. Fol lowing this ini tia l fus ion process , a protein molecule on the sperm acrosome binds to a di rectlyinterlocking protein, ZP3, on the egg. This binding i s bel ieved to ini tiate a series of reactions , including the acrosome reaction, a release ofenzymes by the acrosome, which complete the membrane fus ion; the cortical reaction (see below); and activation of the egg to undergo asecond meiotic divis ion. Fus ion of the ferti l i zing sperm and egg resul ts in haploid nuclei .

The cortica l reaction i s cons idered the analogous process to the acrosome reaction. The binding of sperm to egg resul ts in a release ofca lcium ions , which promote fus ion of cortica l granule membranes with the egg’s plasma membrane. Fus ion expresses factors that cause therelease of an external ly bound, membrane protein on the outer vitelline layer of the egg. Release of this protein forces the vi tel l ine layer awayfrom the zona pel lucida, moving any other sperm away permanently and inhibi ting any other sperm from ferti l i zing the egg. Thepolysaccharide molecule hyalin i s a l so released from the cortica l granules to create a layer around the egg that a lso impedes any furtherferti l i zation.

Oral Contraceptives: The influences of estrogen and progesterone on the menstrua l cycle, ovulation, and implantation have been uti l i zed toa l low the development of various forms of contraception. Es trogen and progestin, the generic name for any synthetic progesteronecompound, molecules inhibi t GnRH pulses , which then decreases FSH and LH secretion, inhibi ting development of ovarian fol l i cles andovulation. The estrogen component in the ora l contraceptive pi l l primari ly suppresses FSH secretion and the progesterone componentsuppresses LH secretion, thereby preventing fol l i cular recrui tment and ovulation, respectively. The combination of estrogen andprogesterone i s a lso used to treat menstrua l disorders by helping to decrease the endometria l l ining and regulate or even prevent menses .The control led effect on the menstrua l cycle i s often used for female patients with pa inful , i rregular, heavy, or temporari ly absentmenstrua l periods .

BREAST DEVELOPMENT AND LACTATIONBreast ti s sue development or thelarche i s a part of secondary sexual changes that occur at puberty because of increased estradiol ,progesterone, and prolactin (see below). Bes ides other changes in body shape, the activi ty of this estrogen via i ts receptor leads to a variableincrease of the adipose cel l s , supporting l igaments and glands that make up the female breast ti s sue.

Lactation requires the action of oxytocin and human placental lactogen hormones , which, a long with prolactin, are discussed immediatelybelow.

OXYTOCIN

Oxytocin i s a nine-amino-acid peptide, including a disul fide bridge formed by two cysteine res idues , which i s released by the posteriorpi tui tary gland. Vasopress in, active in the regulation of tota l body water, di ffers from oxytocin by only two amino acids and i s a lso releasedfrom the posterior pi tui tary gland (Figure 20-8, Chapter 18).

Figure 20-8. Comparison of Oxytocin and Antidiuretic Hormone (ADH, Vasopressin) Structures. The nine-amino-acid peptides oxytocin and ADH(vasopress in) with disul fide bridge as shown. Di ffering amino acids are seen at pos i tions 3 and 8. [Adapted with permiss ion from Katzung BG,et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.]

Binding of oxytocin to i ts receptor activates a Gq protein, which activates phosphol ipase C, leading to ca lcium release and protein kinase C

function. Interestingly, the oxytocin receptor requires both Mg2+ and cholesterol , which are thought to a l losterica l ly activate the receptor.Oxytocin i s involved in the ejection of mi lk from the breasts ’ mi lk glands as wel l as contraction of the uterus during bi rth. Both functions resul tfrom the activation of smooth muscle. Uterine contraction resul ts in a notable increase in the number of oxytocin receptors in the smoothmuscle layer and release of estrogen and prostaglandin F2α. This prostaglandin, which binds to the corpus luteum and helps to lead to i tsdegradation, activates the Gs-coupled pathway, leading to cAMP production via adenyl cyclase. Because of oxytocin’s marked s imi lari ty tovasopress in, i t can a lso increase sodium and water excretion in the kidneys .

PROLACTIN

Prolactin i s a peptide hormone that i s produced in the anterior pi tui tary gland via the action of a speci fic transcription factor ca l led Pit-1.Increased production of prolactin i s due to estrogen, which s timulates Pi t-1 and increases the number of prolactin-producing cel l s . Prolactincan a lso be produced in breast ti s sue as wel l as nerve and immune system cel l s . Prolactin binds i ts receptor on the mammil lary glands andovaries where dimerization of the receptor activates a Janus 2 tyros ine kinase. This leads to subsequent tyros ine phosphorylation,dimerization, and activation of a class of proteins known as signal transducers and activators of transcription (STATs). These activated STAT dimerstravel to the nucleus and augment gene express ion of certa in proteins . Prolactin binding to i ts receptor a lso activates MAP kinases and Srckinase, involved in the regulation of cel l growth and development. The activi ty of both of these additional kinases a lso leads to regulatedgene express ion. Prolactin receptors are a lso found on various other organs and ti ssues throughout the body.

The major function of prolactin i s to s timulate the production of mi lk in the mammary glands , whose enlargement during pregnancy i spromoted by estrogen and progesterone. As noted above, the drop in inhibi tory progesterone levels at the end of a pregnancy i s the fina ls ignal for the beginning of mi lk production. The presence of prolactin after pregnancy a lso s tops the menstrua l cycle through inhibi tion of thepulsati le release of GnRH and, therefore, FSH and LH. Prolactin continues this effect throughout the breastfeeding s tage. The act of suckl ingstimulates the hypothalamus to continue promoting pi tui tary gland secretion of prolactin. Prolactin a lso functions in the lungs to promote theproduction of surfactants in feta l lungs (Chapter 17). In nerve cel l s , prolactin promotes the formation of myelin sheaths (Chapter 19). Fina l ly,prolactin i s thought to counteract the sexual arousa l effects of dopamine and provide postcoi ta l sexual grati fi cation. As a resul t, the refractoryperiod a fter sex may be because of the function of prolactin. In a s imi lar mechanism, overproduction of prolactin may play a role in erecti ledisorder and impotence. The effects of hormones on the production of mi lk or lactation are l i s ted in Table 20-1.

Table 20-1. Hormones Involved in Lactation

Prolactinoma: The presence of otherwise benign growth of prolactin-producing cel l s in the anterior pi tui tary gland, known as a prolactinoma,can lead to severa l concerning and even dangerous s igns and symptoms. The increased number of these cel l s can lead to the worryingproduction of mi lk (galactorrhea) in a nonpregnant woman or even a man. Women can a lso experience i rregular or missed menstrua l cyclesand/or inferti l i ty because of the inhibi tory effects of excess prolactin on GnRH functions . More concerning, though, i s the presence ofheadaches and loss of periphera l vi s ion (bi tempora l hemianops ia) because of the pressure placed on the optic chiasm by the growingtumor. People may have a prolactinoma but wi l l be unaware unti l these mass effect symptoms appear. Treatment of a prolactinoma isusual ly via medications (e.g., bromocriptine or cabergol ine) or neurosurgery.

MALE REPRODUCTIVE SYSTEMThe development of the male reproductive system rel ies on the early activi ty of the sex-determining region (sry) found on the Y chromosome andits subsequent effects on the Wolffian and Mul lerian ducts (see above). In males , s ry and AMH activi ties a long with the cri ti ca l influence oftestosterone resul t in the formation of male sexual characteris tics and the regress ion of the Mul lerian ducts .

TESTOSTERONE

Testosterone i s the primary male s teroid hormone and i s produced in the testes by Leydig cel l s . Testosterone i s produced from cholesterol viathe androgen synthetic pathway noted in Chapter 3. The enzyme 5α-reductase converts testosterone into the much more active DHT (Figure 20-9).Testosterone can a lso be converted to estradiol by the action of the aromatase enzyme. Estradiol i s bel ieved to be important in malereproductive functions but i ts exact role i s s ti l l being elucidated. Women a lso produce smal ler amounts of testosterone from the adrenalglands , ovaries , and, in time of pregnancy, the placenta.

Figure 20-9. Conversion of Testosterone to 5α-Dihydrotestosterone by the Enzymatic Action of 5α-Reductase (see text for further discuss ion). [Adaptedwith permiss ion from Barrett KE, et a l .: Ganong’s Review of Medica l Phys iology, 23rd edi tion, McGraw-Hi l l , 2010.]

Defect of the 5α-Reductase-2 Enzyme: The presence of male or female external geni ta l s tructures i s determined by the presence or absence ofthe testicular hormone, testosterone, and i ts derivative, DHT. Unti l the discovery of DHT, i t was assumed that the development of the malereproductive tract depended enti rely on testosterone. For example, genetic males lacking 5α-reductase-2 activi ty and, therefore DHT, havenormal Wolffian s tructures because this aspect of development i s control led by testosterone. However, the lack of DHT a lso resul ts in anexternal female phenotype, except that the vagina i s incompletely developed because of the actions of AMH. FSH and LH levels are a lsonormal because of normal s igna l ing by testosterone. As a resul t, these genetic males can have male, female, or abnormal geni ta l ia withsubsequent gender identi ty becoming a choice for the patient. Furthermore, puberty wi l l increase testosterone levels , which can beconverted to DHT in sufficient quanti ty by the i soenzyme 5α-reductase-1, potentia l ly ini tiating the development of adul t male characteris tics ,including a penis and scrotum but s ti l l lacking the prostate. For the patient who has chosen a female gender identi ty, surgica l genderconvers ion i s required.

Testosterone and the resul ting DHT function in a variety of di fferent ways throughout the human body. The effects of testosterone on earlydevelopment have been noted above. Starting in puberty, testosterone and other androgens ri se in concentration at the s tart of puberty and,as s teroid hormones , enter into cel l s via pass ive di ffus ion through the plasma membrane with convers ion to DHT via 5α-reductase-2. Severa lsecondary sexual characteris tics in males and females are the resul t of testosterone/DHT, including pubic, axi l lary and additional facia l ,chest and leg ha i r, increased body oi l production and body odor, increased growth (e.g., bone and muscle), enlargement of the penis andcl i toris a long with increased sexual drive and erecti le frequency, changes in facia l bone s tructure, voice changes , and the onset ofspermatogenes is (male) and oogenes is (female). Testosterone a lso contributes to the regulation of platelet aggregation by influence onplatelet thromboxane A2.

Benign Prostatic Hyperplasia (BPH): BPH cl inica l ly a ffects a lmost 50% of men in thei r 50s and over 75% of men in thei r 60s , occurring more oftenin men from western l i fes tyle areas . Al though the exact mechanis tic deta i l s of the benign growth of the prostatic cel l s (s tromal andepithel ia l ) are s ti l l unknown, the effect of androgens as wel l as estrogen are wel l es tabl i shed. DHT i s bel ieved to play a prominent role viabinding as a homodimer to androgen receptors in the s tromal cel l s with resul ting activation of DNA transcription and increased express ionof growth factors for these cel l s . Indeed, DHT binds more tightly to the androgen receptor than does testosterone. 5α-Reductase levels ,respons ible for converting testosterone to DHT, are very high in these affected cel l s , wi th DHT eas i ly a ffecting nearby epi thel ia l cel l s .Es trogen (estradiol ) a l so appears to function in later l i fe, perhaps by sens i ti zing these cel l s to the effects of DHT, a l though this mechanismis even less wel l understood. Ini tia l treatment i s often by “androgen receptor blockers” (name often ends in “os in”), which block theandrogen/DHT receptor, or by di rect inhibitors of 5α-reductase (name usual ly ends in “s teride”). Surgica l or other more aggress ive treatmentmodal i ties may be needed in some patients .

Reproduced with permiss ion from Katzung BG, et a l .: Bas ic and Cl inica l Pharmacology, 11th edi tion, McGraw-Hi l l , 2009.

FSH AND LH

FSH and LH have important roles in the male reproductive system (Figure 20-10) as wel l as in the female. FSH i s cri ti ca l in the development ofthe semini ferous tubules as wel l as sperm (spermatogenes is ). FSH promotes spermatogenesis by binding to i ts receptor located on Sertoli cells,a l so ca l led “nurse cel l s ,” which l ine the tubules .

Figure 20-10. Effects of Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH) on the Testes. LH s timulates the Leydig cel l s to secretetestosterone. FSH s timulates the secretion of androgen-binding protein by the Sertol i cel l s into the lumen, which binds and concentratestestosterone in the semini ferous tubules at the s ight of spermatogenes is . See text for further deta i l s . [Reproduced with permiss ion fromKibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

Sertol i cel l s are primari ly respons ible for the s tructura l and nutrient support of developing sperm, including the fol lowing: Induces meios is of spermatogenic cel l s . Secretes androgen-binding protein, a glycoprotein that binds and concentrates required testosterone near to the developing sperm cel l s . Sertol i cel l aromatase converts testosterone to 17β-estradiol , whose function in the male reproductive system is s ti l l being elucidated. Secretes activin and inhibin, thereby regulating FSH secretion (see above). Produces and secretes AMH (discussed above).

LH increases the activi ty in Leydig cel l s (Figure 20-10) of the cholesterol sidechain cleaving enzyme (Figure 3-9), which converts cholesterol topregnenolone (Chapter 4). As a resul t, Leydig cel l s increase synthes is and secretion of testosterone as wel l as androstenedione. FSH a lsoplays a role by increas ing the number of receptors for LH on Leydig cel l s .

REVIEW QUESTIONS1. How would you describe (a) Wolffian ducts , (b) Mul lerian ducts , (c) sex-determining region of the Y chromosome (s ry), and (d) anti -Mul lerian

hormone?2. What i s the function of each of the fol lowing hormones : (a ) fol l i cle-s timulating hormone (FSH), (b) luteinizing hormone (LH), (c) es trogen, (d)

progesterone, (e) human chorionic gonadotropin (hCG), (f) oxytocin, (g) prolactin, (h) testosterone, and (i ) 5α-dihydrotestosterone (DHT)?3. What i s the bas ic pathway of action for the fol lowing processes : (a ) female development, (b) male development, (c) oogenes is , (d)

spermatogenes is , (e) female menstrua l cycle (including the actions and effects of the various hormones involved), and (f) ferti l i zation?4. What are the roles and importance of G proteins in the receptor-mediated functions of the sex hormones?

SECTION IIIINTEGRATED USMLE-STYLE QUESTIONS AND ANSWERS

QUESTIONS

III-1. A middle-Eastern fami ly presents for eva luation because their infant son died in the nursery with severe hemolys is and jaundice. Thecouple has two prior female infants who are a l ive and wel l , and the wi fe relates that she lost a brother in infancy with severehemolys is induced after a vi ra l infection. The phys ician suspects glucose-6-phosphate dehydrogenase (G6PD) deficiency, implyingdefective synthes is of which of the fol lowing compounds?A. Deoxyribose and nicotinamide adenine dinucleotide phosphate (NADP)B. Glucose and lactateC. Lactose and NADPHD. Ribose and NADPHE. Sucrose and NAD

III-2. Release of which of fol lowing peptides to the blood expla ins the enhanced insul in secretion after a meal rich in carbohydrate?A. Cholecystokinin (CCK)B. GastrinC. Glucose-dependent insul inotropic peptide (GIP)D. SomatostatinE. Vasoactive intestina l peptide (VIP)

III-3. You are asked to review the resul ts for two patients who are s i s ters , both of whom have been diagnosed with a tha lassemia. Mary hasa severe case that requires frequent blood transfus ions . Al ice has mi ld microcytic, hypochromic anemia. One parent was born in Greeceand the other in the Phi l ippines . Both chi ldren express the β-globin gene at 30% of the expected normal level . Mary expresses α-globinin normal amounts . However, Al ice expresses the α-globin at only 50% of normal . This di fference in the α-globin express ion betweenthe s i s ters in some way leads to the di fference in severi ty of the diseases in the s i s ters . Which of the fol lowing explanations accountfor Mary’s more severe tha lassemia?A. Al ice has higher i ron levels than does Mary so that i ron deficiency accounts for the greater severi ty.B. Mary has hemoglobin (Hgb) H disease, which i s s igni ficantly more severe than Al ice’s β-tha lassemia intermedia .C. Mutation in the α-globin gene i s expressed more in Mary’s developing red blood cel l s .D. Precipi tation of α-globin tetramers in Mary’s red blood cel l s ki l l s the developing cel l s .E. Synthes is of ε-globin in Al ice’s developing red blood cel l s compensates for her β-globin gene mutation.

III-4. A 10-year-old female presents with chest pa in and xanthomas over her elbows and knees . Her father died of a heart attack at age 35.Her mother has high cholesterol . Her phys ician suspects heterozygous fami l ia l hypercholesterolemia in the parents with homozygousdisease in the daughter. This disease resul ts from mutations in the receptor for low-dens i ty l ipoprotein (LDL) or the l igand portion ofi ts apoprotein coat. Which of the fol lowing apoproteins might be mutated?A. AIB. B100C. B48D. CIIE. E

III-5. Infants born prematurely are at ri sk for respi ratory dis tress syndrome. In such cases , i t i s common to adminis ter surfactant, the purposeof which i s to a l ter which of the fol lowing properties of water at the a lveolar interface with a i r?A. Dielectric constantB. EvaporationC. Heat of vaporizationD. IonizationE. Surface tens ion

III-6. Ful ly activated pyruvate carboxylase depends on the presence of which of the fol lowing substances?A. Acetyl -coenzyme (CoA) and biotinB. Acetyl -CoA and thiamine pyrophosphateC. Malate and niacinD. Oxaloacetate and biotinE. Oxaloacetate and niacin

III-7. The s trategy for therapy for dopamine deficiency in the substantia nigra of individuals with Parkinson disease i s indicated by which ofthe fol lowing?A. Competi tive inhibi tion of biosynthes is from his ti -dineB. Feedback inhibi tion of dopamine oxidationC. Provis ion of metabol i tes in the a lanine pathwayD. Provis ion of metabol i tes in the tyros ine pathwayE. Stimulation of monoamine oxidase (MAO)

III-8. A teenage female presenting with lack of menstruation (amenorrhea) i s found to have a 46XY karyotype, and deoxyribonucleic acid(DNA) testing shows a dys functional testosterone receptor characteris tic of androgen insens i tivi ty syndrome. Which of the fol lowingstatements accurately describes sex hormones such as testosterone?A. They bind speci fic membrane receptorsB. They cause release of a proteinaceous second messenger from the cel l membraneC. They enhance transcription when bound to receptorsD. They inhibi t trans lation through speci fic cytoplasmic proteinsE. They interact with DNA di rectly

III-9. A patient has compla ined of muscle weakness that has gradual ly worsened. After running an exercise tolerance test and otheranalyses , you conclude that patient has McArdles disease. Which of the fol lowing i s a resul t from this patient’s exercise tolerance testthat would have this speci fic diagnos is as opposed to other muscle carbohydrate disorders?A. Decreased blood glucose due to increased muscle glucose uptake

B. Decreased blood lactateC. Decreased fat oxidation in muscleD. Decreased muscle glycogenE. Decreased use of creatine phosphate

III-10. An elderly patient has been mostly housebound for the winter and has had a poor diet. In the spring, she presents to the emergencyroom (ER) with multiple vi tamin deficiencies . As a resul t of her deficiency in vi tamin C, she wi l l have a biochemica l defect in col lagenmaturation. Which of the fol lowing characteris tics appl ies to this vi tamin C deficiency?A. Deficient activi ty of lysyl oxidaseB. Deficient disul fide bond formation, both intrachain and interchainC. Deficient proteolytic cleavage of disul fide-l inked C-and N-terminal extens ionsD. Inadequate col lagen messenger ribonucleic acid (mRNA) trans lationE. Inadequate hydroxylation of prol ine and lys ine res idues

III-11. Neura l tube defects such as anencephaly and spina bi fida have higher frequencies in certa in populations such as those of Cel tic originand in certa in regions such as South Texas . This suggestion of envi ronmenta l cause produced research showing that deficiency of whichof the fol lowing vi tamins i s associated with the occurrence of neura l tube defects (anencephaly and spina bi fida)?A. Ascorbic acid (vi tamin C)B. Fol ic acidC. Niacin (vi tamin B3)

D. Riboflavin (vi tamin B2)

E. Thiamine (vi tamin B1)

III-12. Which of the fol lowing substances i s secreted by gastric parieta l (oxyntic) cel l s?A. CCKB. GastrinC. Intrins ic factorD. Moti l inE. Somatostatin

III-13. A patient presents with a hemolytic anemia due to a drug treatment. Which of the fol lowing would be a consequence of this disorder?A. Higher percent of erythrocytes that have reta ined their mitochondriaB. Higher percent of reticulocytes in the bloodC. Increase in the amount of feta l Hgb (HgbF) to compensate for the drug effectD. Lower percent of white blood cel l s (WBCs)E. More reticulocytes that have lost thei r mitochondria and nuclei

III-14. A 45-year-old man i s found to have an elevated serum cholesterol of 300 mg percent measured by s tandard conditions after a 12 hr fast.Which of the fol lowing l ipoproteins would contribute to a measurement of plasma cholesterol in a normal person fol lowing a 12 hrfast?A. Chylomicron remnants and very-low-dens i ty-l ipo-proteins (VLDLs)B. Chylomicrons and VLDLsC. High-dens i ty l ipoproteins (HDLs) and LDLsD. LDLs and adipocyte l ipid dropletsE. VLDLs and LDLs

III-15. A comatose infant i s brought to the ER. In the course of the examination, plasma ammonia was found to be elevated 20-fold overnormal va lues . Urine orotic acid and uraci l were both greater than normal . A defect of which of the fol lowing enzymes i s the most l ikelydiagnos is?A. ArginaseB. Argininosuccinate lyaseC. Carbamoyl phosphate synthetase I (ammonia)D. Carbamoyl phosphate synthetase II (glutamine)E. Orni thine transcarbamoylase

III-16. During an overnight fast, which of the fol lowing i s the major source of blood glucose?A. Dietary glucose from the intestineB. Gluconeogenes isC. Glycerol from l ipolys isD. Hepatic glycogenolys isE. Muscle glycogenolys is

III-17. One of the mechanisms of hypersens i tivi ty reactions that i s associated with thrombocytopenia involves antigens promoting theproduction of antibodies , which attach to the patient’s cel l membranes . These cel l s become recognized as foreign and are then taggedwith immunoglobul in (Ig) G or IgM antibodies . This makes these cel l s susceptible to attack by natura l ki l ler (NK) or macrophage cel l s .Which of the fol lowing types of hypersens i tivi ty reactions does this mechanism describe?A. AnaphylacticB. Cel l mediatedC. CytotoxicD. DelayedE. Immune complex

III-18. The arteria l blood gas (ABG) test resul ts for your patient show pH 7.49 (N: 7.35–7.45), pCO2 = 25 mmHg (N: 35–45), and HCO3− = 19 mEq/L (N:

24–28). On the bas is of these resul ts , which of the fol lowing conditions do you predict exis ts in your patient?A. Metabol ic acidos is with increased renal reabsorption of bicarbonateB. Metabol ic a lka los is with increased renal excretion of bicarbonateC. Respiratory acidos is with increased renal reabsorption of bicarbonateD. Respiratory a lka los is with increased renal excretion of bicarbonate

III-19. A patient i s found to have a defect in cycl ic guanos ine monophosphate (cGMP) phosphodiesterase. In which of the fol lowing ways

would this most di rectly affect the visua l cycle in this patient?

A. Fa i lure of Na +–K+-ATPase to function

B. Inward Na +–Ca 2+ channels of the rod cel l membrane remain closedC. Membrane of the rod cel l remains depolarizedD. Requirement for retinol becomes increasedE. Transducin cannot become activated

III-20. Which of the fol lowing describes a mechanism of action of a hypothalamic factor or pi tui tary hormone?A. Antidiuretic hormone (ADH) acts on kidney via Gq, leading to increased intracel lular ca lcium.

B. Corticotropin-releas ing hormone (CRH) acts on corticotropic cel l s by increas ing intracel lular ca lcium.C. Gonadotropin-releas ing hormone (GnRH) acts on gonadotropic cel l s by increas ing intracel lular ca lcium.D. Somatostatin (growth hormone-inhibi ting hormone) acts on somatotrophs via Gs , leading to increased intracel lular cycl ic adenos ine

monophosphate (cAMP).E. Thyrotropin-releas ing hormone acts on thyrotrophs via Gs, leading to increased intracel lular cAMP.

III-21. A 22-year-old woman engaging in a pol i ti ca l protest goes on a hunger s trike on a prominent corner in a ci ty park. Al though food i soffered to her severa l times each day by socia l workers and the pol ice, she refuses a l l offers except for water through the fi rs t 2 weeks .An examination of a sample of this woman’s bra in ti s sue would reveal that her bra in had adapted to us ing which of the fol lowing asfuel?A. Amino acidsB. Free fatty acidsC. GlucoseD. GlycerolE. Ketone bodies

III-22. Which of the fol lowing describes a feature of intestina l absorption of amino acids from the diet?

A. Driven by a Na + gradient from the intestina l lumen to the ins ide of the cel lB. Occurs by s imple di ffus ion

C. Occurs via cotransport with K+

D. Uptake of individual amino acids i s not a feature

E. Uses the same Na +-dependent transporter as glucoseIII-23. Which of the fol lowing s tatements accurately characterizes feta l hemoglobin (HgbF)?

A. Completely replaced prior to bi rthB. Exhibi ts no Bohr effectC. Includes two β-subunits and two γ-subunitsD. Lower affini ty for 2,3-bisphosphoglycerate (BPG) relative to adult HgbE. Lower affini ty for oxygen (O2) relative to adult Hgb

III-24. Coronary artery disease i s a multi factoria l disorder involving occlus ion of the coronary artery with atherosclerotic plaques . Severa lMendel ian disorders affecting l ipid metabol i sm increase susceptibi l i ty for heart attacks , whereas envi ronmenta l factors includesmoking and high-fat diets . A 45-year-old man has a mi ld heart attack and i s placed on diet and s tatin therapy. Which of the fol lowingwi l l be the most l ikely resul t of this therapy?A. High blood cholesterolB. High blood glucoseC. Low blood glucoseD. Low blood LDLsE. Low oxidation of fatty acids

III-25. A col lege s tudent with a normal medica l his tory col lapses during an intramural basketbal l game. His friends think he i s joking and thennotice his blue color and ca l l an ambulance. Resusci tation i s unsuccess ful and the autopsy reveals di lated cardiac chambers withincreased thickness of the ventricular wal l s (hyper-trophic, di lated cardiomyopathy). Electron microscopy of the heart muscle showsabnormal thick fi laments , and the pathologis t suspects a genetic disorder. Genes encoding which of the fol lowing proteins would bemost l ikely to reveal the causative mutation?A. α-ActininB. ActinC. Myos inD. Tropomyos inE. Troponin

III-26. A 14-year-old gi rl i s brought to the cl inic by her father with a compla int of l ightheadedness experienced on the soccer field earl ier in theafternoon. She s tated that she fel t cold and nearly fa inted severa l times , and that the symptoms did not resolve even after she drank apower beverage. On further questioning, her father s tated that she had been very thi rs ty recently, which bothered him because i t meanthaving to make frequent bathroom stops whi le driving on trips . She a lso “eats l ike a horse” and never seems to ga in any weight or growtal ler. Phys ica l examination reveals a thin gi rl who i s at the 30th percenti le for height and weight. A rapid dipstick test reveals glucosein her urine. Eva luation of this gi rl ’s l i ver would reveal an increased rate of which of the fol lowing processes?A. Fatty acid synthes isB. Glycogenes isC. Glycolys isD. Ketogenes isE. Protein synthes is

III-27. In a patient with a type I col lagen mutation, which of the fol lowing groups of diseases would you cons ider in a diagnos is?A. Alport syndrome, Goodpasture syndrome, benign fami l ia l hematuriaB. Col lagenopathies types II and XI, hypochondrogenes isC. Dystrophic epidermolys is bul losa , epidermolys is bul losa acquis i taD. Ehlers–Danlos syndromes types I , I I , VII; os teogenes is imperfectaE. Ehlers–Danlos syndromes types II I , IV; aneurysm

III-28. Which of the fol lowing characterizes nephrogenic diabetes ins ipidus?A. Defect in aquaporin-2B. Defect in the Gq protein

C. Defect in the angiotens in I I receptorD. Increased osmola l i ty of urineE. Mutant (inactive) ci rculating vasopress in (ADH)

III-29. Some cytokines , such as interleukin (IL)-1, activate phosphol ipase C and subsequently activate the nuclear factor-kappa B (NF-κB) gene.Which of the fol lowing receptor classes i s associated with this mechanism?A. Chemokine receptorsB. Ig receptorsC. Transforming growth factor-β receptorsD. Tumor necros is factor (TNF) receptorsE. Type I receptors

III-30. A 35-year-old male presents to your office with a 1-year his tory of shortness of breath. His shortness of breath has increased in severi tyover the past year. Ini tia l ly, his shortness of breath was noticeable only on exertion with moderate activi ty, but over the past 6 monthshe becomes short of breath with mi ld activi ty. Further his tory reveals that the patient has been a two pack per day smoker s ince the ageof 14 years when “he got hooked on cigarettes in high school .” He consumes a lcohol very moderately. On phys ica l examination, he i ss l ightly tachypneic at rest with use of accessory neck muscles during inspi ration. His chest i s barrel shaped and there i s clubbing of thedis ta l fingers . You obta in a chest X-ray that reveals hyperlucency of the lungs and a number of vi s ible blebs . Pulmonary function testingreveals changes of chronic obstructive lung disease cons is tent with emphysema. Because of his age, you order a plasma proteinelectrophores is , which reveals a decreased peak in the α-1 region. You make a diagnos is . Which of the fol lowing s tatements mostl ikely expla ins the emphysema in this patient?A. α-1-Anti tryps in deficiency leading to elevated activi ty of elastase that destroys lung ti ssue.B. Elastase deficiency that causes excess ive accumulation of elastin and clogs the lungs .C. Oxidants from smoking generate reactive oxygen species that activate elastase that destroys lung ti ssue.D. Oxidants from smoking generate reactive oxygen species that activate lung-speci fic col lagenase that destroys lung ti ssue.E. The patient has developed a nonalcohol ic hepati ti s that blocks α-1-anti tryps in secretion, leading to increased elastase activi ty and

destruction of lung ti ssue.III-31. A patient i s found to have a rare disease in which the secretory function of the α-cel l s of the pancreas i s impaired. Di rect s timulation of

which of the fol lowing pathways in l iver wi l l be impaired?A. Ci tric acid cycleB. Glycogenes isC. Gluconeogenes isD. Glycolys isE. Pentose phosphate pathway

III-32. In patients with Niemann–Pick C disease, which of the fol lowing cel lular processes would be defective?A. Cholesterol es teri fi cation in the cytoplasmB. Cholesterol hydroxylation for bi le sa l t formationC. Cholesterol trafficking to the GolgiD. Internal i zation of LDL particlesE. Lyosomal hydrolys is of cholesterol es ter

III-33. Which of the fol lowing describes the correct function for l ingual l ipase?A. De-esteri fi cation of cholesterol es ter l ipid to cholesterol and free fatty acid.B. Hydrolys is of phosphol ipids to free fatty acid and diacylglycerol .C. Primari ly works in the mouth where the pH i s closer to neutra l .D. Produces glycerol and three free fatty acids from triglycerides conta ining medium-chain fatty acids .E. Produces long-chain free fatty acids from the ini tia l hydrolys is of triglycerides .

III-34. How is the relaxed (R) s tate of Hgb s tructura l ly defined?A. Binding of 2,3-bisphosphoglycerate (BPG) to a cleft between the Hgb subunitsB. Binding of carbon dioxide (CO2) to the N-terminus

C. Ionizable his tidines being protonatedD. Subunits being dissociatedE. The pos i tion of i ron in the plane of the heme

III-35. Which of the fol lowing i s the major source of extracel lular cholesterol for human ti ssues?A. AlbuminB. γ-Globul inC. HDLsD. LDLsE. VLDLs

III-36. Which of the fol lowing i s a feature of the enzyme that produces the hormone that peaks shortly before ovulation i s ini tiated in themenstrua l cycle?A. Activated by fol l i cle-s timulating hormone (FSH)B. Activated by luteinizing hormone (LH)C. Consti tutively active to produce estriol (E3) from androstenedione

D. Feedback inhibi ted by estradiol -17β (E2)

E. Forms estrone (E1) from testosterone

III-37. A 4-month-old presents with hypoglycemia and hepatomegaly. Injection of glucagon produces no elevation of blood glucose or bloodlactate, yet a lanine can be converted to glucose. Which of the fol lowing l iver enzymes would most l ikely be defective in this patient?A. Glucose-6-phosphataseB. G6PD

C. Glycogen phosphorylaseD. Pyruvate carboxylaseE. Pyruvate kinase

III-38. Which of the fol lowing diseases wi l l be found in an adult with insufficient amounts of ca lcium and phosphate in the blood?A. HyperparathyroidismB. OsteomalaciaC. Osteoporos isD. Paget’s diseaseE. Rickets

III-39. Which of the fol lowing conditions would cause renin levels to ri se?A. A chronic high-sa l t dietB. Acute treatment with atria l natriuretic peptide (ANP)C. Chronic treatment with an a ldosterone receptor antagonis tD. Treatment with a minera locorticoid receptor agonis tE. Treatment with angiotens in I I

III-40. Which of the fol lowing i s the correct chronologica l order of the major biochemica l events occurring during one cycle of skeleta l musclecontraction and relaxation?1. Actin i s released from the complex2. ATP binds the head of myos in3. Power s troke4. Head of myos in binds access ible actin5. Head of myos in hydrolyzes ATP to ADP and Pi

A. 1, 2, 3, 4, 5B. 4, 2, 5, 3, 1C. 2, 4, 5, 3, 1D. 2, 5, 4, 3, 1E. 5, 4, 3, 2, 1

III-41. Fol lowing traumatic injury, epinephrine i s secreted and subsequently decreases insul in secretion by the β-cel l s of the pancreas . Thisinhibi tion occurs via activation of α2-adrenergic receptors leading to decreased production of cAMP. Al though pancreatic β-cel l s a lsohave β2-adrenergic receptors that increase cAMP, the α2-receptors are more abundant, and with elevated epinephrine in trauma, thislatter effect predominates . Given this information, why would a chi ld infected with pertuss is toxin potentia l ly fa i l to decrease insul insecretion fol lowing a traumatic injury?A. Toxin activates Gs-protein activi ty so that the effect of epinephrine in trauma is offset.

B. Toxin acts l ike a tumor promoter to activate protein kinase C that in turn increases insul in synthes is .C. Toxin di rectly faci l i tates the opening of ca lcium channels so that inward ca lcium movement can promote insul in secretion.D. Toxin inhibi ts cAMP phosphodiesterase to cause an increase in the cAMP concentration.E. Toxin inhibi ts Gi-protein activi ty so that the effect of epinephrine via α2-receptors i s blocked.

III-42. Which of the fol lowing s tatements describe the proteins that accumulate in class ic Creutzfeldt–Jakob disease?A. Bacteria l endotoxin protein that i s infectiousB. Derived from an infectious vi rusC. Infectious protein that can cause cancerD. Infectious protein that i s s imi lar to a normal protein in i ts secondary s tructureE. Intracel lular protein that reverts to an infectious form

III-43. Which of the fol lowing s tatements expla in why fructose 2,6-bisphosphate i s important in regulating glycolys is in the l iver?A. Has a phosphate group with a high negative free energy of hydrolys isB. Is an a l losteric inhibi tor of phosphofructokinase-1C. Is cleaved to triose phosphates in the glycolytic pathwayD. Its formation i s cata lyzed by a glycolytic enzymeE. Provides an intracel lular s igna l that i s sens i tive to changes in blood glucose levels

III-44. Which of the fol lowing enzymes involved in carbohydrate digestion i s released by the exocrine pancreas?A. AmylaseB. DextrinaseC. GlucoamylaseD. LactaseE. Sucrase

III-45. Which of the fol lowing characterizes s ickle cel l disease?A. Aggregation of Hgb tetramers due to a hydrophobic effectB. Arises from a point mutation changing glutamate to lys ine on the β-subunit of HgbC. Binding of BPG to Hgb i s diminishedD. Express ion of the s ickled phenotype involves a hydrophi l i c interactionE. Feta l Hgb (HgbF) i s s igni ficantly affected

III-46. A 28-year-old woman presents to her obstetrician at week 23 of pregnancy, compla ining of extreme fatigue. Eva luations of serum i ronlevel and feta l wel l being are normal , but the nurse notices a weak and i rregular pulse. Chest X-ray reveals an enlarged heart and theelectrocardiogram (ECG) reveals a short PR interva l and prolonged QRS, including a s lurred-up s troke of the R-wave ca l led a δ-wave. TheECG is read as showing Wolff–Parkinson–White syndrome, a condition with ri sks for paroxysmal supraventricular tachycardia . Thephys ician cons iders treatment with ca lcium channel regulators , ba lancing their ri sks to mother and fetus . Contraction of cardiac andskeleta l muscle i s ini tiated by the binding of ca lcium to which of the fol lowing substances?A. ActinB. Actomyos inC. Myos in

D. Tropomyos inE. Troponin

III-47. Which of the fol lowing i s an action of dihydrotestosterone?A. Anabol ic effects on skeleta l muscleB. Development of the prostateC. Development of the testesD. Preventing uterine developmentE. Promoting spermatogenes is

III-48. Which of the fol lowing would patients with primary hyperparathyroidism be unable to suppress (via feedback inhibi tion)?A. 1-Hydroxylase enzyme in vi tamin D metabol i sm by ca lci toninB. 25-Hydroxylase enzyme in vi tamin D metabol i sm by a high blood concentration of active vi tamin D3

C. Action of parathyroid hormone (PTH) on osteo-blasts by sex s teroidsD. Production of ca lci tonin in the thyroid by a high blood concentration of ca lciumE. Production of PTH by a tumor by a high blood concentration of ca lcium

III-49. A patient presents with a deficiency of thiamine. If this patient’s l iver cel l s are compared with normal cel l s , which of the fol lowingsubstances would be produced in the thiamine-deficient cel l in lesser amounts i f the cel l s are only given glucose as a fuel?A. AlanineB. CO2

C. Lactate

D. NADP+

E. Pyruvate

III-50. The ABG test resul ts for your patient show pH 7.31 (N: 7.35–7.45), pCO2 = 25 mmHg (N: 35–45), and HCO3− = 20 mEq/L (N: 24–28). On the bas is

of these resul ts , which of the fol lowing conditions do you predict exis ts in your patient?A. AnxietyB. Chronic obstructive pulmonary disease (COPD)C. Diabetic ketoacidos isD. Hyperammonemia

III-51. Which of the fol lowing i s a feature of the renin–angiotens in system?A. Angiotens inogen synthes ized in, and released by, the juxtaglomerular apparatusB. Renin proteolytica l ly process ing angiotens in I to angiotens in I IC. Angiotens in I binding to the angiotens in receptorD. Angiotens in I I acting as a vasoconstrictorE. Angiotens in-converting enzyme cleaving angiotens in I I to angiotens in I I I

III-52. A postmortem sample of a dementia patient showed evidence of A-β peptide and hyperphosphorylated tau protein depos i ted inplaques . Which of the fol lowing observations i s cons is tent with this postmortem finding?A. Alzheimer dementiaB. Colchicine poisoningC. New variant Creutzfeldt–Jakob diseaseD. Niemann–Pick type AE. Parkinson’s disease

III-53. Creatine phosphate i s a high energy phosphate compound that i s found in skeleta l muscle. Which of the fol lowing s tatements mostl ikely expla ins why there would be l i ttle benefi t to a normal individual to take creatine supplements to boost muscle creatinephosphate concentration?A. Normal creatine phosphate concentration i s sufficient to ini tiate exercise unti l glycogenolys is beginsB. Excess creatine wi l l a l losterica l ly inhibi t the creatine kinase reactionC. Excess creatine intake wi l l cause kidney damage in most individualsD. Creatine phosphate normal ly i s sufficient to ini tiate exercise unti l fatty acid oxidation beginsE. Creatine cannot cross the muscle cel l membrane

III-54. An increased concentration of which of the fol lowing hormones prevents lactation from occurring in pregnancy by blocking the action ofprolactin?A. Androstenedione and progesteroneB. Estradiol and estroneC. Growth hormone and estriolD. Human chorionic gonadotropin (hCG) hormone and progesteroneE. Progesterone and estriol

III-55. Two patients present, one of whom has a defect in glucose-6-phosphatase and the other exhibi ts a defect of fructose 1,6-bisphosphatase. Which of the fol lowing i s most l ikely the condition that would only be found in the patient with the defect in glucose-6-phosphatase?A. Alanine accumulation in the blood fol lowing food deprivationB. Al tered muscle metabol i sm of glucoseC. Glycogen accumulation in the l iverD. Lactic acidos isE. Pentose phosphate pathway i s not functional

III-56. A 7-year-old gi rl has a 1-month his tory of foul -smel l ing diarrhea. Upon further inquiry, the frequency seems to be 4–6 s tools per day. Shehas a lso had trouble seeing at night in the past 2 weeks . Her WBC count i s normal . Phys ica l examination i s enti rely normal .Examination of a s tool sample reveals that i t i s bulky and greasy. Analys is does not reveal any pathogenic microorganisms or paras i tesbut confi rms the presence of fats . Further eva luation of this patient would l ikely reveal which of the fol lowing conditions?A. DiabetesB. Gastrointestina l (GI) infectionC. I lea l disease

D. Insufficient bi le productionE. Lactose intolerance

III-57. Which of the fol lowing would be the earl ies t event in the clotting cascade?A. Binding of platelets to red blood cel l sB. Binding of vi tamin K to endothel ia l cel l surfacesC. Formation of fibrin D-dimersD. Release of ti s sue factor (thromboplastin) by damaged vesselsE. Secretion of von Wi l lebrand factor by platelets

III-58. A patient with myocardia l infarction i s treated with ni troglycerin to di late his coronary arteries . Which of the fol lowing best describesthe action of ni troglycerin?A. Acetyl -CoA and chol ine are condensed to form a neurotransmitterB. Arginine i s converted to a neurotransmitter that activates guanylyl cyclaseC. Guanos ine triphosphate hydrolys is accompl ishes oxidation of LDL proteinsD. Methylation occurs to produce S-adenosylmethio-nineE. Tyros ine i s converted to serotonin

III-59. Which of the fol lowing cytokines i s produced by helper T (Th) cel l s?A. Interferon-αB. IL-1C. IL-2D. IL-3E. IL-5F. IL-10G. Neutrophi l chemotactic factor

III-60. Which of the fol lowing s tatements accurately describes functions of α1-adrenergic receptors?

A. Bronchodi lation and decreased s trength of heart contractionB. Constriction of blood vessels and decreased heart rateC. Increased glycogenolys is and l ipolys isD. Vasoconstriction and reduced sympathetic outflow in the centra l nervous system (CNS)E. Vasodi lation and increased heart rate

III-61. Which of the fol lowing i s an inhibi tory neurotransmitter found exclus ively in the centra l nervous system?A. Acetylchol ineB. GlutamateC. NorepinephrineD. SerotoninE. Substance P

ANSWERS

III-1. The answer is D. Glucose-6-phosphate dehydrogenase (G6PD) i s the fi rs t enzyme of the pentose phosphate pathway, a s ide pathway forglucose metabol i sm whose primary purpose i s to produce ribose and NADPH (Figure 6-7A). Its deficiency i s the most commonenzymopathy. It contrasts with glycolys is in i ts use of NADP rather than NAD for oxidation, i ts production of CO2, and i ts production ofpentoses (ribose, ribulose, and xylulose). Production of NADPH by the pentose phosphate pathway i s crucia l for reduction ofglutathione, which in turn removes hydrogen peroxide. Erythrocytes are particularly susceptible to hydrogen peroxide accumulation,which oxidizes red blood cel l membranes and produces hemolys is . Stresses such as newborn adjustment, infection, or certa in drugscan increase red blood cel l hemolys is in G6PD-deficient individuals , leading to severe anemia, jaundice, plugging of renal tubuleswith released Hgb, renal fa i lure, heart fa i lure, and death. Because the locus encoding G6PD is on the X chromosome, the deficiencyexhibi ts X-l inked recess ive inheri tance with severe affl i ction in males and transmiss ion through asymptomatic female carriers . Ribose-5-phosphate produced by the pentose phosphate pathway i s an important precursor for ribo-nucleotide synthes is (Figure 6-7C).

III-2. The answer is C. CCK i s a peptide hormone whose main effect i s contraction of smooth muscle of the ga l l bla dder and s imultaneoussecretion of pancreatic solutions to increase digestion. However, emptying of the s tomach and gastric acid secretion i s a lso decreasedby CCK as digestion progresses beyond the s tomach. Gastrin s timulates HCl , peps inogen, and intrins ic factor secretion from parieta lcel l s , and peps inogen/renin by chief cel l s of the s tomach. Smooth muscle contraction (i .e., moti l i ty) of the s tomach i s a lso enhanced bygastrin. Somatostatin decreases release of gastrin, CCK, secretin, moti l in, VIP, GIP, and enteroglucagon, leading to decreased s tomachsecretion and contraction. VIP s timulates peps inogen/peps in secretion, di lutes bi le and pancreatic juice, increases bicarbonateproduction in the pancreas , decreases gastrin-induced gastric acid secretion, and increases water secretion in intestine. GIP bes idesincreas ing insul in secretion decreases the release of gastric acid by parieta l cel l s as wel l as smooth muscle contraction (moti l i ty) ofthe s tomach and increases fat metabol i sm by activating l ipoprotein l ipase (see Chapter 11).

III-3. The answer is D. The key to this answer i s the relative proportion of α-globin and β-globin that i s functional in each s i s ter. Normal adultHgb conta ins two α-globin and two β-globin cha ins . Both s i s ters have a s igni ficant deficiency of the β-globin gene to the same extent.Hence, this effect would be the same in both s i s ters . Al ice, unl ike Mary, a lso has reduced express ion of her α-globin. Consequently, incons idering the proportion of α-globin to β-globin in each s i s ter, Al ice has a ratio of approximately 50:30, whereas Mary has a higherproportion of 100:30, that i s , her α-globin exceeds the amount of her β-globin by cons iderably more. Therein l ies the problem for Mary.Because the α-globin i s more than three times the amount of β-globin, i t i s poss ible for α-globin tetramers to form. These tetramersare not soluble and precipi tate in the red blood cel l s , leading to cel l destruction and anemia. In terms of the other poss ible answers ,there i s no evidence of a di fference in i ron levels in the s i s ters . Hgb H disease cannot happen in Mary because this i s a condition inwhich Hgb has four β-globin subunits . Mary has no mutation of α-globin gene as express ion i s normal . Fina l ly, ε-globin i s an embryonicform (see Chapter 14).

III-4. The answer is B. The shel l of apoproteins coating blood transport l ipoproteins i s important in the phys iologic function of thel ipoproteins . Some of the apoproteins conta in s igna ls that target the movement of the l ipoproteins in and out of speci fic ti s sues . B48and E seem to be important in targeting chylomicron remnants to be taken up by l iver. B100 i s synthes ized as the coat protein of VLDLsand marks thei r end product, LDLs , for uptake by periphera l ti s sues . Other apoproteins are important for the solubi l i zation andmovement of l ipids and cholesterol in and out of the particles . CII i s a l ipoprotein l ipase activator that VLDLs and chylomicrons receivefrom HDLs . The “A” apoproteins are found in HDLs and are involved in leci thin–cholesterol acyl transferase regulation. Fami l ia lhypercholesterolemia causes early heart attacks in heterozygotes , particularly in males , and chi ldhood disease in rare homozygotes .

The daughter’s chest pa in was l ikely angina due to coronary artery occlus ion and her skin patches were fatty depos i ts known asxanthomata (Figure 16-8).

III-5. The answer is E. Pulmonary surfactant, composed of leci thin and myel in, i s secreted on a continuous bas is by type II a lveolar cel l s andClara cel l s beginning at approximately 20 weeks of gestation. Pulmonary surfactant has both a l ipid (~90% of tota l ) and a protein (~10%of tota l ) component. About ha l f of the l ipids are dipa lmitoylphosphatidylchol ine with a charged amine group on i ts head group. Theremaining l ipids include phosphatidylglycerol , which modulates the fluidi ty of the surfactant as wel l as cholesterol and other l ipids .The l ipids and proteins in surfactant a l l have the capabi l i ty of interacting with aqueous (hydrophi l i c) or nonaqueous (hydrophobic)envi ronments and i t i s this qual i ty that leads to thei r specia l i zed function in the lung (Figure 17-2). By di rectly interacting with a lveolarwater via thei r hydrophi l i c regions whi le the hydrophobic regions remain in the a i r, pulmonary surfactant creates a myel in meshworkthat l ines the a lveol i—a s trong, intertwined l ipoprotein system that i s ana logous to the myel in sheath of nerve cel l s . This uniquealveolar l ining greatly reduces surface tens ion, a l lowing eas ier expans ion/stretching and col lapse of a lveol i during respiration andthe resul ting changes in pressure. This reduction in surface tens ion makes the work of respi ration much less and reduces the tota lamount of pressure that must be generated for efficient and effective inspi ration and expiration. The pulmonary surfactant a lso helpsa l l the lung a lveol i to expand (inspi ration) and shrink (expiration) at the same rate, thereby reducing the chance for i solatedoverexpans ion and the tota l col lapse of the a lveolar sacs . Al though a l l the other options represent properties of water or solutions ,they have nothing to do with the properties of surfactant.

III-6. The answer is A. Pyruvate carboxylase cata lyzes the convers ion of pyruvate to oxa loacetate in gluconeogenes is : pyruvate + HCO3− + ATP →

oxaloacetate + ADP + Pi. For pyruvate carboxylase to be ready to function, i t requires biotin, Mg2+, and Mn2+. It i s a l losterica l ly activatedby acetyl -CoA. The biotin i s not carboxylated unti l acetyl -CoA binds the enzyme. By this means , high levels of acetyl -CoA s ignal the needfor more oxa loacetate. When ATP levels are high, the oxa loacetate i s consumed in gluconeogenes is . When ATP levels are low, theoxaloacetate enters the ci tric acid cycle. Gluconeogenes is only occurs in the l iver and kidneys .

III-7. The answer is D. Dopamine i s produced from L-3,4-dihydroxyphenyla lanine (L-DOPA), which in turn i s made from tyros ine. Therapy with theL-DOPA precursor increases dopamine concentrations and improves the rigidi ty and immobi l i ty that occur in Parkinson disease.Dopamine i s degraded in the synaptic cleft by MAO-A and MAO-B, producing 3,4-dihydroxyphenyl -aceta ldehyde (DOPAC). DOPAC is in turnbroken down to homovani l l i c acid, which can be measured in spina l fluid to assess dopamine metabol i sm. Inhibi tors of MAO-A andMAO-B have some use in treating Parkinson disease. The metabol i sm of his ti -dine or a lanine i s not related to that of dopamine, butphenyla lanine i s a precursor of tyros ine and L-DOPA (see Chapter 19; Figure 19-7A).

III-8. The answer is C. Al l s teroid hormones , including the sex hormones estrogen, testosterone, and progesterone, can be class i fied as group Ihormones , meaning that they act by binding speci fic cytoplasmic receptors that enter the nucleus and s timulate transcription byspeci fic DNA binding. Most nonsteroida l hormones , for example, epinephrine, are group II hormones that interact with the cel lmembrane and produce a second-messenger effect. The group II hormones , in contrast to s teroids , act in minutes , whereas s teroidhormones require hours for a biologic effect. Recent s tudies have indicated that speci fic cytoplasmic receptors for s teroid hormoneshave an extraordinari ly high affini ty for the hormones . In addition, the receptors conta in a DNA-binding region that i s ri ch in aminoacid res idues that form meta l -binding fingers . Likewise, thyroid hormone receptors conta in DNA-binding domains with meta l -bindingfingers . Like s teroid hormones , thyroid hormones are transcriptional enhancers .

III-9. The answer is A. Patients with McArdles disease are deficient in myophosphorylase, the muscle form of glycogen phosphorylase. Patientsusual ly present in chi ldhood with muscle pa in, fatigue, cramps, and weakness with excess ive myoglobin in the urine, indicative ofrhabdomyolys is during prolonged periods of exercise. Progress ive symptoms and muscle mass loss and weakness are usual ly evidentas the patient ages . If ini tia l exercise intens i ty i s too great, creatine phosphate i s quickly depleted. Because muscle glycogen cannotbe hydrolyzed, i t tends to be at greater concentrations in muscle and exercise cannot el ici t an increase in blood lactate. Diagnos is maybe compl icated by a “second wind” phenomenon exhibi ted by the patients . Evidence suggests that as long as the patient’s ini tia lexercise attempts are of very low intens i ty, within approximately 15 min, exercise can increase the mobi l i zation of glucose transportertype 4 to the plasma membrane. This faci l i tates increased uptake of glucose so the patient can use glycolys is to provide energy forsusta ining the exercise. Indeed patients often show a concomitant decrease in blood glucose that i s often s l ightly elevated inMcArdles ’ patients because of mi ld insul in res is tance. The abi l i ty to achieve second wind a lso a l lows the muscle to begin oxidizingfatty acids .

III-10. The answer is E. Prolyl and lysyl hydroxylases both require vi tamin C as a cofactor. These are i ron-conta ining enzymes , and vi tamin C(ascorbic acid) i s needed to mainta in i ron in i ts reduced (Fe2+) s tate. Al though lysyl oxidase uses the hydroxylys ine product of lysylhydroxylase as a substrate, i ts activi ty wi l l not be deficient. The lysyl oxidase enzyme requires copper and vi tamin B6 as cofactors .There wi l l be less a l lys ine product only because of a reduced amount of substrate. Disul fide bond formation occurs after thehydroxylation s tep and a l though i t wi l l be indirectly affected by vi tamin C deficiency, this process i f not deficient. mRNA trans lation ofcourse occurs prior to the hydroxylation s teps (see Figure 13-2B).

III-11. The answer is B. Spina bi fida , or myelomeningocele, i s a defect of the lower neura l tube that produces an exposed spina l cord in thethoracic or sacra l regions . Exposure of the spina l cord usual ly causes nerve damage that resul ts in para lys is of the lower l imbs andurinary bladder. Anencephaly i s a defect of the anterior neura l tube that resul ts in letha l bra in anomal ies and skul l defects . Fol ic acidis necessary for the development of the neura l tube in the fi rs t few weeks of embryonic l i fe, and the chi ldren of women withnutri tional deficiencies have higher rates of neura l tube defects . Because neura l tube closure occurs at a time when many women arenot aware that they are pregnant, i t i s essentia l that a l l women of chi ldbearing age take a fol ic acid supplement of approximately 0.4mg/day. Frank fol ic acid deficiency can a lso cause megaloblastic anemia because of a decreased synthes is of the purines andpyrimidines needed for cel l s to make DNA and divide. Deficiencies of thiamine in chronic a lcohol ics are related to Wernicke–Korsakoffsyndrome, which i s characterized by loss of memory, lackadais ica l behavior, and a continuous rhythmic movement of the eyebal l s .Thiamine dietary deficiency from excess of pol i shed rice can cause beriberi . Niacin deficiency leads to pel lagra , a disorder thatproduces skin rash (dermati ti s ), weight loss , and neurologic changes including depress ion and dementia . Riboflavin deficiency leadsto mouth ulcers (s tomati ti s ), chei los is (dry, sca ly l ips ), sca ly skin (seborrhea), and photophobia . Because biotin i s widely dis tributed infoods and i s synthes ized by intestina l bacteria , biotin deficiency i s rare. However, the heat-labi le molecule avidin, found in raw eggwhites , binds biotin tightly and blocks i ts absorption, caus ing dermati ti s , dehydration, and lethargy. Lactic acidos is resul ts as abui ldup of lactate due to the lack of functional pyruvate carboxylase when biotin i s miss ing. Vi tamin C deficiency leads to scurvy, whichcauses bleeding gums and bone disease.

III-12. The answer is C. Intrins ic factor i s normal ly produced by the parieta l cel l s of the s tomach and i s essentia l in a l lowing the absorption ofvi tamin B12 in the i leum. CCK i s a peptide hormone produced by cel l s in the duodenum. Its main effect i s contraction of smooth muscleof the ga l l bladder and s imultaneous secretion of pancreatic solutions to increase digestion. Gastrin i s produced by G-cel l s inresponse to presence of undigested proteins and/or dis tens ion of the antrum of the s tomach. Moti l in i s a peptide hormone mademainly in the duodenum and jejunum that increases smooth muscle contraction (fundus , antrum, and ga l l bladder). Somatostatin i sproduced in the s tomach, intestines , and pancreas .

III-13. The answer is B. The anemia i s a resul t of loss of red blood cel l s . To compensate, there i s an increased production of new cel l s . New cel lformation coupled to loss of mature cel l s creates a higher percent of reticulocytes . Erythrocytes a lways have no mitochondria and thereticulocytes a lways lose thei r mitochondria and nuclei . HgbF i s gone within 6 months after bi rth. WBCs count i s unrelated to a drug-

induced anemia.III-14. The answer is C. In the postabsorptional (postprandia l ) s tate, plasma conta ins a l l the l ipoproteins : chylomicrons derived from dietary

l ipids packaged in the intestina l epi thel ia l cel l s and their remnants ; VLDLs , which conta in endogenous l ipids and cholesterolpackaged in the l iver; LDLs , which are end products of del ipidation of VLDLs ; and HDLs , which are synthes ized in the l iver. HDLs are inpart cata lytic because transfer of thei r CII apol ipoprotein to VLDLs or chylomicrons activates l ipoprotein l ipase. In normal patients ,only LDLs and HDLs remain in plasma fol lowing a 12-h fast because both chylomicrons and VLDLs have been del ipidated. Most of thecholesterol measured in blood plasma at this time i s present in the cholesterol -rich LDLs . However, HDL-cholesterol a lso contributesto the measurement. In addition to tota l plasma cholesterol , the ratio of HDL (good) to LDL (bad) cholesterol i s a l so useful forpredicting heart attack ri sks (see Chapter 16).

III-15. The answer is E. Arginase cata lyzes the las t reaction of the urea cycle by cleaving arginine into urea and orni thine. Urea i s secreted fromthe l iver into the blood to be cleared by the kidney for excretion. The orni thine i s regenerated for another turn of the cycle.Argininosuccinate lyase cleaves argininosuccinate into fumarate and arginine in the urea cycle reaction that precedes arginase.Carbamoyl phosphate synthetase I i s an ini tia l reaction associated with the urea cycle s tep that cata lyzes the production ofcarbamoylphosphate from ATP, NH3, and CO2. This carbamoylphosphate i s then condensed with orni thine to produce ci trul l ine in areaction cata lyzed by orni thine transcarbamoylase. In the pyrimidine synthetic pathway, carbamoyl phosphate i s a lso produced in areaction cata lyzed by carbamoyl phosphate synthetase II that uses glutamine instead of NH3 as a ni trogen source. When orni thinetranscarbamoylase i s deficient in the l iver, carbamoyl phosphate accumulates and moves from the mitochondria l matrix into thecytoplasm. In the cytoplasm, this carbamoyl phosphate i s used to produce pyrimidine bases (orotic acid and uraci l ) that are producedin an uncontrol led manner with the excess being excreted in the urine (Figure 5-9A; Chapter 18).

III-16. The answer is D. In the absorptive phase fol lowing a meal , the major source of glucose i s glucose taken di rectly from the intestine intothe blood system. Much of this glucose i s absorbed into cel l s and, in particular, into the l iver via the action of insul in, where i t i ss tored as glycogen. Once the effects of daytime eating have subs ided and a l l the glucose from absorption has been s tored, the normalovernight fast begins . During this period, the major source of blood glucose i s hepatic glycogen. Through the effects of glycogenolys is ,which are mediated by glucagon, hepatic glycogen i s s lowly parceled out as glucose to the bloodstream, keeping blood glucose levelsnormal . In contrast, muscle glycogenolys is has no effect on blood glucose levels because no glucose-6-phosphatase exis ts in muscleand hence phosphorylated glucose cannot be released from muscle into the bloodstream. Fol lowing a more prolonged fast or in theearly s tages of s tarvation, gluconeogenes is i s needed to produce glucose from glucogenic amino acids and the glycerol released byl ipolys is of triglycerides in adipocytes . This i s because the l iver glycogen i s depleted and the l iver i s forced to turn to gluconeogenes isto produce the amount of glucose necessary to mainta in blood levels (Chapter 10).

III-17. The answer is C. The mechanism provided describes type II that i s a l so known as antibody dependent. The anaphylactic or a l lergy (type I)reaction involves repeat exposure to certa in a l lergens that leads to binding of IgE to i ts receptor on mast cel l s or basophi l s . Bindingreleases his tamine; leukotrienes B4, C4, and D4; pros-taglandin D2; platelet; eos inophi l ; and neutrophi l factors . Immune complex (typeIII) reaction occurs when antigen binds antibodies to produce immune complexes with complement proteins . Complexes depos i t insmal l blood vessels , joints , and glomerul i el i ci ting inflammatory response. Neutrophi l s and platelets cause ti ssue damage. Cel l -mediated or delayed (type IV) reaction i s associated with cytotoxic T cel l s recognizing an antigen and attacking the ti ssue, whereas Th1cel l s release cytokines , which attract monocytes and macrophages that cause most of the cel lular damage (see Table 15-3).

III-18. The answer is D. The patient’s pH i s above normal so this has to be an a lka los is . The low pCO2 i s indicative of increased expiration of

CO2. On the bas is of carbonic anhydrase reaction , this i s an example of respi ratorya lka los is . Increased expiration of CO2 (i .e., hyperventi lation) drives the reaction toward the left, thereby decreas ing the formation of

both protons (H+) and bicarbonate. The bicarbonate levels are decreased, indicating that the kidney has markedly decreasedreabsorption (increased excretion) of bicarbonate. This decreased reabsorption of bicarbonate, a base, by the kidney compensates forthe a l ready bas ic pH (Chapters 17 and 18).

III-19. The answer is C. Nutri tional vi tamin A (retinol esters ) i s converted in the retina to 11-cis-retina l , an i somer of a l l -trans-retina l forms byretina l i somerase. 11-cis-Retina l then covalently attaches to a lys ine res idue on the visua l protein ops in. The resul ting rhodops inmolecule becomes a G-protein-coupled receptor with the “l igand” for activation being l ight. When l ight s tikes the retina, photoexci tedrhodops in binds to a multi subunit membrane protein ca l led transducin which, in turn, activates a cGMP phosphodiesterase by caus ingdissociation of inhibi tory subunits . The phosphodiesterase cata lyzes the destruction of cGMP (cGMP → GMP) near the membrane of therod cel l . Normal ly, cGMP keeps inward Na +–Ca 2+ channels open to mainta in membrane depolarization. When cGMP levels fa l l , inwardNa +–Ca 2+ channels close, thereby lowering both intracel lular Na + and Ca 2+. The fa l l in Na + el i ci ts hyper-polarization caus ing the rod cel lto release less glutamate neurotransmitter. The reduced amount of this inhibi tory neurotransmitter generates an electrica l impulse tothe occipi ta l lobe of the bra in that triggers the perception of l ight (Figure 19-13).

III-20. The answer is C. Release of GnRH by the hypothalamus resul ts in activation of a speci fic GnRH receptor (GnRHR) located in the pi tui tarygland. This receptor i s a membrane-bound Gq-protein-coupled s timulator of phosphol ipase C, which resul ts in ca lcium release andprotein kinase C activation via convers ion of plasma membrane phosphatidyl inos i tol into inos i tol triphosphate and diacylglycerol . CRHis released from the hypothalamus and binds to a receptor on corticotrophs that acts via Gs-protein-coupled s timulation of adenylcyclase leading to production of cAMP. ADH works via a s imi lar mechanism as CRH. TRH, l ike GnRH, works via Gq protein. Somatostatinworks via Gi-protein-coupled inhibi tion of adenyl cyclase (Chapter 20).

III-21. The answer is E. This woman has created a sel f-imposed s tarvation through her hunger s trike. During s tarvation, many fuel sources arerecrui ted to support bodi ly functions , including protein degradation, which suppl ies amino acids as gluconeogenic precursors , andtriglyceride degradation, which yields glycerol , free fatty acids , and, eventual ly, ketone bodies . The bra in normal ly prefers glucose asi ts main fuel , so no adaptation i s needed. During s tarvation, changes in bra in gene express ion upregulate severa l enzymes to enableuse of ketone bodies as fuel . No matter how long the fast las ts , the bra in cannot use glycerol , amino acids , or free fatty acids as di rectfuel sources (see Chapter 10).

III-22. The answer is A. After digestion, amino acids and very smal l peptides are coabsorbed with sodium via group-speci fic amino acid orpeptide active transport systems in the apica l membrane. At least five dis tinct brush border transport systems exis t that are class i fiedas fol lows: (a ) neutra l amino acids (uncharged a l iphatic and aromatic), (b) bas ic amino acids and cystine (Cys–Cys), (c) acidic aminoacids (Asp, Glu), (d) imino acids (Pro), and (e) dipeptides and tripeptides . The mechanisms for concentrative transepithel ia l transportof L-amino acids and dipeptides are analogous to that for Na-dependent glucose absorption. The driving force for the Na +-dependenttransport i s derived from the maintenance of low intracel lular levels of Na + by the action of the Na +–K+-ATPase. Hydrolys is of ATPprovides energy to export three Na + in exchange for two K+. Thus , the high gradient of Na + between the intestina l lumen and thecytoplasm provides the driving force for active transport of amino acids , dipeptides , and tripeptides (see Chapter 11).

III-23. The answer is D. HgbF i s composed of two α-globin and two γ-globin proteins . It has a lower affini ty for 2,3-BPG than HgbA because of aserine at pos i tion 143 in the γ-globin gene instead of a his tidine res idue. This decreased affini ty for 2,3-BPG resul ts in HgbF having anincreased affini ty for O2 relative to adult and a shi ft in the O2 di ssociation curve to the left. Thus , HgbF i s able to extract O2 from HgbA

in the placenta and then del iver the O2 to the feta l ti s sues . The feta l ti s sues s ti l l produce protons and CO2 to faci l i tate the release ofO2 a t the ti ssues (Bohr effect). The shi ft to the left i s precise, however, because though i t a l lows the HgbF to “s tea l” O2 from thematernal Hgb, i t i s not so s igni ficant that the feta l ti s sues cannot remove the O2 for thei r own ti ssues . HgbF pers is ts for about 6months after bi rth. It i s for that reason that defects in the β-globin may go undetected for severa l months (see Chapter 14).

III-24. The answer is D. Statins act as feedback inhibi tors of 3′-hydroxy-3′-methylglutaryl -CoA (HMG-CoA) reductase, the regulated enzyme ofcholesterol synthes is . Effective treatment with a s tatin, a long with a low-fat diet, decreases levels of blood cholesterol . The loweringof cholesterol a lso lowers the amounts of the l ipoprotein that transport cholesterol to the periphera l ti s sues , LDL. Because l ipids , l ikecholesterol and triglycerides , are insoluble in water, they must be associated with l ipoproteins for transport and sa lvage betweentheir major s i te of synthes is (l iver) and the periphera l ti s sues . Those l ipoproteins associated with more insoluble l ipids thus havelower dens i ty during centri fugation, a technique that separates the lowest dens i ty chylomicrons from VLDLs (VLDLs with pre-β-l ipoproteins ), LDLs (LDLs with β-l ipoproteins ), intermediate-dens i ty l ipoproteins , and HDLs (HDLs with α-l ipoproteins ). Each type ofl ipoprotein has typica l apol ipoproteins such as the apo B100 and apo B48 (trans lated from the same mRNA) in LDL. LDL i s involved intransporting cholesterol from the l iver to periphera l ti s sues , whereas HDL i s a scavenger of cholesterol . The ratio of HDL to LDL i s thusa predictor of cholesterol depos i tion in blood vessels , the cause of myocardia l infarctions (heart attacks ). The higher the HDL–LDLratio, the lower the rate of heart attacks (see Chapter 16).

III-25. The answer is C. Cardiac and skeleta l muscles are s imi lar in that both are s triated and conta in two kinds of interacting protein fi laments .The thick fi laments conta in primari ly myos in, whereas the thin fi laments conta in actin, troponin, and tropomyos in. The thick and thinfi laments s l ide past one another during muscle contraction (Figure 12-2). Myos ins are a fami ly of proteins with heavy and l ight cha ins ,and muscle myos ins function as ATPases that bind to thin fi laments during contraction. Congenita l defects in muscle fi laments ,potass ium channels , and the l ike that affect cardiac contracti l i ty are substantia l contributors to these tragic and unexpected diseasecategories—important reasons for annual and trans i tional (sports , precol lege) phys ica l examinations .

III-26. The answer is D. This gi rl ’s symptoms are cons is tent with extreme hyperglycemia, which i s cons is tent with her excess ive thi rs t(polydips ia), urination habi ts (polyuria), and appeti te (polyphagia). Her neurologic symptoms are probably secondary to ketoacidos is ,l ikely resul ting from type 1 diabetes . The finding of glucose spi l lover into her urine s trongly supports this conclus ion. An acutehyperglycemic condition due to type 1 diabetes i s characterized by a near absence of insul in with unopposed glucagon action,particularly in the l iver. So both gluconeogenes is and ketogenes is are elevated in such patients . Al l the other processes l i s ted wouldbe operating at reduced activi ty relative to thei r levels in the presence of a higher insul in–glucagon ratio (Chapter 10).

III-27. The answer is D. Alport syndrome, Goodpasture syndrome, and benign fami l ia l hematuria are associated with type IV col lagenmutations . Col lagenopathies types II and XI and hypochondrogenes is are associated with type II col lagen mutations . Dystrophicepidermolys is bul losa and epidermolys is bul losa acquis i ta are associated with type VII col lagen mutations . Ehlers–Danlos syndromestypes II I and IV and aneurysm are associated with type II I col lagen mutations (Chapter 13).

III-28. The answer is A. Nephrogenic (kidney-associated) diabetes ins ipidus , a condition of poor fluid ba lance regulation and notable for largeoutput of very di lute urine, i s closely associated with mutations of the aquaporin-2 channel . Under normal conditions , a decrease inblood volume or an increase in osmola l i ty triggers release of ADH (vasopress in) from the posterior pi tui tary to s igna l mobi l i zation ofaquaporin-2 channels in the kidney to increase water reabsorption. A defect in the Gs protein that mediates ADH effects in the kidneycould lead to nephrogenic diabetes ins ipidus . However, a defect of Gq protein would only affect the vasopressenergic effect. Adefective angiotens in I I receptor would lead to decreased secretion of a ldosterone that in turn would cause a decreased reabsorptionof sodium with a subsequent loss of water. However, this i s not nephrogenic because the primary defect i s in the zona glomerulosacel l s of the adrenal cortex. A mutated ADH would be cons idered a neurogenic (bra in associated) or “centra l” diabetes ins ipidus (seeChapter 18).

III-29. The answer is B. The receptor class ca l led Ig receptors , including the IL-1 receptor, activates phosphol ipase C and subsequent NF-kB geneactivation. Types I and II receptors , which include many of the major cytokines and interferons , both function via a Janus kinasemechanism. TNF receptor group uti l i zes an intermediary protein termed TNF receptor type 1-associated death domain, which transmitsconformational changes from the receptor to TNF receptor-associated factor 2, which interacts with severa l other s igna l ing peptides tomediate programmed cel l death (apoptos is ) via NF-κB gene activation or by the activator protein 1, a transcription factor related to c-Fos and c-Jun. The chemokine receptor group functions via Gq proteins , leading to the release of intracel lular ca lcium that el ici tsdi rected chemotaxis . The tumor growth factor-β receptors are serine–threonine kinases whose phosphorylation can lead to theproduction of cAMP, cGMP, diacylglycerol , and/or activated ca lmodul in, which subsequently lead to cel l functions and DNA express ion(see Chapter 15).

III-30. The answer is A. This patient has a deficiency of α-1-anti tryps in. α-1-Anti tryps in i s a plasma protein of approximately 400 amino acids . Itmigrates in the α-1 region on a plasma protein electrophores is and i s the major component of this fraction. Normal ly, i t serves as theprimary serine protease inhibi tor in the ci rculation, particularly of elastase. Because the lack of α-1-anti tryps in a l lows for increasedelastase activi ty, this condition resul ts in a s low but s teady degradation of extracel lular fibri l s , particularly elastin. Thus , the lungsundergo proteolytic damage. This i s particularly prominent in the lung and leads to premature onset of emphysema. This process i smarkedly accelerated by smoking so that avoidance of smoking i s a priori ty. On the α-1-anti tryps in, a methionine res idue (Met358) i scri ti ca l for binding proteases to i t. In the lungs of smokers , the s ide cha in of this methionine res idue i s oxidized to methioninesul foxide. Thus , any (α-1-anti tryps in that remains becomes ineffective as a protease inhibi tor. Consequently, individuals with α-1-anti tryps in deficiency who smoke have reduced antiproteinase function from both causes and therefore show more severe symptomsthan nonsmokers with the disease. The increased severi ty i s reflected in greater proteolytic destruction of lung ti ssue largely byelastase (see Chapter 17).

III-31. The answer is C. Glucagon i s a hormone produced and secreted by the α-cel l s of the pancreas in response to decreases in blood glucose.This hormone s timulates gluconeogenes is in the l iver by increas ing the level of cAMP, which in turn activates the cAMP-dependentprotein kinase. This enzyme inactivates pyruvate kinase by phosphorylation and thus inhibi ts glycolys is . Glyco-genes is and glycolys isin l iver are inhibi ted by glucagon. Pentose pathway and ci tric acid cycle are not di rectly affected by glucagon (see Chapter 10).

III-32. The answer is C. Cholesterol in cel l s may be synthes ized de novo or taken up via LDL particles . A defect in the latter process leads tofami l ia l hypercholesterolemia. Once the LDL particles are endocytosed, they are processed in the lysosome in part by an acidcholesterol es ter hydrolase (ACEH) that removes the ester creating free cholesterol . Patients with Wolman disease have defectiveACEH. The free cholesterol i s then transferred from the lysosome to the Golgi us ing Niemann-Pick C (NPC) protein. It i s this protein thatis defective in patients with Niemann–Pick C disease. Cholesterol in the Golgi i s further processed by esteri fi cation by acyl -CoA–cholesterol acyl transferase and the cholesterol es ter accumulates in droplets in the cytoplasm. Unl ike other ti s sues , l i ver has theunique abi l i ty to get rid of cholesterol by convers ion to bi le sa l ts via the fi rs t key reaction cata lyzed by the enzyme cholesterol 7α-hydroxylase (see Chapter 19).

III-33. The answer is E. Lingual l ipase ini tiates hydrolys is of long-chain triglycerides into glycerol and free fatty acids , which continues into andthrough s tomach. Optimal activation requires acidic (~pH 4) envi ronment, so vast majori ty of activi ty i s in s tomach. Lingual l ipase i ssecreted by the dorsa l surface of the tongue (Ebner’s glands). In infants , breast mi lk comes equipped with mi lk l ipase that i ssynthes ized in the mammary cel l and exported by exocytos is . Mi lk l ipase i s unique because i t i s s timulated by bi le acids and thereforeis inactive unti l i t reaches the infant’s intestina l lumen, hydrolyzes triglycerides at a l l three pos i tions , and conveniently prefers to

cleave off the medium-chain fatty acids that are enriched in mi lk (see Chapter 11).III-34. The answer is E. Hgb exis ts in an equi l ibrium between a tense (low O2 a ffini ty; gives up O2 eas i ly to ti s sues) and a relaxed s tate (high O2

affini ty). In the deoxy (tense) form, the subunits are rotated 15° relative to the oxy form (see Figure 14-4). At a more molecular level , theR- and T-s tates of Hgb can be defined by the pos i tion of the i ron, relative to the plane of the heme. In the T-s tate, the i ron i s pul ledaway from the plane of the heme, making i t more di ffi cul t for O2 to bind. In the R-s tate, the i ron i s “pul led” (by O2) into the plane,making i t eas ier for O2 to bind. Hence, the R-s tate of Hgb has a higher affini ty for O2 than does the T-s tate, whether or not O2 i s

present. O2 s tabi l i zes the R-s tate, whereas , the T-s tate i s s tabi l i zed by H+, CO2, and BPG. Al though these latter factors s tabi l i ze the T-s tate, they do not s tructura l ly determine the shi ft to the T-s tate (Figure 14-5).

III-35. The answer is D. The uptake of exogenous cholesterol by cel l s resul ts in a marked suppress ion of endogenous cholesterol synthes is .Human LDL not only conta ins the greatest ratio of bound cholesterol to protein but a lso has the greatest potency in suppress ingendogenous cholesterogenes is . LDLs normal ly suppress cholesterol synthes is by binding to a speci fic membrane receptor thatmediates inhibi tion of hydroxymethylglutaryl (HMG) coenzyme A reductase. In fami l ia l hypercholesterolemia, the LDL receptor i sdys functional , wi th the resul t that cholesterol synthes is i s less respons ive to plasma cholesterol levels . Suppress ion of HMG-CoAreductase i s atta ined us ing inhibi tors (s tatins ) that mimic the s tructure of mevalonic acid, the natura l feedback inhibi tor of the enzyme(see Chapter 16).

III-36. The answer is A. E2 peaks just before ovulation begins and i s formed from testosterone by aromatase. This i s the main estrogen innonpregnant, ferti le females . Aromatase in the granulosa cel l s of the ovaries i s activated by FSH. There i s no feedback inhibi tion ofaromatase. Control of estrogen production occurs via feedback control of release of FSH and LH from the pi tui tary. Al though LHincreases production of E2, i ts effect i s on the production of the testosterone precursor in the ovarian theca cel l s . E3 i s the mostabundant estrogen during pregnancy and i s formed in the placenta and feta l adrenal glands and l iver. E1, produced by aromatase fromandrostenedione in adipose cel l s , i s found predominately in menopausa l women as wel l as in men (see Chapter 20).

III-37. The answer is C. The presentation of hepatomegaly and hypoglycemia i s immediately suggestive of a l iver glycogen s torage disease.Glucagon injection normal ly should increase glycogenolys is , leading to glucose production. However, in this patient, nei ther glucosenor lactate i s produced, suggesting an inabi l i ty to hydrolyze glycogen at a l l . Glucose-6-phosphatase and pyruvate carboxylase are bothassociated with synthes is of glucose from a lanine and this i s normal in the patient.

Neither G6PD nor pyruvate kinase i s involved in glucose production (see Chapter 10).III-38. The answer is B. When osteoblasts are unable to form hydroxyapati te or when sufficient ca lcium and/or phosphate are not ava i lable, the

disease of osteomalacia resul ts . In chi ldren, this condition i s known as rickets . Osteomalacia , l i tera l ly meaning “bone softness ,” hasnormal amounts of organic col lagen matrix but deficient minera l i zation unl ike osteoporos is (see below), where normal ly minera l i zedbut decreased bone matrix i s the problem. As a resul t, patients suffering from osteomalacia have weak and eas i ly fractured bones .Most often, osteomalacia/rickets i s caused by deficient vi tamin D ei ther in the diet (i .e., poor intake or poor intestina l absorption) orsecondary to low sun exposure/absorption. Other causes include kidney or l iver disease (or other disorders that affect vi tamin Dmetabol i sm and/or absorption) decreased phosphate levels , cancers , and medication s ide effects (e.g., anticonvulsant medications).Symptoms of osteomalacia include bone pa in (often s tarting in the lumbar region of the spine, pelvis , and legs ) and related muscleand nerve weakness/numbness . Laboratory tests show low ca lcium levels in serum and urine (often accompanied by low serumphosphate). Hyperparathyroidism can be caused by low blood ca lcium but phosphate would be normal or elevated. Osteoporos is i s abone condition in which the amount of bone minera l i s s igni ficantly lowered, leading to an a l tered and weakened bone matrix and amarkedly increased ri sk of fracture. In females , i t occurs after menopause because of loss of estrogen and can occur in older menbecause of loss of testosterone. Paget’s disease of the bone, a lso known as ostei ti s deformans , i s a condition of excess ive boneturnover (breakdown and reformation) resul ting in bone deformities , pa in, decreased s trength, and resul ting arthri ti s and fractures . Agenetic l inkage has been suggested as the poss ible role of a paramyxovirus , a l though no convincing evidence has been found (seeChapter 13).

III-39. The answer is C. Renin i s a proteolytic enzyme produced in the juxtaglomerular cel l s of the renal a fferent arteriole. Renin release i scontrol led in severa l ways : (1) Baroreceptors in the kidney are s timulated by decreased renal arteriolar pressure. (2) Low bloodpressure s timulates cardiac receptors that activate the sympathetic nervous system and the resul ting catecholamines s timulate thejuxtaglomerular cel l s via β1-adrenergic receptors . (3) The macula densa, which i s composed of cel l s adjacent to the juxtaglomerular

cel l s , i s s timulated by a decrease in the ci rculating concentration of Na + or Cl −. Once the renin–angiotens in system is activated,angiotens in I I triggers the release of a ldosterone, a miner-a locorticoid, that binds to i ts receptor in the kidney caus ing increasedsodium reabsorption to correct the problem be i t a drop in Na or pressure. Hence, an agonis t of the minera locorticoid receptor woulddecrease renin secretion. Likewise, providing angiotens in I I wi l l cause a ldosterone secretion and hence lower renin release. Incontrast, blocking the a ldosterone receptor with an antagonis t would prevent Na reabsorption, leading to increased renin. A chronicsa l t diet would repress renin secretion. ANP opposes the renin–angiotens in system and would hence reduce renin secretion (seeChapter 18).

III-40. The answer is E. In the relaxation phase of skeleta l muscle contraction, the head of myos in hydrolyzes ATP to ADP and Pi, but the productsremain bound. When contraction i s s timulated (via the regulatory role of ca lcium in actin access ibi l i ty), actin becomes access ible andthe actin–myos in–ADP–Pi complex i s formed. Formation of the complex resul ts in release of Pi. This i s fol lowed by release of ADP, whichis accompanied by a conformational change in the head of myos in. This conformational change resul ts in the power s troke in whichactin fi laments are being pul led past the myos in about 10 nm toward the center of the sarcomere. Another molecule of ATP i s now ableto bind the head of myos in, forming an actin–myos in–ATP complex. The ATP-bound myos in has a low affini ty for actin and thus actindissociates from the complex (see Chapter 12).

III-41. The answer is E. Bordetella pertussis produces and releases an enzymatica l ly active, protein toxin respons ible for the i l lness . Released ini ts inactive form, pertuss is toxin binds to receptors on a cel l membrane and i s transported via the Golgi apparatus to the endoplasmicreticulum. Upon activation, the toxin adds ADP molecules to the α-subunits of Gi proteins and, being inhibi ted by the ADP ribosylation,are unable to s top adenyl cyclase production of cAMP. The resul ting a l tered s ignal ing leads to a variety of cl inica l mani festations . Incontrast to pertuss is toxin effects , epinephrine binding to α-2-adrenergic receptors increases Gi protein activi ty and normal ly lowerscycl ic AMP production. In trauma, action via α-2-adrenergic receptors consequently lowers the concentration of cAMP, leading todecreased secretion and synthes is of insul in. This effect in trauma is important because secretion of insul in would lower bloodglucose levels that would be detrimenta l to resusci tation fol lowing trauma. Al though the other options l i s ted would interfere with theepinephrine effect in trauma, they are unrelated to how the toxin functions (see Chapter 17).

III-42. The answer is E. The “prion hypothes is” asserts that proteins , devoid of nucleic acids , can themselves be infectious agents . Infectiousagents require horizonta l transmiss ion into an uninfected host, and subsequent propagation of further infectious agents within thathost. Protein-only infectivi ty has been expla ined with some fascinating biochemistry. Prion proteins (PrPs ) are respons ible for a host oftransmiss ible spongi form encephalopathy including Creutzfeldt–Jakob disease, ovine scrapie, and bovine spongi form encephalopathy(BSE, a lso known as mad cow disease). Prion infectivi ty requires the endogenous express ion of PrPs , which are normal cel lularconsti tuents , anchored to the surface of neurons . The infectious agent in these diseases i s an amyloidogenic form of PrP, ca l led PrP-res

(for protease-res is tant), which i s a variant of PrP that i s in a di fferent conformation (e.g., a s l ightly soluble protein with β-s trandcharacter). PrP-res induces a conformational change in the normal cel lular PrP protein, turning i t into a PrP-res . This convers ion ofotherwise wel l -behaved proteins involves a conformational change from α-hel ix to β-s trand. Notably, PrP-res i s s table to heat, so BSE-ta inted meat i s s ti l l infectious after cooking. Transmiss ion of spongi form encephalopathies between species can occur, as evidencedby transmiss ion of BSE to humans . Transmiss ion depends upon sequence homology between the transmitted PrP-res and theendogenous PrP and hence occurs with very low frequency. However, once the native host form of PrP i s converted to the PrP-resconformer, i t seeds further ol igomer formation. The ol igomeric or “protofibri l ” s tage may be the most toxic to neura l cel l s . Largeamyloid fibri l s and inclus ion bodies or plaques may be less toxic or even protective. The best therapeutic s trategy may therefore beone that i s focused on early prevention of ol igomer formation (see Chapter 9).

III-43. The answer is E. Fructose 2,6-bisphosphate i s an a l losteric activator of l iver glycolys is at the phosphofructokinase-1 reaction and aninhibi tor of gluconeogenes is at the fructose 1,6-bisphosphatase reaction. Fructose 2,6-bisphosphate i s made by a bi functional enzymethat conta ins both phosphofructokinase-2 and fructose 2,6-bisphosphatase activi ties depending on the nutri tional s tate. In the fedstate, insul in promotes formation of this regulator by activating the kinase activi ty of the bi functional enzyme, whereas glucagonpromotes removal of the regulator by activating the protein’s phosphatase activi ty. The fructose 2,6-bisphosphate can only beconverted to fructose-6-phosphate and i s not cleaved (see Chapter 10).

III-44. The answer is A. Amylase i s released by the exocrine pancreas to ini tiate random digestion of amylose and amylopectin cha ins producingmaltotriose, maltose, amylose, glucose, and ol igosaccharides . Dextrinase, glucoamylase, lactase, and sucrase are a l l loca l i zed on thesurface of the smal l intestina l epi thel ia l cel l s and involved in very speci fic hydrolys is reactions of products derived from amylaseaction (see Chapter 11).

III-45. The answer is A. Sickle cel l disease i s caused by a recess ive, s ingle-nucleotide mutation in the β-globin gene of Hgb (Figure 14-7A),inheri ted from both parents (i .e., homozygous). This mutation resul ts in the substi tution of the normal glutamic acid (negative charge)with a va l ine (hydrophobic) at amino acid 6. Sickle red blood cel l s bind O2 normal ly and the oxygenated red blood cel l s have a normalbiconcave shape. However, when the s ickle Hgb unloads i ts O2, the normal conformational change exposes the va l ine at pos i tion 6 tothe surface creating a “hydrophobic patch.” The hydrophobic patch on one deoxygenated Hgb molecule can interact with thehydrophobic patch on a second Hgb molecule creating s ti ff Hgb polymers (Figure 14-10A–B). These internal polymers cause the redblood cel l to s ti ffen and adopt abnormal shapes including a crescentic or “s ickle” shape that gives the disease i ts name (Figure 14-10C). Because the internal s tructure of the Hgb i s unchanged, effects of H+, CO2, and 2,3-BPG are reta ined.

III-46. The answer is E. Ca lcium ions are the regulators of contraction of skeleta l muscle. Ca lcium is actively sequestered in sarcoplasmicreticulum by an ATP pump during relaxation of muscle. Nervous s timulation leads to the release of ca lcium into the cytosol and ra isesthe concentration from less than 1 mM to about 10 mM. The ca lcium binds to troponin C. The ca lcium–troponin complex undergoes aconformational change, which i s transmitted to tropomyos in and causes tropomyos in to shi ft pos i tion. The shi ft of tropomyos in a l lowsactin to interact with myos in and contraction to proceed. Mutations affecting proteins involved in muscle contraction can present withlow muscle tone and developmenta l delay in chi ldhood, as chronic muscle cramps or fatigue, or with cardiomyopathies due toweakened heart muscle. As cardiac muscle contraction i s coordinated by electrica l conduction from the s inus and atrioventricular (AV)nodes , muscle protein abnormal i ties can a lso interfere with cardiac rhythm. A speci fic mutation in troponin I can causecardiomyopathy and the i rregular cardiac rhythm known as Wolff–Parkinson–White syndrome (see Chapter 12; Figure 12-4).

III-47. The answer is B. The roles of dihydrotestosterone include development of the prostate, penis , scrotum, and geni ta l skin. In contrast,testosterone during development promotes formation of the epididymis , vas deferens , ejaculatory duct, and seminal ves icles . Duringpuberty, i t i s respons ible for secondary sex characteris tics , including muscle growth, and spermatogenes is . Development of the testesis determined by s ry (sex-determining region of the Y-chromosome). Mul lerian inhibi tory factor accounts for duct regress ion in malesso that the uterus does not develop (see Chapter 20).

III-48. The answer is E. Primary hyperparathyroidism refers to the uncontrol led overproduction of PTH. The 1-hydroxylase enzyme in vi tamin Dmetabol i sm is located in the kidney and i s activated by PTH not by ca lci tonin. The 25-hydroxylase enzyme is not regulated by vi taminD3. Al though sex s teroids (estrogen/ testosterone) can block the action of PTH, this effect i s not lost in these patients . Instead theamount of PTH that i s produced overwhelms these effects of sex s teroids . Production of ca lci tonin would be high because of elevatedblood ca lcium and would not be suppressed (see Chapter 13).

III-49. The answer is B. In the mitochondria , thiamine i s required for functioning of both the pyruvate dehydrogenase reaction (pyruvate + CoA +NAD+ → acetyl CoA + ATP + CO2) and the ci tric acid cycle α-ketogluta-rate dehydrogenase reaction (α-ketoglutarate + CoA + NAD+ →succinyl -CoA + ATP + CO2). Lactate, a lanine, and pyruvate would a l l actua l ly be increased because of the inabi l i ty of the cel l s to oxidize

pyruvate. NADPH production i s unrelated to metabol i sm of pyruvate. Arguably decreased oxidation of pyruvate would increase NAD+

concentration that, i f anything, would ra ise the NADP+ level (see Chapter 10).III-50. The answer is C. The patient’s pH i s below normal so this has to be a condition associated with an acidos is . The remaining i ssue i s

whether i t i s metabol ic or respi ratory. On the bas is of the carbonic anhydrase reaction , ametabol ic acidos is wi l l overproduce protons (H+) such as in a lactic acidos is or a diabetic ketotic cri s i s . This lower pH drives thereaction toward the left, both lowering bicarbonate and increas ing production of CO2. To compensate for the metabol ic acidos is , thelungs wi l l compensate by increas ing respiration to blow off the excess CO2, hence accounting for the decreased pCO2. In a respi ratoryacidos is , pCO2 wi l l be elevated because of hypoventi lation as in COPD. Anxiety causes hyperventi lation that wi l l decrease pCO2 and bythe carbonic anhydrase reaction a lso lower the proton concentration, thereby ra is ing the pH (i .e., respi ratory a lka los is ). In the case ofhyperammonemia, a base ammonia wi l l cause a metabol ic a lka los is with a ri se in pCO2 to reta in acid.

III-51. The answer is D. When the renin–angiotens in system is activated, renin i s released. Renin release i s control led in severa l ways : (1)Baroreceptors in the kidney are s timulated by decreased renal arteriolar pressure. (2) Low blood pressure s timulates cardiac receptorsthat activate the sympathetic nervous system and the resul ting catecholamines s timulate the juxtaglomerular cel l s via β1-adrenergicreceptors . (3) The macula densa, which i s composed of cel l s adjacent to the juxtaglomerular cel l s , i s s timulated by a decrease in theci rculating concentration of Na + or Cl −. Renin i s a proteolytic enzyme that cleaves angiotens inogen to angiotens in I . Angiotens inogen i ssecreted by the l iver. Convers ion of angiotens in I to angiotens in I I i s cata lyzed by angiotens in-converting enzyme. Angiotens in I I i s theactive form that binds to a receptor in the adrenal cortex triggering a ldosterone synthes is and secretion. Additional ly, to acutely ra iseblood pressure, angiotens in I I binds to receptors in the vascular system caus ing constriction (see Chapter 18).

III-52. The answer is A. Bra ins from Alzheimer disease (AD) patients conta in interneuronal amyloid plaques and neurofibri l lar tangles . Amyloidplaques cons is t predominantly of amyloid-β (Aβ) proteins , a long with smal ler amounts of other proteins , such as α-synuclein andpreseni l l in. The predominant amyloidogenic form of Aβ i s 42 amino acids long (des ignated Aβ42) and i s an extracel lular proteolyticproduct of amyloid precursor protein (APP), present in the plasma membranes of neurons . APP i s cleaved by proteases ca l ledsecretases . Cleavage by secretases α and γ releases two soluble fragments of APP, nei ther of which i s amyloidgenic. However, cleavageby secretases β and γ releases the Aβ42 fragment. The membrane-spanning region of Aβ42 i s primari ly α-hel ica l , a fter cleavage andrelease from the membrane, i t undergoes a trans i tion to a β-s trand conformation. Once in this conformation, Aβ42 i s normal ly cleared

rapidly. If the Aβ42 i s not cleared, i t can nucleate plaques from vi rtua l ly a l l other Aβ42 proteins that i t contacts . As these plaques form,they res is t proteolys is and transport and serve to trap future Aβ42 proteins as they are formed. Early onset fami l ia l AD i s caused bymutations in certa in proteins part of the active secretase γ complex, resul ting in excess ively high production of Aβ42. APP mutations areusual ly in or near the Aβ42 portion of APP and presumably make APP a better substrate for cleavage by secretases β or γ, or i t makesAβ42 more l ikely to undergo the α-hel ix to β-s trand convers ion (see Chapter 19).

III-53. The answer is A. Creatine phosphate i s used in the fi rs t approximately 5 sec of exercise to immediately replace ATP that i s being used.The reaction creatine phosphate + ADP → creatine + ATP i s cata lyzed by the enzyme creatine kinase. This enzyme l ies at equi l ibrium sothat i t ins tantaneous ly responds when ATP concentration begins to fa l l . There i s no other regulation of this reaction except by massaction effect caused by changes in concentrations of reactants and products . The 5 sec of creatine phosphate use are sufficient toa l low the muscle to activate glycogenolys is so that metabol i sm of glucose uni ts can begin to provide the energy to susta in exercise. Inan aerobic muscle, ini tia l use of glycogen can a lso provide time to mobi l i ze fatty acids as the muscle fuel but creatine phosphatealone cannot “buy” enough time. Creatine i s produced in sufficient amounts by l iver that secretes to the ci rculation for uptake andsubsequent phosphorylation by muscle. Creatine must be replaced by l iver on a regular bas is because muscle converts creatine tocreatinine for excretion. One concern with taking creatine supplements i s that i t can cause dehydration so that extra water must beconsumed when taking i t. There are instances where creatine supplements may be especia l ly beneficia l—in patients with a deficiencyof creatine due to l iver enzyme defect or in McArdles disease patients who cannot mobi l i ze glycogen and hence can benefi t from theadditional ava i labi l i ty of creatine phosphate unti l increased glucose uptake and fat mobi l i zation can occur (see Chapter 12).

III-54. The answer is E. During pregnancy, the primary estrogen produced i s E3. Binding of estrogen to the progesterone receptor causes aconformation change that helps activate the receptor so that estrogen i s essentia l for progesterone/progesterone receptor functions .Estrogen a lso acts to increase the tota l number of progesterone receptors , thereby ampl i fying progesterone effects . During pregnancy,progesterone, together with E3, inhibi ts lactation and smooth muscle contraction; decreased progesterone levels are one potentia ltrigger for labor and a lso ini tiate mi lk production. E2 i s the main estrogen in nonpregnant, ferti le females . Androstenedione i s anadrenal androgen that in females and males can serve as precursor to the synthes is of E1 in fat cel l s . Growth hormone has no l ink tolactation inhibi tion or onset. hCG is a heterodimeric glycoprotein hormone produced by newly ferti l i zed embryos and, subsequently bythe placenta. hCG mainta ins the corpus luteum and causes increas ing production of progesterone but i s not di rectly related toinhibi tion of lactation (see Chapter 20).

III-55. The answer is C. Both glucose-6-phosphatase and fructose 1,6-bisphosphatase are enzymes in the gluconeogenic pathway. However, onlyglucose-6-phosphatase i s a lso required for the convers ion of glycogen to glucose. Hence, only the patient with the glucose-6-phosphatase defect wi l l show glycogen accumulation. In early s tarvation, lactate provides approximately 50% of the carbons forglucose synthes is . Its decreased use for this purpose in both patients can lead to lactic acidos is . Simi larly, a lanine i s the key aminoacid precursor for gluconeogenes is and hence i ts use to make glucose wi l l be affected in both patients . Because glucose-6-phosphatase i s not found in muscle, only the patient with the fructose 1,6-bisphosphatase defect could show a l tered muscle glucosemetabol i sm. Fina l ly, these defects would not affect function of the pentose phosphate pathway (see Chapter 10).

III-56. The answer is D. This patient’s greasy, foul -smel l ing s tools indicate s teatorrhea. Her vis ion problems may be a mani festation of vi taminA deficiency due to fat malabsorption. The most l ikely explanation i s bi l iary insufficiency, that i s , decreased bi le sa l t productionleading to poor emuls i fi cation of dietary fats . Active i lea l disease i s a poss ibi l i ty, but the WBC count would l ikely be elevated unlessher condition was in remiss ion. GI infection i s less l ikely due to the absence of pathogenic organisms in her s tool . Lactose intolerancecan produce diarrhea but not s teatorrhea (see Chapter 11).

III-57. The answer is D. The clotting cascade cons is ts of a series of ordered enzymatic s teps whose end resul t i s the formation of a clot. Thereare two l imbs of the clotting cascade: the extrins ic pathway and the intrins ic pathway. The extrins ic pathway rel ies on the exposure ofti ssue factor by an injury, which then activates Factor VII, the most abundant of the clotting factors . The intrins ic pathway s tarts whenFactor XII, an inactive serine protease encounters exposed col lagen from an injured blood vessel . Factor XII i s transformed into anactivated serine protease. Activated Factor XII cleaves inactive Factor XI into activated Factor XI. This in turn cleaves the inactive form ofFactor IX, and the activated Factor IX transforms inactive Factor VII to i ts active form. Later events in the actua l formation of the clotinvolve platelets , von Wi l lebrand factor, ca lcium interacting with endothel ia l surfaces , and formation of D-dimers (see Chapter 14;Figure 14-15).

III-58. The answer is B. Ni troglycerin causes release of ni tric oxide (NO), which activates guanyl cyclase, produces cGMP, and causesvasodi lation. NO is formed from one of the guanidino ni trogens of the arginine s ide cha in by the enzyme NO synthase. NO has a shortha l f-l i fe, reacting with O2 to form ni tri te and then ni trates that are excreted in urine. Coronary vasodi lation caused by ni troglycerin i sthus short l ived, making other measures necessary for long-term rel ief of coronary occlus ion. The neurotransmitter formed bycondensation of acetyl -CoA and chol ine i s acetylchol ine, which does not play a role in di lation of coronary arteries (see Chapter 16).

III-59. The answer is D. IL-3 i s produced by Th cel l s to promote growth and di fferentiation of precursors of neutrophi l s , eos inophi l s andbasophi l s , mast cel l s , platelets , red blood cel l s , as wel l as monocytes , macrophages , and dendri tic cel l s . IL-3 a lso promotes his taminerelease from mast cel l s . IL-1 costimulates Th lymphocytes but i s produced by monocytes , macrophages , B lymphocytes , dendri tic cel l s ,and fibroblasts . IL-2 i s produced by Th1 cel l s and i s involved in growth, prol i feration, and further activation of T and B lymphocytes andNK cel l s . IL-5 produced by Th2 and mast cel l s s timulates growth and di fferentiation of activated B cel l s and eos inophi l s to increase Igsecretion, especia l ly IgG and IgA. Neutrophi l chemotactic factor (IL-8) produced by macrophages , epi thel ia l , and endothel ia l cel l spromotes chemotactic movement of neutrophi l s to a s i te of infection and inflammation as wel l as intracel lular s igna l ing, andincreased metabol i sm and his tamine release by neutrophi l s . IL-10 i s produced by cytotoxic T cel l s among severa l others and i tsfunctions include inhibi tion of antigen presentation and cytokine production by macrophages and Th1 cel l s . Fina l ly, inter-feron-αmade by WBCs (leukocytes ) promotes the development of fever by release of prostaglandin-E2, reduces pa in by interacting with μ-opioid receptor, and s timulates macrophages and NK cel l s to ki l l vi ruses (Table 15-2).

III-60. The answer is B. α1-Receptors are postsynaptic adrenoreceptors located in smooth muscle, eye, lung, blood vessels , gut, and thegenitourinary system. Stimulation of these receptors activates a Gq protein. These s ignals lead to exci tation, which i s exhibi ted bymydrias is , bronchodi lation, constriction of blood vessels , increased s trength of heart contraction (pos i tive inotropy), and decreasedheart rate (negative chronotropy). α2-Receptors are located chiefly on the presynaptic nerve terminals and Gi-protein activation, whichinhibi ts adenyl cyclase activi ty. It produces vasoconstriction and reduces sympathetic outflow in the CNS. β1-Receptor s timulationactivates Gs protein, which increases adenyl cyclase activi ty. This action resul ts in pos i tive chronotropy (heart rate increase),dromotropy (conduction of the impulse through the heart’s AV node), and inotropy (force of heart contraction). β2-Receptors are mostlypostsynaptic adrenoceptors located in smooth muscle and glands . They a lso activate adenyl cyclase via a Gs protein but a lso inhibi tionof the same enzyme by a Gi protein. The di rectly oppos i te effects are bel ieved to a l low di fferent functions in speci fic cel l and ti ssuelocations . The β2 s timulation relaxes smooth muscle resul ting in vasodi lation and bronchodi lation as wel l as release of insul in andthe induction of gluconeogenes is (Chapters 8 and 19).

III-61. The answer is D. Serotonin i s a genera l ly inhibi tory monamine type neurotransmitter found in the CNS. Acetylchol ine can be exci tatory orinhibi tory and i s found in the CNS, neuromuscular junction, and many autonomic nervous system (ANS) synapses . Glutamate i s found in

the bra in and spina l cord and i s exci tatory. Norepinephrine, such as serotonin, i s a monoamine neurotransmitter but can be exci tatoryor inhibi tory found in the CNS, sympathetic ANS synapses , and nearly a l l ti s sues . Substance P i s a neuropeptide that i s genera l lyexci tatory and found in the spina l cord, bra in, sensory neurons , and GI tract (see Chapter 19).

SECTION IVAPPENDICES

APPENDIX IBIOCHEMICAL BASIS OF DISEASES

Contributing Editor: Harold Cross, MD, PhDCofounder, Windows of Hope, (www.wohproject.org) Professor and Director, Medica l Student Teaching, Department of Ophthalmology and

Vis ion Sciences , Col lege of Medicine, Univers i ty of Arizona, Tucson, Arizona, USA

This appendix presents selected examples of inheri ted diseases affecting bas ic biochemica l processes . Diseases are categorized as per thei rinvolvement with the four bas ic categories of biochemica l molecules (amino acids/proteins , carbohydrates/ glycoproteins , l ipids/glycol ipids ,and nucleic acids/deoxyribonucleic acid). Additional categories of mitochondria l enzymes and diseases affecting bi l i rubin, blood clotting,s teroid hormones , and vi tamins/minera ls/electrolytes are a lso provided. Inheri tance i s predominately autosomal recess ive unless otherwisestated. Minor variations of these genetic diseases may not be noted.

For further information, the reader i s referred to the Onl ine Mendel ian Inheri tance in Man® (www.ncbi .nlm.nih.gov/omim/), “acomprehens ive compendium of more than 12,000 human genes and genetic phenotypes” and Windows of Hope (www.wohproject.org), “apopulation-based medica l project dedicated to the detection, characterization, and treatment of inheri ted heal th problems.”

AMINO ACID SYNTHESIS/DEGRADATION

AMINO ACID TRANSPORT

UREA CYCLE DISORDERS

STRUCTURAL PROTEINS

CARBOHYDRATES

GLYCOGEN STORAGE

MITOCHONDRIAL ENZYMES (EXCLUDING UREA CYCLE AND FATTY ACID OXIDATION)

LIPIDS AND FATTY ACID OXIDATION ERRORS

NUCLEOTIDE METABOLISM

DEFECTIVE DNA

BILIRUBIN METABOLISM

BLOOD CLOTTING FACTOR DEFECTS

STEROID HORMONE SYNTHESIS

VITAMINS/MINERALS AND ELECTROLYTES

APPENDIX IIBIOCHEMICAL METHODS

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) [SODIUM DODECYL SULFATE (SDS)/NON-SDS]PAGE i s a laboratory technique des igned to separate proteins according to thei r s i ze, shape, and a lso charge. Proteins for PAGE are usual lyprepared by placing them in an anionic (negative charge) detergent mixture, which, a long with heating to approximately 60°C, denatures theprotein, breaks any cysteine–cysteine disul fide bonds , and creates a fa i rly l inear protein s tructure. PAGE can a lso be run with an addition ofSDS. SDS, i f used, i s included in the detergent mixture and ass is ts in dis rupting the proteins ’ secondary and tertiary s tructures . In addition, SDSbinds to each peptide cha in in a ratio of one SDS per two amino acid res idues , thereby adding a negative charge proportional to the peptidelength. The addition of SDS to PAGE samples , therefore, a lso makes the proteins ’ shapes and native charges i rrelevant; only the tota l lengthand the resul ting SDS charge matters .

Polyacrylamide gel i s composed of l inear acrylamide molecules , cross -l inked by bisacrylamide via the cata lytic actions of ammonium persulfateand tetramethylethylenediamine (TMED). Cross -l inking forms a web-l ike polyacrylamide lattice with pores of approximately the same s izethroughout. Varying the amounts of acrylamide, bisacrylamide, and aqueous solution a l lows scientis ts to accurately vary the pore s i ze and,therefore, the relative abi l i ty to separate di fferently s i zed protein molecules .

After polymerization, the gel i s placed in an electrophores is device and immersed in a buffer with a cathode (pos i tive charge) at the top andan anode (negative charge) at the bottom. The protein samples are “loaded” onto individual gel lanes formed by a removable comb. A trackingdye i s usual ly included in the samples to monitor the progress of the proteins through the gel .

The electric field causes the negatively charged proteins (charge usual ly enhanced by the proportional negative charge of SDS molecules ) tomove toward the anode proportional to thei r length (and, therefore, charge). Movement through the gel i s not di rectly proportional to theovera l l charge, though, because the larger proteins wi l l encounter much more di ffi cul ty in moving through the pores because of thei r s i ze andl inear rigidi ty. Al though the length of each peptide increases the electrica l force moving i t down the gel , the “fi l tering” action of the gel poreswi l l a l low smal ler molecules to travel further down the gel , whereas larger molecules remain near the top.

(A) The PAGE system employs the electric charge deployed between a positive electrode (anode) and negative electrode (cathode).(B) Proteins are loaded(origin) onto polyacrylamide gel, which is exposed to this electric field. Negatively charged proteins are driven toward the anode, whereas positively chargedproteins will remain nearer to the cathode. The relative movement of each protein depends on its specific charge determined by its amino acid composition.When SDS is not used (“native PAGE”), the secondary-to-quaternary structure of each protein also influences the movement through the acrylamide gelmatrix. When SDS is used, higher order structure is eliminated, separation by charge predominates. [Reproduced with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

When the tracking dye approaches the bottom of the gel , the electrica l field i s s topped and the gel i s s ta ined us ing a variety of chemica lsor solutions , which bind to proteins (e.g., Coomassie blue, silver) to a l low visua l i zation in the gel . Proteins of known molecular weight, referredto as “molecular markers,” are normal ly run in a separate lane to a l low di rect comparison with the proteins from the experimenta l sample.PAGE gels can be used further for western blotting (see below) and other biochemica l techniques .

SDS or non-SDS PAGE i s often used during the biochemica l i solation of a s ingle protein from ti ssue or protein mixtures , a l though i t can a lsobe used cl inica l ly for diagnostic and/or treatment purposes . Because of i ts abi l i ty to dissolve molecules , SDS i s sometimes used in enemas

as a laxative.

Markers of known molecule weight (kDa) are loaded in one gel lane (left) to allow characterization of proteins separated on the other lanes of the gel.[Reproduced with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Two-dimensional (2D) PAGE i s performed in two di rections on the same sample, offering improved separation for complex mixtures ofmolecules . In this technique, samples are exposed to an electric current for a fi rs t separation. The gel i s then turned 90° and a current i s aga inappl ied with an a l ternative buffer, which affects the movement of the samples di fferently. As a resul t, a second separation i s achieved.Examples include molecular weight separation fol lowed by separation based on the molecules ’ overa l l pH, known as the “isoelectric point.”2D electrophores is i s used in many cl inica l conditions as a preparatory s tep before western blotting (see below) for tests such asconfi rmation of human immunodeficiency vi rus (HIV) and hepati ti s B infection, the detection of prions in Creutzfeldt–Jakob disease, a lsoknown as “mad cow disease,” and diagnos is of Lyme disease, among others .

Samples are run via normal SDS–PAGE (shown in vertical direction), which separates the proteins based on size, turned 90°, and run a second time viaisoelectric focusing (shown in horizontal direction) technique with a pH gradient, which separates the proteins based on total pH of the protein (see text).Running the same sample by these two different techniques allows increased separation of complex mixtures of proteins and/ or separation of proteins ofthe same size, but differing isoelectric points. [Reproduced with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion,McGraw-Hi l l , 2009.]

IMMUNOASSAYSImmunoassay i s a generic term for the appl ication of antibodies to biochemica l testing. These techniques paved the way for a wide variety oftechniques of separation, measurement, and pos i tive identi fi cation of biologica l molecules , and include radioimmunoassays (RIA), enzyme-linked immunosorbant assay (ELISA or EIA), and western blotting.

Al l immunoassays involve the addition of an antibody speci fic for a known molecule (antigen) to a solution presumably conta ining, at leastin part, that molecule. If the antigen i s present, the antibody wi l l bind speci fica l ly and, i f properly chosen and prepared, s trongly. Monoclonalantibodies wi l l normal ly bind the tightest and with the greatest speci fici ty; polyclonal antibodies are usual ly weaker and less speci fic in thei rbinding patterns . Selective precipi tation or chromatography (see below) can be used to i solate and puri fy the antibody–antigen complex asneeded.

If a chemica l or another label has been previous ly attached to the antibody, i t can be used to detect the presence and, in many cases , thequanti ty of the antigen. Examples of these detection techniques include fluorescent molecules , enzymes that produce a particular color when

provided an appropriate substrate, radioactive or magnetic labels , and even gold particles , which coalesce to form a visua l precipi tate.Al ternatively, another antibody with a radioactive detector molecule, which selectively binds to the fi rs t antibody, can be added later in thetest. Immunoassays are used in a multi tude of cl inica l tests , especia l ly for the detection of infections in blood, urine, cerebra l spina l fluid,and so on.

RIA

The fi rs t immunoassay developed was the RIA. In this test, a molecule of interest, such as a hormone, has a radioactive molecule (e.g., iodine-125, carbon-14, or hydrogen-3) attached to i t. The researcher a lso has an antibody (monoclonal or polyclonal ), which speci fica l ly recognizesand binds to this hormone.

A measured amount of the antibody and a measured amount of the pure hormone (both radiolabeled and unlabeled forms) are added toassay tubes with concentration of the unlabeled hormone greatly exceeding that of the radiolabeled hormone. Because only a smal l amountof antibody i s added and i ts capaci ty to bind the hormone i s l imited, the antibody-binding s i tes wi l l be saturated (i .e., a l l occupied; see figurebelow) As a resul t, only a fraction of the tota l amount of hormone wi l l be actua l ly bound to the antibody. Given that the antibody cannotdi fferentiate between labeled and unlabeled hormones , both forms of the hormone wi l l compete for the l imited number of binding s i tes onthe antibody. When a smal l amount of unlabeled hormone (i .e., from standard or patient sample) i s added to the assay, then only a smal lamount of the bound-labeled hormone i s displaced from the antibody. As the amount of unlabeled hormone increases , the amount ofradiolabeled hormone bound to the antibody decreases cons iderably.

A variety of techniques can be used to separate unbound hormone from the antibody-bound hormone. Therefore, di rect measurement ofbound-labeled hormone can be eas i ly made. Us ing known quanti ties of unlabeled hormone, i t i s poss ible to construct a s tandard curve whichcompares the amount of radiolabeled hormone bound to the antibody with the amount of pure unlabeled hormone added to the tube. Byreplacing pure hormone with a patient sample, the concentration of the hormone in the sample can then be di rectly determined.

A molecule of interest (A) can be detected by placing a radioactive label (yellow starburst) onto a protein (B), which is known to bind to (A). Importantexamples of protein (B) include hormones and monocolonal and polyclonal antibodies. [Adapted with permiss ion from Mescher AL: Junqueira ’s Bas icHis tology Text and Atlas , 12th edi tion, McGraw-Hi l l , 2010.]

A specific antibody to the hormone is incubated with the radiolabeled hormone. A standard curve is created by introducing known concentrations of unlabeledhormone to displace the radiolabeled hormone. The amount of radioactivity remaining is a function of the unlabeled hormone concentration. When unknownsamples are used, the measurement of the radioactivity remaining allows the hormone concentration to be determined from the standard curve. [Adaptedwith permiss ion from Kibble JD and Halsey CR: The Big Picture: Medica l Phys iology, 1st edi tion, McGraw-Hi l l , 2009.]

One drawback of this method i s that RIA measures the immunologic activi ty, not the biologica l activi ty, of a hormone. Idea l ly, both abioassay and RIA are eva luated. However, in a cl inica l s i tuation, with a few rare exceptions , us ing the RIA provides sufficiently accurateinformation on hormone levels in the patient sample. The advantages of RIA are sens i tivi ty and speci fici ty as wel l as rapid and economicevaluation of patient blood or urine samples .

ELISA OR EIA

The principle of RIA i s the bas is of newer methods of measurement, for example, ELISA or EIA. The ELISA technique uses two di fferentantibodies with varying binding affini ty to a molecule that the researcher wishes to detect or s tudy. An example i s a polyclonal or amonoclonal antibody made aga inst the same molecule. Normal ly, a microti ter plate, conta ining multiple test wel l s coated with the polyclonalantibody (not shown in figure), i s used. The test samples are placed in the wel l s and the antigen, i f present, i s a l lowed to bind to theantibody. After a sui table time period, the remainder of the test sample i s removed and the wel l s are washed to remove nonspeci fica l lybound molecules . Next, a monoclonal antibody aga inst the same antigen i s added to the wel l s and a l lowed to bind to the antigen molecule.The use of polyclonal and monoclonal antibodies usual ly a l lows multiple binding to the same antigen molecule. Detection techniques arethen used to determine the presence and quanti ty of the antigen being tested. One common method i s to use an enzyme attached to theantibody, which produces a colored product. After the binding of the second antibody, addi tion of the enzyme’s substrate creates a colorreaction, which can often be quanti tated (see the figure below). Detection us ing radioactive or fluorescent molecules attached to the secondantibody can a lso be uti l i zed. ELISA i s used for a wide variety of cl inica l tests (e.g., identi fi cation of HIV, a l lergens , and/or drugs), especia l ly i fthe concentration of a certa in protein or other antigen must be determined. The ELISA technique i s a lso the bas is for home pregnancy tests ,where the hormone binds fi rs t to an antibody–enzyme complex with detection and quantization via a second antibody conta ining a dye that i sproduced by an enzyme.

Antibody to an antigen being tested is applied to the sample. Following binding, the antigen can be detected either via an attached enzyme on the primaryantibody (direct method, left) or by the addition of a secondary antibody with an attached enzyme (indirect method, right). When substrate is added to theantibody–antigen sample, the enzyme produces either a fluorescent or colored product, allowing easy measurement, which allows both quantitative andqualitative assessment of the original antigen. [Adapted with permiss ion from Mescher AL: Junqueira ’s Bas ic His tology Text and Atlas , 12th edi tion,McGraw-Hi l l , 2010.]

CHROMATOGRAPHY

THIN LAYER (PAPER) CHROMATOGRAPHY (TLC)

TLC or paper chromatography rel ies on a thin layer of materia l (e.g., paper, cel lulose, s i l i ca , and others ) with varying absorbance for molecules .Al though often used for nonbiologica l molecules , TLC can be uti l i zed for protein and l ipid separation and/or identi fi cation. A smal l volume ofeach sample for analys is i s placed in vertica l lanes close to one end of the TLC layer, and that end i s partia l ly immersed in a solvent, ensuringthat the sample i s not covered by the solvent. This solvent i s drawn through and up the TLC materia l by capillary action. As i t travels , i t carriesthe sample molecules up with i t at rates depending on the molecule and solvent properties , including the level of adsorption onto thechromatography layer and the solubi l i ty of the sample in the l iquid (see the fol lowing figure). As a resul t of the varying sol id and l iquid layeraffini ties , separation of a mixture of molecules resul ts . The TLC layer i s then “developed” for molecule detection via chemica l or spectroscopicmeans . In the case of a s ingle, puri fied molecule, the dis tance traveled on a speci fic TLC with a speci fic solvent can provide identi fi cation bycomparison with known standards . Two-way TLC can a lso be used, in which after separation by one solvent, the sheet i s turned 90° andexposed to the actions of a second type of solvent. Two-way TLC offers improved separation for some molecules .

(A) Diagram of TLC equipment, illustrating TLC support medium, solvent (light brown), point of application of three samples (black dots), and direction ofmovement (indicated by arrow) of molecular samples as the solvent moves up the medium. (B) Actual TLC plate showing known sample of G (left lane) andL (middle lane) and separation of a mixture of these two proteins into the component parts (right lane). [Adapted with permiss ion from Naik P:Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

COLUMN CHROMATOGRAPHY

Column chromatography uti l i zes the same principles of TLC, but in a three-dimens ional column system that a l lows much larger amounts ofsample to be separated and s tudied. A glass tube or “column” i s fi l led with a selected materia l , ca l led stationary phase, which has particularchemica l qual i ties (e.g., hydrophobic, hydrophi l i c, anionic (–) charges , cationic (+) charges , etc.). Molecules wi l l bind to the s tationary phase,depending on their individual chemica l qual i ties . For example, a protein with a s trong hydrophi l i c external character wi l l not want to bind to ahydrophobic column materia l , but wi l l want to bind to a hydrophi l i c one. The variation in binding of each molecule leads to separationbecause molecules that bind less wi l l travel more quickly through the column and vice versa . Often, the molecules wi l l bind to the s tationaryphase unti l another l iquid solvent, ca l led mobile phase or eluent, wi th s tronger hydro-phobic/hydrophi l i c or anionic/cationic qual i ties i s runthrough the column. Sometimes , sequentia l eluents , each with a di fferent chemica l qual i ty, are run through the column, each caus ing selectedmolecules to unbind and flow out of the column. The materia l that passes through the column is col lected in smal l samples of a knownvolume, ca l led fractions , for further s tudy. Often, molecules in the fractions are monitored by l ight [e.g., ul traviolet (UV)] absorption or otherdetection methods .

GEL FILTRATION CHROMATOGRAPHY

Gel filtration chromatography, a variation of column chromatography, i s used for the biochemica l separation of biologica l molecules , mainlyproteins and nucleic acid s trands , depending on the s i ze and shape of the molecule. The technique usual ly preserves biologica l function. Gelfi l tration uses beads formed from polyacrylamide (see above), dextrans , or agarose (see Chapter 2) with pores of approximately equal s i ze. Theparticular pore size of each type of gel fi l tration bead depends on their exact materia ls and di fferent fabrication techniques whosecharacteris tics a l low researchers to optimize column chromatography for varying samples .

Biologica l samples are appl ied to the top of the column in a smal l volume. A solution i s run through the column carrying the samplethrough the gel beads . Smal l and/or predominately compact globular molecules are able to enter the bead pores and, therefore, have a largevolume ava i lable to them. This large volume resul ts in a s lower trans i t time through the column. Larger particles or ones with a noncompact orl inear s tructure are unable to enter the bead pores and travel more quickly through the smal ler volume between the beads . The moleculars ize and shape of each biologica l molecule appl ied to the column di rectly determine how fast or s low i t travels through and out (elution) ofthe column (see figure below). This varying speed resul ts in separation.

ION-EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography, another type of column chromatography, takes advantage of charged groups (e.g., ions) that are part of the gel beadmateria l . Di fferent biologica l molecules interact more or less with these chemica l groups (e.g., a more negative molecule wi l l be attracted topos i tively charged gel beads), a l tering thei r speed of elution and resul ting in separation. Some molecules wi l l bind so s trongly to the chargedgel beads that a solution with a s tronger ionic charge must be used to displace or “elute” them.

Small molecules (blue) are able to enter the gel beads, increasing their available volume and slowing down the speed of travel through the column. Largermolecules (red) are unable to enter the gel beads and travel more quickly through and out of the column. As a result, molecules separate and elute from thecolumn at a rate proportional to their molecular weight/size. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee BrothersMedica l Publ i shers (P) Ltd., 2009.]

Gel beads with a positive ionic charge attract negatively charged molecules (blue) more than positively charged ones (orange). This different affinity, basedon charge, allows separation of various molecules. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica lPubl i shers (P) Ltd., 2009.]

AFFINITY CHROMATOGRAPHY

Affinity chromatography works much l ike ion-exchange chromatography (above), but takes advantage of speci fic molecules that are part of orprevious ly attached to the gel bead materia l . As an example, antibodies coupled to the gel materia l are sometimes used for very speci ficbinding to the des i red molecule. Other examples of a ffini ty chromatography include the use of gel -bound substrates or coenzymes. Thebiologica l molecules remain bound to the gel bead unti l a second solution of changed ionic s trength or pH i s appl ied to affect thei r releaseand elution.

Regardless of the particular type of column chromatography used, eluted biologica l molecules are col lected in smal l , ordered volumeal lotments , known as fractions , and the molecules are detected by spectroscopic or chemica l identi fi cation methods .

HIGH-PERFORMANCE/PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

HPLC i s a technique used for enhanced and precise separation of even smal l amounts of biologica l and nonbiologica l molecules . HPLCcolumns uti l i ze variations of the separation principle behind column chromatography, depending on variable attractive and repuls ive forceson the gel materia l and their interaction with the biologica l molecules being s tudied. HPLC a lso uti l i zes smal ler columns than regular columnchromatography, with more specia l i zed gel materia ls . The smal ler columns and resul ting tighter packing of the gel materia l in HPLC combinedwith the use of a pump to propel the column solution (sometimes organic solvents ) ei ther in a s ingle solution or as a gradient of di fferentsolutions under high pressure lead to fast, efficient, and more selective separation of molecules that may not be poss ible with othertechniques . Methods to detect these molecules once they are eluted, including l ight absorbance, fluorescence, electrochemica l , and others ,are s imi lar to those uti l i zed in column chromatography.

PROTEIN AND DEOXYRIBONUCLEIC ACID (DNA)/ RIBONUCLEIC ACID (RNA) PRECIPITATIONThe selected precipi tation of biologica l molecules , including proteins and DNA/RNA, i s often used during the preparation, analys is , andconcentration of biochemica l samples . Al l methods rely on the varying solubi l i ty of these molecules in di fferent aqueous or nonaqueoussolutions , depending on their hydro-phobic, hydrophi l i c, and other biochemica l properties . The particular amino acid R-groups (primarystructure) as wel l as the resul ting secondary, tertiary, and quaternary s tructures (Chapter 1) of a protein determine i ts interactions with watermolecules or organic solvents and, therefore, how soluble i t wi l l be in those solutions . Because proteins have di fferent primary s tructures ,each wi l l have unique solubi l i ty in a particular solvent and wi l l a l low separation by varying precipi tation techniques .

One such technique that i s often used i s referred to as “salting out” and often uses the chemica l ammonium sulfate (a l though other sa l ts canbe used). Us ing this technique, the concentration of ammonium sul fate in a mixture of proteins i s gradual ly increased, resul ting in greaterbinding of water by the increas ing ion concentration. As the increas ing number of ions draw the water away from the proteins , i ts abi l i ty tokeep them in solution decreases . Proteins may then s tart to aggregate together to protect thei r hydrophi l i c and/or hydrophobic portions . If theprocess continues , the proteins wi l l come out of the solution (precipi tate). The particular hydrophi l i c/hydrophobic characteris tics of eachprotein wi l l make i t and i ts identica l molecules precipi tate at the same time, resul ting in separation. As each protein has a set sa l tconcentration at which i t precipi tates , this technique can be eas i ly reproduced as part of a molecular puri fi cation process . Other techniquesus ing organic, polymeric, and meta l sa l t solutions , including ones that take advantage of the overa l l pH of a protein (as determined by i tsamino acid R-groups), are used in a s imi lar fashion. Usual ly, the proteins can be redissolved and can reta in thei r biologica l functions .

DNA and RNA molecules (Chapter 4) can be s imi larly precipi tated us ing ethanol or isopropanol via a s imi lar mechanism. Water normal lysurrounds these molecules , interacting with the charged phosphate backbone and keeping the DNA or RNA in solution. The gradual addi tionof ethanol (usual ly about 64% ethanol/water) blocks the water–phosphate backbone interactions and leads to precipi tation of the DNA orRNA. Smal ler nucleic acid molecules may require longer exposure to ethanol or other techniques to ful ly precipi tate. After precipi tation,centri fugation i s used to bring the insoluble nucleic acid molecules together in a smal l mass , ca l led a “pel let,” and the remaining solutioncan be removed. The DNA or RNA can be resuspended in an aqueous solution and can normal ly reta in ful l biologica l activi ty. Ethanolprecipi tation can a lso be used for polysaccharides (Chapter 2) or methanol for proteins (Chapter 1).

DNA AND RNA SEQUENCINGDNA and RNA sequencing have advanced the understanding of genes and gene products exponentia l ly s ince their development. Furtheradvances and refinement of the DNA sequencing technology a l lowed determination of the complete human genome in 2003, a task that unti lthen was deemed a lmost imposs ible. Sequencing of the nucleotides of DNA and RNA rely on many of the same biochemica l techniquesa l ready discussed above.

The Maxam–Gi lbert method of sequencing was developed in the 1970s and i t rel ies on radioactive label ing and chemica l cleavage of theDNA being examined. The labeled fragments are separated by gel electrophores is , and the sequence i s interpreted from the resul ting X-rayfi lm. Al though this sequencing technique has fa l len out of favor in comparison with the Sanger technique (see below), i t i s s ti l l used inspecia l ty appl ications including DNA footprinting, in which the interaction of DNA and DNA-binding proteins can be determined.

In 1975, the Sanger technique, often referred to as “chain-termination” method, was developed, and i t i s s ti l l used, with someimprovements , today. The key to this method i s the appl ication of dideoxynucleotide triphosphates (ddATP, ddGTP, ddCTP, and ddTTP), which lack5′ and 3′-hydroxyl (OH) group and, therefore, block the addition of further nucleotides . This cha in termination by the dideoxynucleotidesresul ts in DNA fragments of various lengths , which can be separated by gel electrophores is .

More speci fica l ly, the DNA to be sequenced i s reduced to a s ingle-s trand DNA template by heat denaturation. The sample i s divided intofour parts to which DNA polymerase and a l l four of the regular dATP, dGTP, dCTP, and dTTP nucleotides are added a long with one of thedideoxynucleotides per sample. The DNA polymerase uses the s ingle-s trand template to form a second s trand. At various times , thedideoxynucleotide ends DNA repl ication, but only at the point where that dideoxynucleotide i s added to the growing s trand. As a resul t,multiple length s trands , a l l ending in the same dideoxynucleotide, are produced. When the ddATP, ddGTP, ddCTP, and ddTTP samples areseparated by gel electrophores is , each on i ts own gel lane, an eas i ly read sequence of fragments di rectly denotes the DNA sequence (seebelow).

(A) DNA to be sequenced is reduced to single strands by heat denaturization and used to produce four separate samples, G, A, T and C, with DNA polymerase,deoxynucleotides, and one of the four dideoxynucleotides (ddGTP, ddATP, ddTTP, or ddCTP), respectively. (B) The DNA polymerase produces DNA strands ofvarying length, each terminated by the specific dideoxynucleotide. (C) These strands are separated by gel electrophoresis and the sequence read from bottom(shortest fragment) to top (longest fragment) from the gel (example sequence shown on gel: AGTCTTGGAGCT). [Adapted with permiss ion from MurrayRA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Standard DNA sequencing a l lows researchers to determine the nucleotide code for DNA up to 1,000 bases long. Other techniques a l lows impler, fas ter, and more complex sequencing. DNA restriction endonuclease enzymes , often ca l led “restriction enzymes,” cut DNA at veryspeci fic nucleotide sequences . The use of di fferent restriction enzymes , each cutting the DNA at very di fferent points , produces di fferent butoverlapping fragments of the same DNA molecule. Sequencing of each restriction enzyme fragment and subsequent comparison andappropriate overlap of the sequences a l low characterization of DNA that may be too long for the s tandard technique. Variations in label ing

the fragments , including a technique in which the dideoxynucleotides are labeled, and techniques for better fragment preparation andseparation have a lso a l lowed eas ier and even machine-automated DNA sequencing of longer and longer nucleotides cha ins approaching5,000 bases . These improved techniques were cri tica l in the abi l i ty to sequence the human genome.

RNA sequencing rel ies on the abi l i ty to use reverse transcriptase to produce a double-s tranded DNA molecule (often referred to ascomplementary DNA or cDNA) from an RNA sequence of interest. Fol lowing the production of the analogous DNA molecule, sequencing i s doneas described above and then interpreted for the RNA molecule. However, RNA sequencing i s more problematic than DNA sequencing becauseof the fact that RNA is much less s table. Simi lar techniques are used to produce a cDNA l ibrary—a col lection of DNA that codes for a l lmessenger RNA (mRNA) conta ined in a particular cel l type. cDNA l ibraries are usual ly establ i shed for s imple organisms wherein the numberof mRNA molecules i s l imited. cDNA l ibraries are a lso used in cloning (see below).

SOUTHERN, NORTHERN, AND WESTERN BLOTSDNA Southern and RNA northern blots are techniques combining PAGE and a variation of TLC/paper chromatography (described above).Southern blotting was developed by Dr. Ed Southern and i s named in his honor. The subsequent adaptation of the technique to RNA andproteins (described below) resul ted in the analogous names of northern and western blotting. A method was a lso developed to detect post-trans lational modi fications to proteins (eastern blotting), a l though the term has fa l len somewhat out of favor.

SOUTHERN BLOTTING

Southern blotting i s used to identi fy and i solate a speci fic DNA sequence and can a lso quanti tatively determine the number of times a speci ficsequence occurs in a particular cel l type (e.g., how many repeats and/or copies of a gene in a particular organism or cel l type of interest). Theprocedure begins with i solation of DNA via precipi tation and column puri fi cation techniques fol lowed by exposure to restriction enzymes ,which cut the long DNA molecule into smal ler and more manageable pieces . The DNA is next electrophoresed for s i ze separation on anagarose gel . The gel i s next removed from the gel electrophores is equipment and the DNA is transferred from the gel to a piece of nylonmembrane whose pos i tive charge binds the negatively charged DNA molecules (e.g., phosphate groups of the backbone). The transfer takesplace via s imple capi l lary action by placing the membrane on top of the gel in a sui table transfer buffer. After transfer, heat or UV radiation i sappl ied to the membrane to cova lently attach the DNA molecules to the membrane, and hybridization probes (DNA, RNA, or syntheticol igonucleotides) with radioactive, fluorescent, or chemica l detection capabi l i ties are used to identi fy a speci fic DNA strand sequence.

The basic blotting techniques (Southern, northern, and western) are illustrated, including gel separation, transfer to paper, and detection. The techniques ofnorthern and western blotting are further explained in the text (see below). The specific DNA, RNA, or protein of interest is detected and identified by anyone of a variety of techniques discussed above. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus trated Biochemistry, 28th edi tion,McGraw-Hi l l , 2009.]

DNA molecules , separated on a gel and prior to transfer to the nylon membrane, can be visua l i zed us ing the chemica l ethidium bromide (EtBr).EtBr revers ibly fi ts or “intercalates” between nucleotide base pa i rs and becomes capable of fluorescing i f i l luminated by UV l ight. Because theinterca lation of EtBr i s revers ible, identi fied and functional DNA can be col lected by cutting out the particular band of interest from theagarose or polyacrylamide gel . Interca lation a l ters the secondary and tertiary s tructures of the DNA molecule and, therefore, DNA repl icationand express ion. As a resul t, interca lation of DNA is involved in the action of severa l chemotherapeutic drugs (e.g., doxorubicin, daunorubicin,and dactinomycin). For the same reason, EtBr can be a potent carcinogen i f not handled correctly.

NORTHERN BLOTTING

Northern blotting a l lows researchers to determine the level of production (and, therefore, control of express ion) of a particular mRNA byquanti tative determination of the relative amount of that particular mRNA relative to tota l mRNA. Varying influences on both tota l and speci ficmRNA express ions such as development, diseases (e.g., cancers ), and/or experimenta l techniques (including drug development) can,therefore, be ascerta ined. Northern blotting s tarts with the i solation of RNA from a cel l wi th appl ication of a ti s sue or cel l sample to an oligo(dT) cellulose affinity column, which wi l l bind only to the polyadenosine nucleotide tails found in RNAs (see Chapter 9), whereas other molecules runthrough the column unbound. Other techniques including chemica l extraction and magnetic bead i solation are a lso used. The ini tia lseparation/puri fication of the RNA provides a markedly puri fied sample of RNA.

The RNAs are then appl ied to an agarose gel with formaldehyde added to promote l inearization of a l l RNA molecules (see figure above).PAGE gels with urea added can a lso be used for smal ler RNA sequences . The RNA on the gel i s then transferred to a nylon membrane whosepos i tive charge binds the negatively charged RNA molecules (e.g., phosphate groups of the backbone). This transfer i s accompl ished in amanner analogous to Southern blotting (see above) and then heat or UV radiation i s appl ied to the membrane to create cova lent l inkagesbetween the RNAs and the nylon. Much l ike Southern blot detection, speci fic “hybridization probes” (DNA, RNA, or arti fi cia l ly constructedol igonucleotides) of known sequence and labeled radioactively, fluorescently, or with enzymes for chemica l detection are then used toidenti fy particular RNA s trands of interest. The use of northern blotting for the quanti tation of mRNA abundance has been mainly replaced toa great extent by quanti tative reverse-transcription polymerase chain reaction (RT-PCR) (described below), which i s much more sens i tive andrel iable.

WESTERN BLOTTING

Western blotting i s a biochemica l technique, adapted from a s imi lar technique for DNA of Southern blotting, for detection and identi fi cation ofspeci fic proteins . Like Southern and northern blotting, western blots take advantage of the same immunoassay techniques described above,coupled with those of PAGE separation. Western blotting begins with a PAGE gel (one or two dimens ional ) to separate a mixture of proteins(see the figure above). The unsta ined gel i s removed from the gel electrophores is equipment and placed in a specia l apparatus that a l lowstransfer of the proteins , aga in us ing an electric current, from the gel to a piece of nitrocellulose or polyvinylidene difluoride membrane. Thismembrane s trongly binds to and immobi l i zes the proteins in each band so they cannot di ffuse, thereby mainta ining the origina l separationwhi le blotting and identi fying the sample. The membrane can then be used for detection of a speci fic protein us ing antibodies as discussedin the Immunoassays section above. Detection i s aga in via enzyme-l inked, fluorescent tag, radioactive-label , or other methods as discussedabove.

PCRPCR i s a powerful technique developed in the 1980s to produce multiple copies (i .e., thousands to mi l l ions ) of a particular DNA sequence. PCRrel ies on repeti tive cycles of (a ) partia l “melting” of double-s tranded DNA, (b) appl ication of short DNA primer sequences (two primers speci ficfor one particular gene, but for DNA repl ication in both the 5′ → 3′ and 3′ → 5′, sense and antisense di rections), and (c) adding DNA polymeraseand a l l four DNA nucleotides (dA, dG, dC, and dT) to copy that gene. The process ends with cool ing of the DNA to a l low reanneal ing of thedouble-s tranded DNA. Subsequent melting/primer, polymerase, and nucleotides/cool ing cycles a lso act on any newly produced DNA, resul tingin an exponentia l ampl i fi cation of the DNA primer’s gene target with every cycle. An agarose gel with EtBr i s often used to veri fy theampl i fi cation. PCR i s used in a vast number of varying molecular biologica l and medica l techniques , including ampl i fi cation of DNA clones(see below) and/or hybridization probes for northern or Southern blotting (see above). In addition, PCR enables eas ier gene analys is oforganisms and ti ssues/cel l s , genetic determinations to include the s tudy of genetic diseases and paterni ty/heredi tary testing, detection oflow levels of infective bacteria l or vi ra l organisms, and forens ic determination of individuals at crime scenes (genetic fingerprinting) whenonly trace amounts of a DNA sample are ava i lable.

Quantitative PCR a l lows the determination of relative amounts of DNA strands and further extends PCR’s diagnostic and scienti fi ccapabi l i ties and appl ications . Fina l ly, the technique of RT-PCR a l lows the ampl i fi cation of DNA from RNA strands by making a cDNA copy us ingreverse transcriptase. This enables the quanti fi cation of mRNA abundance for speci fic genes , and has largely replaced northern blotting asthe method of choice for mRNA determinations .

Each PCR cycle includes DNA denaturization of a segment of double-stranded DNA with a segment to be amplified to produce a mixture of single-strandedDNA, annealing with specific DNA primers, and, finally, polymerization by the addition of polymerase and nucleotides (G, C, A, and T). Prior to the start of thenext sample, the replicated DNA is allowed to reanneal to again form double-stranded structures. Further cycles will exponentially reproduce the DNA strandof interest. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

CLONINGCloning i s a term used in two senses ; fi rs t, the process to produce identica l copies of an organism and, second, for the copying of DNAsequences . Only the latter wi l l be discussed here. The abi l i ty to clone a DNA fragment a l lows researchers to produce multiple copies of aselected gene or portion of DNA (e.g., via PCR), but a lso a l lows them to s tudy nonexpressed portions of DNA, including promoter andregulation regions and DNA that i s not expressed. Indeed, the abi l i ty to select and ampl i fy any particular DNA sequence for s tudy i s anessentia l part of the science of molecular biology.

The bas ic approach to cloning of any piece of DNA includes (a) cutting the DNA into an appropriately s i zed fragment via restriction enzymes;

(b) inserting the DNA into another specia l piece of DNA ca l led a vector, which conta ins an appropriate promoter region to a l low DNArepl ication and/or transcription; (c) introducing (transfection) these DNA fragment/vector DNA molecules into an appropriate cel l type forampl i fi cation of the DNA; and, fina l ly, (d) screening the resul ting cel l s to find and confi rm the identi ty of the DNA fragment of interest via areporter or marker gene (e.g., one that produces antibiotic res is tance or an enzyme producing a colored substrate). Once a clone i s i solatedand confi rmed, i t then offers a powerful tool for the researcher as noted above.

[Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd., 2009.]

The s ize of the DNA fragment that can be introduced into a cloning vector and s ti l l a l low the vector to function i s l imited. As a resul t, thefragmentation of a large DNA to a sui table length i s an important fi rs t s tep in cloning. This fragmentation can be done us ing very speci fic orrandom restriction enzymes. Cloning vectors are speci fic, ci rcular pieces of DNA (plasmids) that may have originated from bacteria or vi ruses orthat have been arti fi cia l ly produced. They have the abi l i ty to enter into cel l s and to be expressed in that cel l , ei ther separately or byincorporating themselves into that cel l ’s DNA. Cloning vectors a lso have speci fic DNA sequences where known restriction enzymes can beuti l i zed to cut open the vector and insert DNA fragments under wel l -control led laboratory conditions . Therefore, a speci fic restriction enzymeis used to cut open the vectors ; newly fragmented DNA sequences of interest are added and DNA l igase repairs the vector, which now includesthe DNA fragment. Transfection of these cloning vectors into cel l hosts i s performed by various techniques that wi l l not be discussed here.

Vector DNA (left) can be incorporated into human DNA (right) by the use of specific restriction enzymes (e.g., BamH1 or EcoR1) that produce DNA fragmentswith particular end, base-pair sequences that can subsequently be joined by DNA ligase. The ability to produce specific recombinant DNAs is an importantresearch and diagnostic tool. [Reproduced with permiss ion from Naik P: Biochemistry, 3rd edi tion, Jaypee Brothers Medica l Publ i shers (P) Ltd.,2009.]

Because the process may involve somewhat random fragmentation of the DNA and transfection i s often of low efficiency, a screening methodmust be used to identi fy those cel l s that conta in a functioning clone. The cloning vectors wi l l often express a particular marker (e.g., anenzyme that produces a colored product, as described above, or confers res is tance to an antibiotic) that a l lows the researcher to identi fy thecel l s with these working clone vectors . Fina l ly, cel l s that appear to conta in the DNA fragment of interest in a functional vector may be used forPCR ampl i fi cation (see above) and DNA sequence analys is (see above) of the clone/DNA fragment to confi rm that a success ful clone has beenproduced.

FLOW CYTOMETRYFlow cytometry, developed ini tia l ly in 1968, i s an important scienti fi c and medica l research appl ication that has a lso begun to ga in a role incl inica l treatments . This method takes advantage of fluorescent label ing of antibodies (as uti l i zed in severa l procedures above) and theabi l i ty to exci te these fluorescent molecules via lasers . Exci tation a l lows the detection (via a digi ta l detector) of individual cel l s or molecules(e.g., chromosomes) at an extremely fast rate (up to thousands of particles/ second). Particles (i .e., cel l s or large molecules ) can a lso bedetected by l ight scattering (often used to determine cel l type) at the same time. Multiple lasers and multiple detectors can be arranged so asto a l low the operators to s tudy cel l s labeled with severa l antibodies/fluorescent probes . Flow cytometers have been used to s tudychromosomes, DNA, RNA, proteins , cel l activation, enzyme activi ty, pH, intracel lular concentration of selected ions , membrane fluidi ty andapoptos is , and multidrug-res is tant cancer cel l s . Medica l fields such as hematology, oncology, pathology, genetics , and other specia l tiesuti l i ze flow cytometry extens ively. More advanced flow cytometry even a l low sorting of each individual cel l or molecule based upon theirscattering and/or labeled fluorescence characteris tics .

A reporter gene containing a particular enzyme [e.g., chloramphenicol transferase (CAT) whose activity can be easily detected by established testingmethods] is recombined with a test gene of interest, precipitated, and, finally, transfected into cells. The cells are cultured, then harvested and assayed forCAT activity to identify cells that have received a copy of the test gene. [Adapted with permiss ion from Murray RA, et a l .: Harper’s I l lus tratedBiochemistry, 28th edi tion, McGraw-Hi l l , 2009.]

Principles of Flow Cytometry See text for further details.

LABORATORIESCl inica l laboratory testing rel ies on severa l biochemica l techniques for the assay of important phys iologica l molecules found in the humanbody. The l i s t/table below reviews these techniques for severa l common tests .

BIOCHEMICAL BASIS FOR CLINICAL LABORATORY TESTS (CHEMISTRIES)

APPENDIX IIIORGANIC CHEMISTRY PRIMER

OVERVIEW Know and be fami l iar with the speci fic roles of the s ix important elements in l iving organisms. These are:

• Carbon• Hydrogen• Nitrogen• Oxygen• Phosphorous• Sul fur

Be fami l iar with the important functional groups that are formed by the s ix bas ic elements and understand their genera l s tructura l andfunctional roles in biology and medicine.• Hydrogen (partia l ly charged and ion)• Hydroxyl group• Carboxyl ic acid group• Amine group• Phosphate group• Sul fur-sul fur bond• Aldehyde group• Ketone group

INTRODUCTIONAlthough there are over 100 elements known, only a few are regularly seen in nature. In fact, less than a thi rd of the elements found on earthare found in some l i fe form, with only s ix elements seen in a l l l i ving organisms. These elements are carbon (C), hydrogen (H), ni trogen (N),oxygen (O), phosphorus (P), and sul fur (S). These s ix “biologica l” elements can be remembered as C—H—N—O—P—S that sounds l ike “chin-ups .” These s ix elements can bond with themselves (e.g., C—C and O—O) and with each other (C—H, O—H, and C—N). Specia l bonding patternslead to charged H atoms as wel l as hydroxyl , carboxyl , amine, high-energy phosphorus , and s trong sul fide-bonded groups . These moleculesserve s tructura l and functional roles as wel l as specia l i zed roles in very speci fic biologica l actions and reactions for functions in the humanbody (Table 1).

TABLE 1. The Six Bas ic Elements of Li fe

THE SIX ORGANIC ELEMENTS (C, H, N, O, P, AND S)

CARBON (C)

Main s tructura l element of a l l l i ving ti ssue, C usual ly forms four bonds with other elements . C—C bonds (e.g., sugar or fat molecules ) conta inenergy used in metabol i sm. C i s found in a lmost a l l biologica l molecules .

HYDROGEN (H)

Hydrogen can exis t with both a partia l charge and a ful l ionic charge, making i ts roles both varied and versati le. H i s an important s tructura lelement but i s often a lso seen in accessory roles , helping to form important functional groups and molecular s tructures . Functional ly, H cantransfer from one molecule to another or be by i tsel f as H+ (see next section). H forms one bond with another element. H i s found in mostbiologica l molecules .

NITROGEN (N)

Involved in many energy transfers and specia l i zed s tructures . N usual ly forms three or four bonds with other elements . N i s important inbiologica l molecules including amino acids/ proteins , complex carbohydrates , and l ipids and in nucleic acids .

OXYGEN (O)

Involved in many energy-related reactions . O usual ly forms two bonds with other elements . O i s found in a lmost a l l biologica l moleculesincluding amino acids/proteins , carbohydrates , l ipids and nucleic acids , as wel l as multiple other chemica l and biologica l molecules .

PHOSPHORUS (P)

Involved in energy s torage. P usual ly forms four bonds with other elements . P i s most importantly found in deoxyribonucleic acid (DNA) andribonucleic acid (RNA) and the nucleotides that form them.

SULFUR (S)

Involved in specia l i zed s tructures . S usual ly forms two bonds with other elements . S i s found in two amino acids and i s a lso seen in complexcarbohydrates and l ipids .

BIOCHEMICAL FUNCTIONAL GROUPS (H, OH, COOH, NH3, PO3, S—S, COH, AND )

From H bonding in proteins and DNA to S—S covalent bonding, these s ix atoms can combine, ei ther a lone or with trace inorganic elements andcofactors , to form severa l biologica l ly functional groups that are the key to l i fe. Some of the essentia l functional groups are discussed belowand summarized in Table 2.

TABLE 2. Biochemica l Functional Groups

HYDROGEN (PARTIALLY CHARGED AND IONIC FORMS, H+)

Although H plays an important role in i ts nonionized form, i ts further role in l iving creatures i s far too important to not mention here. H atomsinvolved in bonds often unfa i rly share the energy of the bond. When this happens , the atoms forming that bond wi l l be ei ther partia l lynegative or partia l ly pos i tive. H often acquires a partia l pos i tive charge, which serves as the bas is for H bonding and the s tabi l i zation ofprotein s tructure, DNA’s hel ica l form, and even biologica l membranes . If the H completely loses i ts electron, i t becomes a H ion (H+) wi th acharge of +1 and goes into solution. H+ i s an eas i ly transferrable atom that helps drive a myriad of biologica l reactions that are l i tera l ly toonumerous to mention. The role of H, both partia l ly charged and as an ion, i s explored in various chapters .

HYDROXYL GROUP (—OH–)

The pa i ring of an O and a H, referred to as a hydroxyl group (OH−), forms an important molecular component that plays numerous roles inbiology. Water (H2O, a lso known as H—O—H) has a OH− and a separate H+ and these negative and pos i tive charges help water to dissolve

other molecules and serve in biologica l functions . The OH– a l so combines with other functional groups to form proteins , DNA and RNA, andfunctional membranes , and often serves as the essentia l group in an enzyme activi ty. When a OH– i s on any biologica l molecule, i tsimportance, ei ther s tructura l ly or functional ly, i s usual ly high.

CARBOXYL GROUP (—COOH)

A C with a double-bonded O and a separately bonded OH is a carboxyl group (COOH). The H partia l ly dissociates from this group forming COO–

and H+ both of which are reactive in thei r own right. The COOH, much l ike the OH– di scussed above, i s involved in numerous biologica lreactions including formation of amino acid to amino acid bonds ; formation of the DNA and RNA backbone s tructure; the metabol i sm ofsugars ; and the formation of complex l ipid s tructures conta ined in membranes , hormones , and other specia l i zed l ipid-based molecules . If aCOOH is on a molecule, important s tructura l or functional reactions often occur.

AMINE GROUP (—NH2)

A N and two H plus an a lkyl group form the important biologica l group referred to as a primary amine. Bonds to one or two additional a lkylgroups (e.g., C) ins tead of H create a secondary and tertiary amine, respectively. When converted to an amino group, the amine wi l l often havea partia l pos i tive charge (NH3

+). With a change in the bonding of the N atom, an amine group can separate and become ammonia (NH3), whichis important in protein and amino acid waste el imination (i .e., urine). Ammonia , especia l ly in excess , a l so shows up in many i l lnesses as animportant clue in diagnos is and treatment. Amine groups are another clue to the location of important biologica l processes . The addition of afourth H atom to the amino group forms the pos i tively charged, cation ammonium (NH+

4), a l so important in many reactions and medica lconditions in humans .

PHOSPHATE GROUP (PO3 AND PO4)

The phosphate group, composed of one P and ei ther three or four O, i s the energy-carrying molecule of biology, speci fica l ly when l inked toanother phosphate group. The major functional component of adenos ine triphosphate, the phosphate group i s a lso involved in forming thebackbone s tructure of DNA and RNA, and i s seen in many extracel lular s igna l ing processes that ini tiate DNA synthes is and the production ofspeci fied proteins .

SULFUR–SULFUR BONDS (—S—S—)

Two S atoms can l ink together with one bond to form one of the s trongest l inkages in biology. S—S bonds are important in many proteinstructures that need to have high res is tance to breakage. Whi le S—S bonds are not as preva lent as the other groups l i s ted above, they are veryimportant in certa in s tructures found in both heal thy and diseased persons .

ALDEHYDE GROUP (—COH)

Like COOH, the a ldehyde group (COH) conta ins a double-bonded O, but the remaining bond i s to a H atom. Al though not as reactive as theCOOH, COHs are s ti l l important s tructura l and functional groups in human biology.

KETONE (— )

The ketone group ( ) conta ins a double-bonded O l ike the COOH and COH, but i ts C i s bonded only to two other C. Al though thisel iminates the reactive OH– found in the COOH, i t a l so makes this group very reactive because of an inequal i ty of charge between the C and O

atoms. As a resul t, the C i s very susceptible to bonding with a number of other atoms. are seen in multiple biologica l moleculesimportant to man, including acetone, acetoacetate, amino acids , carbohydrates , fatty acids , and ketone bodies .

SUMMARYSix important atomic elements—C, H, O, N, P, and S—are found in every l iving creature and form the s tructure of biologica l molecules . Inaddition, these elements form important functional groups , including H (partia l ly charged and ionic), OH−, COOH, amine, P, S—S, COH, and

. Understanding these s imple s tructures gives better ins ight and understanding of the fundamenta l chemica l and biochemica lreactions of the human body.

INDEX

Note: Page numbers fol lowed by “f” and “t” indicate figures and tables , respectively.

α–l inkages , 18α-hel ix, 10α-l inked hel ica l s tructure, 18α1-adrenergic receptors , 319α-1-anti tryps in, 255α-1-anti tryps in (AAT) protease inhibi tor deficiency, 343tα-1-anti tryps in deficiency, 255β2-agonis ts , 256–257β-l inkages , 18β-l inked l inear cha in, 18β-ketothiolase/acetyl -CoA acyl transferase, 84β-Lactam antibiotics , 58β-N-acetylhexosaminidase, 91β-oxidation cycle, 84β-turn s tructure, 5017β-estradiol (E2), 301ω-3 acid [eicosapentaenoic acid (EPA)], 29ω-6 acids (dihomogamma-l inolenic acid (DGLA), 2910-carbon, 15-carbon (farnesyl pyrophosphate), 9211-deoxycorticosterone, 3416-carbon fatty acid pa lmitate, 27, 28f2, 3-bisphosphoglycerate, 59, 2072,3-diphosphoglycerate, 2072, 3-enoyl -CoA hydrase, 842, 4-dienoyl -CoA reductase, 862′-deoxyribose sugars , 503-ketoacyl -CoA thiolase, 8430-carbon squalene molecule, 923-HMG-CoA, 91–9211-β-Hydroxylase deficiency, 366t11-β-Hydroxysteroid dehydrogenase type 2 deficiency, 367t17-α-hydroxylase deficiency, 299, 367t18-Hydroxylase deficiency, 368t21-Hydroxylase (sa l t-los ing type), 368t3-β-Hydroxysteroid dehydrogenase deficiency, 366t3-hydroxy-3-methyl -glutaryl -CoA (3-HMG-CoA), 913-hydroxyacyl -CoA dehydrogenase, 843-hydroxyacyl -CoA dehydrogenase deficiency, 843′-hydroxyls , 5011-cis-retina l , 294

AA (retinol ), 144tABO Blood Groups , 29Absent β-oxidation of VLCFA, 87Abeta l ipoproteinemia (Bassen–Kornzweig syndrome), 351tABG test, 318Absent A-oxidation of VLCFA, 87Acarbose, 76Aceta ldehyde dehydrogenase, 228Acetazolamide, 208Acetoacetate, 88Acetone, 88Acetyl coenzyme A (CoA), 69, 80, 136Acetyl -CoA carboxylase, 80, 136

regulation of, 80fAcetyl -CoA fragment, 84Acetylchol ine (Ach), 173, 174, 248, 256, 290, 295Acetylsa l icyl i c acid, 214Achlorydia , 154Acid–base ba lance, 251, 254–255, 268, 277

NH3 and, 277–278Acidic keratins , 183tAcidos is , 254Acros in, 305Acrosome reaction, 305Acrylamide molecules , 374Actin, 168, 169fActin-binding proteins (ABP), 171Actin fi laments , 11

skeleta l muscle, 11Activators , 56Activin, 300, 309Active enzymatic polymers , 80, 80fActive transport, 98Active vi tamin D (ca lci trol ), 35fAcute lymphoblastic leukemia, 114bAcute rejection, 234Acute respiratory dis tress syndrome (ARDS), 259–260Acyl -CoA dehydrogenase deficiencies , 354tAcyl -CoA dehydrogenase enzyme, 84Adaptive immune system, 222Addisonian cri s i s , 275Adenine (A), 38Adenos ine deaminase, 42

deficiency and gene therapy, 42Adenos ine diphosphate (ADP), 214molecules , 262Adenos ine monophosphate (AMP), 39Adenos ine triphosphate (ATP), 39, 65, 132, 152, 168, 209, 236, 270

in muscles , sources of, 179fAdenyl cyclase, 100, 101f, 273Adrenal androgens , 35Adrenoleukodystrophy (ALD) (Addison– Schi lder Disease or Siemerl ing–Creutzfeldt Disease), 87Adherens junctions , 177Adhes ion, 213ADH (vasopress in), 306fAdrenal insufficiency (Addison’s disease), 275Adrenergic receptors , 289Affini ty chromatography, 380Affini ty of the enzyme for the substrate, 56, 56fAggregation, 213Alanine, 5t, 135

cycle, 135transaminase, 135

Aldolase reductase, 140bAldosterone, 34Airways , 251Albinism, 334tAlbumin, 157t, 202Aldehyde dehydrogenase, 151tAldehyde group (COH), 397, 397tAldosterone, 275–276, 275f

and disease, 275effect on pH, 276and potass ium-sparing diuretics , 275

Al i ski ren, 270Alka l ine phosphatase, 194, 200Alka los is , 254Alkaptonuria , 335tAl losteric effector, 206, 207Al losteric regulation, 59, 59fAlpers ’ disease, 350tAlpha-actinin, 171Alpha-amylase, 151Alport’s syndrome, 264Als trom syndrome, 12, 182Alti tude s ickness , 208, 272Alveolar sacs , 253Alveol i , 251, 253, 253f

col lapse during respiration, 253Alzheimer’s diseases , 94, 182Amelogenin, 150Amine group (–NH2), 397, 397tAmino acid, 1, 3, 130, 156t, 165t, 269t. See also Enzymes

bas ic s tructure of, 6fcomponents of various biologica l molecules , 11tdefined, 4enzyme’s primary s tructure, 56intestina l absorption of, 314peptides and proteins , 3R-group, 3sequence, 170, 170fsources and fates , 60f

synthes is of, 60fsynthes is/degradation of, 334t–339t

Amino acid degradation, 61–62pathways , 61f

Amino acid metabol i sm, 61–62, 130fdiseases of, 62t

Amino acid res idue, 7, 7fAmino acid synthes is , 61, 60fAminopterin, 40Amphotericin B, 95bAmiodarone, 260Ammonia (NH3) toxici ty, 278Ammonium persul fate, 374Amylase, 18Amylopectin, 18Amylose α-bond, 18Anaerobic respi ration, 135Androgens , 35

binding protein, 309insens i tivi ty syndrome (AIS), 299, 312, 369treceptor blockers , 308, 308f

Androstenedione, 35Anemia, 203Aneurysm, 246Angiostatin, 218Angiotens in, 240Angiotens in-converting enzyme (ACE), 273

inhibi tors , 248Angiotens inogen, 159tAngiotens inogen/angiotens in I and II , 270, 273

convers ion of angiotens inogen, 273feffects of body, 274t

Angiotens in receptor blocker, 273Anoxia , 247Anthracos is , 260tAntibacteria l antibody functions , 223fAntibacteria l compounds , 151tAntibiotic use and clostridium difficile, 166Antibody, 222–223, 222f, 223f, 224t

structure of, 222fAntibody molecules

structure of, 222fsummary of, 224t

Anticodons , 108Antidiuretic hormone, 34, 306fAntigen, 222

dendri tic cel l presentation of, 229fT-cel l receptor recognition of, 225f

Antigen-presenting cel l s , 256Antiglomerular basement membrane disease, 264Antimicrobia l enzymes , 151tAnti -Mul lerian hormone (AMH), 298Anti thrombin II I , 301Aplastic anemia, 203Apol ipoprotein, 242Apoproteins , 33, 253Aquaporin-2 (AQP-2), 99, 276Aquaporins , 99Arachidate (20-carbon), 24fArachidonic acid, 29, 51Arginase 1, 278tArginase deficiency (argininemia), 341tArginine, 5tArgininosuccinate lyase, 278tArgininosuccinate synthetase (ASS), 278t

deficiency (ci trul l inemia, types I and II), 341tArgininosuccinic aciduria/acidemia, 342tArrestin, 294Arteria l blood gas (ABG) test, 314Arteriostenos is , 246Artery, 241fArti fi cia l pulmonary surfactants , 261Asbestos is , 261tAscorbic acid, 194, 213

Asparagine, 5tAsparagine amino acid / “N-glycosylation”, 76Aspartic acid, 5tAspergi l los is fungal infections , 262Aspirin, 214, 248

in treatment of myocardia l infarction, 248Asthma, 254, 256

β-adrenergic theory of, 256asthmatic type I hypersens i tivi ty response, 257tenvironmenta l factors , 256genetic component to, 256and immunologica l diseases , 256severe cases , 256treatment, 256–259

Ataxia telangiectas ia (AT), 360tAtelectas is , 253Atheroma, 246Atheroscleros is , 246, 247f

formation and infarction, 247ftreatment of, 246

Atheroscleros is formation and infarction, 247fAtopic dermati ti s , 192tATP, 66–68. See also Adenos ine TriphosphateATP s tructure with i ts magnes ium cofactor, 68fATP synthase, 70

cyl indrica l -shaped F0 protein, 70F1 headpiece s ta lk, 70F1 headpiece subunit, 70

ATP7A enzyme, 57Atres ia , 300Atria l natriuretic peptide (ANP), 274

receptors , 239Atrophic gastri ti s , 163Atrophy, 175Attractants , for neutrophi l s and eos inophi l s , 257tA type proanthocynidin, 267Autoimmune diseases , 231Autoimmune hemolytic anemia, 203Autosomal recess ive polycystic kidney disease, 12, 182Axonemal dynein, 180Azidothymidine (AZT), 107, 107bAzi thromycin, 110b

BB1 (thiamine), 144tB2 (riboflavin), 144tB3 (niacin), 144tB5 (pantothenic acid), 144tB6 (pyridoxinepyridoxa l , pyridoxamine), 144tB7 (biotin), 144tB9 (fol ic acid), 144tB12 (cyanocobalamin), 144tBacteroides , 163Barbi turates , 295Bardet–Biedl syndrome, 12, 182Bari tos is , 261tBarrett’s esophagus , 154Barters ’s syndrome (Gi telman variant), 370tBarth syndrome, 94. See also X-l inked diseaseBartter syndrome, 267Basa l cel l carcinoma, 113bBasa l lamina, 264

disorders , 264Bas ic protein s tructure, a ffecting factors , 7–9

amino acid compos i tion, 7fina l destination, 7fina l modi fications , 7functional s i tes , 7specia l amino acids , 7

Basophi l s , 228Bauxi te fibros is , 261tBenign prostatic hyperplas ia (BPH), 308Benzodiazepines , 295

Benzothiazepines , 236, 238f, 238tBerger’s disease, 266tBeryl l ios is , 261tBeta-blockers , 239Bicarbonate, 269tBicarbonate ions , movement of, 255Bicarbonate ions production by pancreas , 161fBi functional enzyme, 134Biguanide medications , 71Bi le sa l ts , 24, 33

sequestrants , 33in triglycerides metabol i sm, role of, 160f

Bi le synthes is , 157, 157tBi l i rubin measurement, 155Binding of the effector, 59, 59fBiochemica l functional groups , 396Biotin (biotinidase) deficiency, 370tBisacrylamide, 374Bisphosphonates , 197Bladder, 266Bleomycin, 260Blood

calcium and phosphate levels , control of, 200fclotting, 213, 214f

cascade, 214, 215, 216, 216ffibrin meshwork, 216, 217fplasmin and clot dissolution, 218–219, 219fplatelet plug formation, 213–214, 215fplatelet plug formation and clot formation, di fference between, 217, 218regulation of clot formation, 218, 218f

components of, 202–203, 202fimpact of O2–CO2 exchange, 254inadequate O2 del ivery, phys iologic response to, 209inadequate synthes is of hemoglobin (Hgb) molecule, 205, 206ti ron

ferri tin, 211, 212hepcidin in regulation of, 212–213metabol i sm, 211, 212ftransferrin, 211, 212f

O2 bindingal losteric binding of, 205, 206–207, 207fregulation of, 207–209, 207f, 208frelaxed hemoglobin (Hgb), 205, 205ftense hemoglobin (Hgb), 205, 205foverview of, 201–202

RBC functions , 203, 203f, 204f, 205fseparation of, 202fs ickle cel l disease (SCD), 209–211, 210f

Blood glucose, 313concentration, 140f

Blood vessels , 179t, 240, 241f, 242fcompos i tion of, 241fpermeabi l i ty, 240

Bloom syndrome, 361tB lymphocytes , 227Bohr effect, 207Bone, 199t. See also Connective ti ssue and bone

components of, 187, 194–197, 194f, 195f, 196fformation, markers of, 200fractures and heal ing, 198growth and remodel ing, 197–198role in human body, 185s ia loprotein, 194

Bone matric, minera l i zation in, 195Bone s ia loprotein, 194Bordetella pertussis, 262Bowman’s capsule, 2642,3-BPG to deoxyhemoglobin, binding of, 208fBradykinin, 273Bra in Natriuretic Peptide (BNP), 239, 276Breathing muscles , 253Bronchi , 251Bronchiole tubes , 252

Bronchiti s , 255–256Bronchodi lators , 258fBrunner’s glands , 164tB-type natriuretic peptide (BNP), 239Byss inos is , 261t

CCalbindin, 199Calci ferol , 145tCalci tonin, 198, 199t, 279

effects on target organs , 199tCalci triol , 279Calcium, 146t, 269t

activation of smooth muscle cel l s , 237fCalcium channel blockers (CCBs), 236, 237, 239f

classes and actions , 238tCalcium-induced ca lcium release (CICR), 171, 176Calcium level regulation, 199, 199t, 200fCalcium oxa late, 279Calcium phosphate, 279

Calcium release channels , 240Calor (warmth), 30Cal lus , 198Calmodul in, 173, 240

activation, 178Calsequestrin, 173Campylobacter jejuni, 287Canal icular, 196Cancer therapies , 180Carbamate, 207Carbamoyl phosphate synthetase I (CPS-I), 62, 278t

deficiency, 342tCarbohydrate digestion, 317Carbohydrate intolerance, 18Carbohydrate metabol i sm, 156t

overview of, 66fCarbohydrate molecules

breakdown (catabol i sm), 65synthes is (anabol i sm), 65

Carbohydrates , 15, 130and ferti l i zation, 21protein molecules , 18biochemica l Roles of, 15tdefini tion, 15structura l configuration, 15–16, 16f

Carbon (C), 396, 396tCarbon–carbon double bonds , a l teration of, 86fCarbon dioxide (CO2), el imination of, 253Carbon dioxide (CO2) in red blood cel l s , fate of, 208fCarbonic acid, 254Carbonic anhydrase, 195, 207, 254, 254f

inhibi tor, 208Carbon monoxide (CO) poisoning, 204Carboxylate, 269tCarboxyl group (COOH), 397, 397tCarboxyl ic acid group, 24Cardiac cycle, 239–240Cardiac markers , 247Cardiac muscle, 168f, 175–176, 176f

structure, 176fs tructure and function, 236, 236f, 237f, 238f, 238t

Cardiol ipin (Diphosphatidylglycerol ), 94Cardiomyocytes , 175Cardiovascular system, 253

atheroscleros is , 246, 247fblood vessels , 240, 241f, 242fcardiac cycle, 239–240cardiac muscle s tructure and function, 236, 236f, 237f, 238f, 238tendogenous cholesterol/l ipoprotein metabol i sm and transport, 241, 243f, 244f, 245f

HDL, 244, 245fintermediate-dens i ty l ipoprotein (IDL) and LDL, 243–244VLDL, 241, 242, 243

heart attack, 247–248overview of, 235

s inoatria l and trioventricular nodes , 236, 237Carni tine pa lmitoyl transferase I (CPT I), 82, 82fCarni tine pa lmitoyl transferase (CPT) I deficiency, 83, 352tCarni tine pa lmitoyl transferase (CPT) I I deficiency, 83, 353ttreatment of, 83Carni tine trans locase deficiency, 352tCarrier protein, 98Carti lage dis tribution in adults , 186fCaspofungin, 75Cataract, 264Catecholamines , 137–138, 290–292, 291fCatheps in enzymes , 195Catheps in K enzyme, 195Cel l -mediated immunity, 222

and humoral immunity, 226fCel l cycle, 114, 114t

microtubules , 114bphases , 114t

Cel l s , 152tCel lulose, 18, 19fCel lulose β-bond, 18Centra l nervous system (CNS), 152, 319Centriole/basa l body, 182Centroacinar emphysema, 255Centronuclear/ myotubular myopathies (CNM), 173Cephal ic phase of eating, 150Ceramide/sphingol ipids synthes is , 27, 28f, 90Cerebros ides , 27, 28fCGMP-speci fic phosphodiesterase type 5, 272Chal icos is , 261tChaperones , 110Charged R-groups , 4Chemokine receptor group, 229Chemotherapy, 260

agents , 40Chief (zymogenic) cel l s , 154Chloride, 145t, 269tCholecystokinin, 293Cholecystokinin (CCK), 153t, 161, 164tCholesterol , 24, 30–32, 246

derived hormones , 100tefflux regulatory protein (CERP), 244ester, 32, 32f, 242esterase, 162texogenous and endogenous pathways of, 243ffunctions of, 30–32,ga l l s tones , 161interaction, 95fmetabol i sm, 32molecule, 32, 32fregulation of, 92s idechain cleaving enzyme, 309synthes is , 91–92, 92fthree-dimens ional s tructure, 32

Chol inesterase, 174Chondrocytes , 186Chondroi tin, 20, 76, 193Chondroi tin sulphate, 194Chromium, 147tChromosomes (right) and a gene, relationship between, 45fChronic bronchi ti s , 255–254Chronic granulomatous disease, 73Chronic kidney disease (CKD), 270Chronic obstructive pulmonary disease (COPD), 254Chronic rejection, 234Churg–Strauss syndrome, 262Chylomicrons , 242

metabol ic fate of, 244fChymotryps in, 162tChymotryps inogen, 162tCigarette smoking and bronchi ti s , 256Ci l ia , 181, 181f, 182Ci l ia/flagel la , defects in, 12Ci l iopathies , 12

Citric acid cycle, 65, 68–69regulation of, 69

Clara cel l s , 253Clari thromycin, 110bCl inica l laboratory tests , 388t–394tClofibrate, 246Cloning, 42, 384–386, 385f

construction of recombinant DNA, 386fClot dissolution, 219Clot formation, 213, 215f

by platelets , 215regulation of, 218, 218f

Clotting. See under BloodClotting cascade, extrins ic and intrins ic pathways of, 216fClotting cascade event, 318Clotting factors , 301Cobalt, 147tCoal worker’s pneumoconios is , 261tCockayne syndrome, 361tCoenzyme A (CoA), 57Cofactors , 56Col lagen, 10, 50

fibers , formation of, 188fpeptides , 189fs tructure of, 187fsynthes is and scurvy, 194types , 190t–192t

Col lagen-based connective ti ssue, 189fCol lagen fibers , 186

formation of, 188fCol lagen IV, 255Col lecting duct, 268Column chromatography, 378Common fatty acids found in humans , 27fCommon phosphol ipids found in humans , 26fCommon unsaturated fatty acids , 24fCompact bone matrix, 198Competi tive inhibi tion, 58Complement deficiency and disease, 233Complement system

alternative pathway, 232, 232f,class ica l pathway, 232, 232f

Complex l ipids , metabol i sm of, 88–92Congenita l lobar emphysema, 255Conjugated bi le acids , 33Connecting capi l lary, 241fConnective ti ssue, 179t, 186–187, 186f, 187f–188f, 189f

col lagen-based, 189fConnective ti ssue and bone. See also Bone

bone, components of, 187, 194–197, 194f, 195f, 196fbone formation, markers of, 200bone growth and remodel ing, 197–198calcium level regulation, 199, 199t, 200fconnective ti ssue, 186–187, 186f, 187f–188f, 189f

col lagen types , 190t–192toverview of, 185resorption, 200

Conn syndrome, 275Copper, 146tCopper deficiency, diseases of, 57Cori cycle, 135, 135fCorona radiata layer, 305Coronary artery disease, 314Corrected ca lcium, 200Cortica l reaction, 305Corticosteroids (adrenal gland), 29, 34, 261

for asthma, 259for ILDs , 260

Cortisol , 275Cortisone, 275Cow’s mi lk, excess consumption of, 213CPT diseases , 83Cranberry Juice, 267C-reactive protein (CRP), 159t

Creatine kinase (CK), 247Creatine phosphate, 174, 270, 318Creatinine, 266Creutzfeldt–Jakob disease, 111b, 317, 375Crigler–Najjar syndromes, 364tCrohn’s disease, 262Cryptogenic organizing pneumonia , 261tCushing’s syndrome, 90Cycl ic adenos ine monophosphate (cAMP), 100, 101f, 150, 237, 262, 271, 273, 299, 303Cycl ic guanos ine monophosphate (cGMP), 271, 276, 294

defect, 314NO-mediated production of, 272

Cycl in–CDK heterodimer complex, 115Cycl in-dependent kinase (CDKs), 114Cyclooxygenase (COX)-1 or -2, 29, 214, 248Cysteine knot, 187Cystic fibros is (CF), 262, 371tCystic fibros is transmembrane conductance regulator (CFTR), 262Cysteine, 4, 6f, 6t, 279Cystine “disul fide” bond, 4, 7fCystinuria , 340tCysti ti s , 267Cytidine synthes is , 40fCytidine triphosphate (CTP), 39, 88Cytokines , 257tCytokines , 229Cytomegalovi rus , 287Cytoplasmic dynein, 180Cytoskeleta l proteins , comparison of, 182fCytostatics , 231Cytos ine (C), 39Cytotoxic T cel l s , 224

DD-3-hydroxybutyrate, 88Danger s igna ls , 222D-dimers , 219Decreased HMG-CoA reductase activi ty, 92Deletion mutation, 113Deep vein thrombos is (DVT), 219Defective androgen receptor, 299Deferas i rox, 211Degenerative arthri ti s , 193Dehydration, 210Dementia , 318Demyel inating disorders , 283Dendri tic cel l s (DCS), 227, 228–229, 229f, 230t–231t

presentation of antigen, 229fDenta l plaque formation, 150Deoxy form of UMP (dUMP), 39Deoxyribonucleic acid (DNA), 38Deoxyhemoglobin, 208fDeoxyribose, 39Dermatomyos i ti s , 261tDesaturase enzyme, 81Desmin, 177, 183tDesmopress in, 217Desquamative inters ti tia l pneumonia , 261tDetoxi fication, 157, 157tDHT, 307–308Diabetes Mel l i tus (DM), 138–139Diabetic neuropathy, 143bDiabetic nephropathy, 266tDiacylglycerol , 24, 25fDiaphragm dysfunction, 254Diarrhea, 318Dietary measures , 246Digestive system

gal l bladder, 160–161large intestine and anus/rectum, 163, 166fl iver, 155, 156f–159f

l ipid metabol i sm in, 159–160, 160fmouth, 150–152, 151t, 152toverview of, 149, 150f

pancreas , 161, 161f, 162tsmal l intestine, 161, 163, 163f, 164t–165tstomach, 152–155, 152f, 153t, 155f

chief (zymogenic) cel l s , 154enterochromaffin-l ike cel l (ELC), 154G cel l s , 154hormone production from, 152fmucus (neck) cel l s , 152parieta l (oxyntic) cel l s , 153–154

prostaglandin E2, 154, 155fDigestive tract, functions of, 179tDihydropyridine, 236, 238t

receptor, 176Dihydrotestosterone, 317Di l tiazem, 238tDipa lmitoylphosphatidylchol ine, 253Disaccharides , 50

in human biology, 16fDis ta l convoluted tubule, 268, 268fDis ta l tubule, 265f, 267Diuretic medications , 267Diva lent meta l transporter (DMT), 211, 212DNA

bas ic s tructure of, 44tdouble-hel ix s tructure of, 42sequences of A’s , G’s , C’s , and T’s of genes , 42

DNA and RNA sequencing technique, 380–381, 381fDNA binding moti fs , 112tDNA hel icase, 106DNA molecules , 42DNA polymerase, 106DNA repl ication, 106–107DNA Southern and RNA northern blots , 382–383, 382fDNA topoisomerase, 106Dolor (pa in), 30Dopamine, 236, 237, 287–288Dopamine deficiency, 312Double hel ix, 42Drummond’s syndrome (blue diaper syndrome), 340tDubin–Johnson syndrome, 364tDuodenum, 161Dynamin, 173Dynein, 180, 181fDystrophin, 9

EEating disorders , 293Ecchymoses , 213Edema, 203Effector molecule, 59Ehlers–Danlos syndrome (EDS) (cutis hyperelastica), 190t, 343tEicosanoid, 29

overview of, 31tsynthetic pathway, 30f

Eicosatrienoic acid, 51Elastase, 162tElastic fibers , 187, 188, 189f

quaternary s tructure of, 189fElastin microfibri l s , 189fElectrocardiogram (ECG), 239Electrolytes , 151tEl l iptocytos is , 96, 96bELISA technique, 377Elongation factors , 110bEmbolys is , 248Emphysema, 255

centroacinar, 255congenita l lobar, 255panacinar, 255

Endocrine pancreas , 161Endonuclease enzymes , 113Endocrine pancreatic hormones , 162tEndogenous cholesterol/l ipoprotein metabol i sm and transport, 241, 243f, 244f, 245fEndometrios is , 302

Endothel ia l cel l , 253Energy production and use in muscles , 178, 179f, 180Enhancer proteins , 111Enoyl -CoA hydrase, 84Enoyl -CoA hydratase, 84Enoyl -CoA i somerase, 86Entamoeba his tolytica , 95Enteric vi ruses , 287Enterochromaffin-l ike cel l (ELC), 154Enteroglucagon, 153tEnzyme activi ty

a l losteric regulation of, 59, 59fEnzyme-based diseases , 56Enzyme complexes with more than subunit (“multimeric”), 59Enzyme deficiencies , 18Enzyme kinetics , 56Enzyme reactions , 11, 56–57, 58

cofactors , 57, 57fmaximum rate of the reaction, 57number of molecules of substrate, 57

Enzyme regulation, 58competi tive, 58competi tive and noncompeti tive inhibi tion, 58fnoncompeti tive, 58uncompeti tive, 58

Enzymes , 11, 55, 56–61cata lyzation, speed or “rate”, 58digestive, 150, 151tfeedback regulation, 58

metabol i sm, 58product, 56, 56fregulation, 58

Eos inophi l s , 227–228Epinephrine, 80, 236, 240, 316Epithel ia l cel l , 253Epitope, 222Epstein–Barr vi rus , 287ER-bound ribosomes, 110Erecti le dys function (ED), 272Ergosterol , 95bErythrocytes , 202Erythromycin, 110bErythropoies is , 202Erythropoietin (Epo), 202, 278–279

regulation of red blood cel l production by, 279fEscherichia coli, 27Essentia l amino acids , 4, 25, 60Essentia l fatty acids , 25Essentia l unsaturated fatty acids , 82Essentia l myos in l ight cha ins , 171Esteri fied cholesterol molecule, 32fEstradiol , 300Estriol (E3), 301Estrogens , 301, 307t

classes of, 301ffunctions , 301

Estrone (E1), 301Ethambutol , 75Even-chain fatty acid, 84Exci tation-contraction coupl ing, 171–173, 172fExocrine functions , 161Exocrine pancreatic enzymes , 162f, 162t“Expressed sequences” or “exons , 42External sphincter, 266Extracel lular cholesterol , 316Extracel lular matrix (ECM), 196Extracel lular matrix s tructure, 22fExtracel lular s igna l -related kinases , 299Eye, 179tFFabry disease, 353tFaci l i tated protein channels , 97f, 98Factor V Leiden, 219, 365t

Fal lopian tubes , 11Fami l ia l hypercholesterolemia, 245Fanconi anemia, 361tFarber l ipogranulomatos is , 353tFarmer’s lung, 261tFasciotomy, 174Fast-twitch fibers , 174Fatty acid cha in double bonding, 24fFatty acid cha ins , 84

carboxyl (CO2–) group, 84

Fatty acid degradation, 82–88, 85fFatty acid elongase, 81Fatty acid elongation, 81Fatty acid metabol i sm, 80–88Fatty acid oxidation, 140tFatty acid packs , 25Fatty acid synthase, 81, 82fFatty acid synthes is , 80–81, 140t–141t

malonyl -CoA production, 81Fatty acid-derived prostaglandins , 265Fatty acids , 24, 24fFatty acids , 178

short-cha in, 163Fatty acyl -CoA molecule, 82Fatty s treak, 246Female reproductive system

breast ti s sue development and lactation, 306–307, 307testrogens , 301FSH, 299–300GnRH, 299hCG, 302–303LH, 300menstrua l cycle, 303, 304fprocess of ferti l i zation, 305, 305fprogesterone, 301–302

Fenestrations , 264Fermentation, 163Ferric reductase, 211Ferri tin, 111b, 211, 212, 213Ferroportin, 212Ferti l i zation, 305Feta l red blood cel l s , 159tFibrates , 246Fibri l -associated col lagens , 191t, 192tFibri l l in, 187Fibrin, 213Fibrin meshwork, 216, 217fFibrinogen, 216

to fibrin, convers ion of, 217fFibrinolytic system regulation by protein C, 218fFibrodysplas ia oss i fi cans progress iva (FOP), 194Fibrous proteins , 8Five-carbon i sopentenyl pyrophosphate, 92Flagel la , 181, 181f, 182Flavin adenine dinucleotide (FAD) cofactor molecule, 84Flavin adenine dinucleotide, reduced (FADH2), 57Flow cytometry, 386, 387fFluoroquinolones , 166Focal segmenta l glomeruloscleros is , 266tFol l i cle-s timulating hormone (FSH)

female, 299–300male, 308f, 309

Fragi le X syndrome, 362tFredrickson class i fi cation, 245Friedreich ataxia , 362tFructose, 165tFructose 2, 6-bisphosphate, 317Fructosuria (hepatic fructokinase deficiency), 344tFuel homeostas is during s tarvation, 35Fumarase deficiency, 350t

GG proteins , 93, 99, 199, 262, 297

G-protein receptors , 101t, 262, 273GABA, 292, 293f, 295GAG carbohydrates , 76

GAGs, 76functions of, 78tGalactokinase deficiency (ga lactosemia II), 345tGalactorrhea, 306Galactosamine, 20Galactose, 16, 17fGalactose-based glycosphingol ipid molecules , 27Galactose epimerase deficiency (ga lactosemia II I), 345tGalactosemia, diagnos is of, 51Galactosemia (class ic) (ga lactosemia I), 345tGal l bladder, 160–161GalNAc Carbohydrate, 28fGangl ios ides , 29, 90Gaseous waste products , el imination of, 252Gastric inhibi tory peptide (GIP), 153tGastric parieta l (oxyntic) cel l s , 313Gastric ulcers , 154Gastrin, 153tGastroenteri ti s , 163Gastroesophageal reflux disease (GERD), 154Gaucher’s disease (GD), 353tG cel l s , 154Gel fi l tration chromatography, 378–379, 379fGene express ion, 113Genome, 42Gi lbert syndrome, 364tGitelman syndrome, 267Gl ia l fibri l lary acidic protein, 183tGlobin cha ins , 203Globos ide, 28fGlobular proteins , 8Glucagon, 80, 136–137Glucocorticoids , 34–35, 52, 138Glucokinase, 66Gluconeogenes is , 65, 71–72, 140t–141t

metformin, 71pathway of, 72fprimary regulation of, 71

Glucosamine, 20, 76Glucose, 16Glucose-6-phosphatase, 71Glucose-6-phosphate dehydrogenase (G6PDH) deficiency, 73

moderate G6PDH deficiency, 73treatment of, 73

Glucose-6-phosphate, 66, 134–135Glucose–Alanine cycle, 135fGlomerular disorder diseases , 266Glomerular fi l tration rate (GFR), 270Glomerulonephri ti s , 264Glomerulus , 264Glucagon, 293Glucocorticoids , 231Glucosamine, 193Glucose, 269tGlucose-6-phosphatase, 318Glucose-6-phosphate dehydrogenase deficiency (G6PDH), 311, 345tGlucose-6-phosphate i somerase, 151tGlutamic acid, 5t, 292, 293fGlutamine, 5tGlutathione transferase, 151tGlyceryl trini trate, 242fGlycine, 292Glycine encephalopathy, 335tGlycoca lyx, 265Glycera ldehyde-3-phosphate, 66Glycerol , 24Glycerol -3-phosphate dehydrogenase, 70–71Glycine, 5tGlyco- and tauro-bi le acids , 33Glycogen, 18, 65

and the plant s tarch forms amylopectin and amylose, 19f

Glycogen s torage disease (GSD), 18, 76, 77t, 180, 347tGSD (type Ia ) (Von Gierke disease), 347tGSD (type II I) (Cori or Forbes disease), 348tGSD (type II) (Pompe disease), 348tGSD (type IV) (Andersen disease; amylopectinos is ), 348tGSD (type VI) (Hers disease), 349tGSD (type VII) (Tarui disease), 349tGSD (type V) (McArdle disease), 349t

Glycogen s torage disease type VII / Tarui ’s disease, 66“class ic” variant, 66“late onset” variant, 66infanti le, 66

Glycogen synthase kinase 3, 302Glycogen synthes is , 75, 75f, 140t–141tGlycogenin, 75Glycogenolys is/glycogen breakdown, 75–76, 76f

debranching enzyme, 75glucan transferase enzyme, 75inhibi tors , 76, 140t–141trepeti tive removal of glucose res idues , 75

Glycol ipids , 24, 27, 32Glycolys is , 65, 66–68, 69, 140t–141t

energy production, 66includes 10 enzyme steps , pathway of, 67freaction of, 66phosphorylation, 66

Glycoproteins , 18, 76, 152tbiochemica l roles of, 78tphosphorylated, 150

Glycosaminoglycans (GAGs), 20, 193, 264and proteoglycan associated with col lagen, 20fbiologica l role of, 21t

Glycosphingol ipid molecules , 27, 29Glycosphingol ipid, 27Glycosyl transferases , 75

inhibi tors , 75Glycyrrhizin, 275Gonadotropin-releas ing hormone (GnRH), 299Gonads , 297Goodpasture’s syndrome, 261t, 264, 266tGout, 41Gram-negative bacteria , 233Gram-pos i tive bacteria , 58Guanine (G), 38Granulocyte colony-s timulating factor, 230tGranulocytemacrophage colony-s timulating factor, 230tGranulosa cel l s , 300Granulys in, 224Granzyme, 227Guanylyl cyclase, 239, 276, 294Guanos ine monophosphate (GMP), 39Guanos ine triphosphate (GTP), 69Gui l la in–Barré Syndrome (GBS), 287Gustin, 150Gut flora , 163, 166

HH-substance, 29Hageman, 218Hair and cystine double bonding, 4Hard clot formation, 217fHartnup disease (neutra l aminoaciduria), 340tH bonding, 396HDL. See High-dens i ty l ipoprotein (HDL).Heart

attack, 247–248sectional view of, 236f

“Heat shock” proteins , 110Hel ix–loop–hel ix, 112tHel ix–turn–hel ix, 112tHelper T lymphocytes , 224Helper T (Th) cel l s , 319Hematology, 202Heme

breakdown of, 160fmolecule, 203, 204fsynthes is of, 204f

Hemoglobin (Hgb)to bi l i rubin, degradation of, 160fbreakdown of, 157tdiseases , 206tinadequate synthes is of, 205, 206tO2 binding of oxygen to, 205fsummary of diseases , 206tsynthes is and s tructure of, 204, 204f

Hemoglobinopathies , 96, 96bHemolys is , 210, 211Hemolytic anemia, 313Hemophi l ia , 217Hemophi l ia A, B, and C, 365tHemorrhage, 213Hemos iderin, 211Henoch–Schönlein purpura, 266tHeparin, 20, 218Hepatomegaly, 316Hepcidin, 212, 213

action of, 214fin inflammatory response, 214fin regulation of i ron, 212–213

Hereditary fructose intolerance (a ldolase B deficiency), 346tHereditary glomerulonephri ti s , 266tHeterodimeric glycoprotein hormone (hCG), 302–303Hetero-ol igomer, 9Heterotrimers , 216Hexamer, soluble, 216Hexokinase, 66Hexoses fructose, 16Hgb, relaxed (R) s tate of, 316HgbF, 314High-dens i ty l ipoprotein (HDL), 241, 244, 245fHigh-performance/pressure l iquid chromatography (HPLC), 380

H ion (H+), 396–397, 397trole in blood, 254

His tamine (H2), 153, 228, 257treceptors , 98b

His tidine, 4, 6t, 228His tidinemia, 335tHis tocompatibi l i ty, 234His tone acetyl transferases (HATs), 111His tone deacetylases , 111His tones , 104HMG-CoA reductase, 92Homocystinuria , 336tHomo-ol igomer, 9Hormonal involvement, in sexual di fferentiation, 298fHormone-respons ive breast cancers , 302Hormone respons ive elements , 301Hormones

affecting s tomach, 153tproduction by digestive system, 155fproduction from stomach, 152f

Human chorionic gonadotropin (hCG), 299Human immunodeficiency vi rus (HIV), 95Human papi l lomavirus (HPV), 229Humoral hyperca lcemia of mal ignancy (HHM), 199Humoral immunity, 222, 226f, 227Huntington’s Chorea (HD), 293Hyal in, 305Hyal ine membrane, 260Hyal ine membrane disease, 254Hyaluronic acid, 20Hydrogen (H), 396, 396tHydrophi l i c l ipids , 23Hydrophi l i c (water loving) R-groups , 4Hydrophobic (s ticky) patch, 209, 210fHydrophobic (water hating) R-groups , 4Hydrophobic “ta i l ,” 36

Hydrophobic l ipids , 23Hydrophobic regions , 253Hydrophobic substrate, 56Hydrophobic, s igna l ing molecules , 100tHydroxyapati te, 187, 195fHydroxyl groups (OH), 16, 399Hydroxylys ine or hydroxyprol ine res idues/“O-glycosylation”, 76Hydroxyurea, 211Hyperacute rejection, 234Hyperammonemia, 278Hyperammonemia–hyperorni thinemia–homocitrul l inuria (HHH syndrome), 340tHyperbi l i rubinemia, 155Hyperca lcemia, 267Hypercapnia , 254Hypercholesterolemia, 312Hyperdibas ic aminoaciduria type 2, 278tHyperglycerolemia (glycerol kinase deficiency), 346tHyperparathyroidism, 317Hypersens i tivi ty reactions , 233–234, 233t–234t, 313Hyperva l inemia, 336tHypoalbuminemia, 202Hypochloremia, 267Hypochloridia , 154Hypoglycemia, 247, 316Hypokalemia, 267Hypomagnesemia, 267Hyporeflexia , 287Hypoventi lation, 254Hypoxanthine, 247Hypoxia , 208, 209Hypoxia , 254

inducible factor, 209

IIdiopathic pulmonary fibros is , 261tIgA nephropathy, 266tImmune cytokines , 230t–231tImmune system

antibody, 222–223, 222f, 223f, 224tantigen, 222cel l s associated with

basophi l s , 228B lymphocytes , 227cytokines , 229dendri tic cel l s (DCS), 228–229, 229f, 230t–231teos inophi l s , 227–228innate immune system, 229, 231monocytes and macrophages , 227natura l ki l ler (NK) cel l s , 227neutrophi l s , 227T lymphocytes , 223–227, 225f, 226f

complement system, 232–233, 232fhypersens i tivi ty reactions , 233–234, 233t–234toverview of, 222

Immunoassays , 376Immunodeficiencies , 222Immunoglobul in (Ig), 256

antibodies , 151t, 203, 222Immunologica l memory, 222Immunophi l in inhibi tors , 231Immunoreceptor tyros ine-based activation moti f (ITAM), 223Immunosuppressants , 231Increased ectopic (tubal ) pregnancies/male inferti l i ty, 12Individual amino acids , 61Infant respiratory dis tress syndrome (IRDS), 254Infective diseases , of respi ratory system, 262Inflammation, 30Inflammatory rubor (redness ), 30Inhalation anesthetics , 295Inhibin, 300, 309Inhibi tory neurotransmitter, 319Inhibi tors , 56Innate immune system, 222, 229, 231

Inos ine (I), 38Insertion mutation, 113Insul in, 81, 159t

secretion and peptides , 311Integrin cel l surface matrix receptor, 196fIntegra l proteins , 96Integrin receptor, 195Interleukin (IL), 197, 226, 230t, 255Intermediate-dens i ty l ipoprotein (IDL), 243–244, 245, 245fIntermediate fi laments (IF), 167, 182–183, 182f, 183tInternal urethra l sphincter, 266“Intervening sequences” or “introns , 42, 45fIntersti tia l lung diseases (ILDs), 254, 261Intestina l ca lcium absorption, 199Intestina l lumen ferric, 212fIntestine, 199tIntravenous anesthetics , 295Iodine, 146tIon channel , 100tIonophores , 98bIon-exchange chromatography, 379, 379fIonized ca lcium, 200Ipratropium bromide, 257–258Iron, 146t. See also under Blood

deficiency, 203deficiency anemia, 206t, 213

Iron response element (IRE), 111bIron transport

overview of, 212fby transferrin, 212f

Ischemia, 247Isoleucine, 5tIsova leric acidemia, 336tIsozymes/isoenzymes , 59

JJanus Kinase (JAK), 2, 102f, 209, 224, 278Juxtaglomerular (JAG) cel l s , 265

KKartagener Syndrome, 12, 182Keratin, 51, 183tKeratinocytes , 229Kernicterus , 73, 155Ketoacid derivatives , 51Ketoacidos is , 140, 141f, 142fKetone bodies , 88Ketone body synthes is/degradation, 88, 91f“Kinked” cis double bond, 24Krebs cycle, 69Ketone group (CPO), 397t, 398Ketotic hyperglycinemia, 336tKidney, 199t

bas ic anatomy, 264fand bicarbonate excretion, 255impact of O2–CO2 exchange, 254role in synthes is of active vi tamin D, 279

Kidney s tones (nephrol i thias is ), 279Kines ins , 180, 181fKinin–kal l ikrein system, 273Kininogen, 215Knobloch syndrome, 191t, 192tKrabbe disease, 354tKupffer cel l s , 159t

LL-3-hydroxylacyl -CoA dehydrogenase, 84Labrador lung, 261tLactate, 135Lactate dehydrogenase, 135, 180Lactate threshold, 180Lactic acid, 135f, 175, 178Lactoferrin, 151tLactoperoxidase (sa l ivary), 151t

Lactose, 52intolerance, 18

Lambert–Eaton syndrome, 174Lamel lar bone, 198Lamina, 105Lamina rara interna, 264Lamina rare externa, 264Laminin, 264Laminopathies , 106Large intestine

and anus/rectum, 163, 166ffunctions of, 166f

Larynx, 252Leci thin, 253Leci thin cholesterol acyl transferase (LCAT), 244Lectin pathway, 232Leigh syndrome, 350tLens rupture, 264Leptin, 293Leptomycins , 104, 104bLesch–Nyhan syndrome (LNS), 41, 52, 358tLeucine, 5t

zipper, 112tLeukocyte-speci fic tyros ine kinase (Lck), 223Leukotriene antagonis ts , 258–259Leukotriene-associated bronchoconstriction, 258Leukotriene B4, 257tLeukotrienes , 29, 257tLeydig cel l s , 298, 309“Lick and fl ip” activi ty, 61Liddle’s syndrome, 275Lightheadedness , 315Linear trans double bond, 24Lingual l ipase, 151t, 316Linoleic acid, 51Lipid metabol i sm, 79, 156t

in l iver, 159–160, 160fLipid molecules , 23

bas ic functions , 24double bonds , 25

Lipid rafts , 96, 97fLipid synthes is in selected ti ssues , 81Lipid-derived hormones , 34, 79Lipid-derived molecules , 23Lipids , 23, 79, 130, 165tLipid transport and metabol i sm, 243fLipolys is , 141tLipoprotein-associated phosphol ipase, 246Lipoprotein disorders , 244Lipoprotein l ipase (LPL), 242Lipoprotein s tructure, 32fLipoproteins , 32, 139, 141t, 246

bas ic characteris tics , 33tmetabol i sm and transport, 241, 243f, 244f, 245f

5-l ipoxygenase, 258Liver, 155, 156f–159f

l ipid metabol i sm in, 159–160, 160fLong-chain 3-hydroxyacyl -CoA dehydrogenase (LCHAD) deficiency, 84Long-chain acyl -CoA dehydrogenase (LCAD), 87Loop diuretics , 267Lordos is , 197Low-dens i ty l ipoprotein (LDL), 88, 159L-type ca lcium channels , 176, 239Lungs , 251, 252

and acid–base ba lance, 254–255, 255fand O2–CO2 exchange, 254–255structure of, 252f

Lutea l phase, 299f, 300Luteinizing hormone/choriogonadotropin receptor (LHCGR), 303Luteinizing hormone (LH)

α- and β-subunit, 300female, 300male, 308f, 309ovulation prediction by, 300

Lyme disease, 375Lymphocyte function-associated antigen (LFA), 229Lymphoid inters ti tia l pneumonia , 261tLymph vessels , 179tLys ine, 5tLys inuric protein intolerance, 278tLysophosphol ipase, 162tLysozyme, 151t

MMacrol ides , 110bMacrophage phagocytic functions , 262Macrophages , 227Mad cow disease, 111b, 375Magnes ium, 146t, 269tMajor his tocompatibi l i ty complex (MHC), 225fMale inferti l i ty, 182Mal ignant melanoma, 113bMalonyl -CoA, 80, 81, 82f, 136

production by hormones , 80fManganese, 146tMannose-binding lectin pathway, 232MAP kinases , 306Maple syrup urine disease, 337tMarfan syndrome, 344tMatrix meta l loproteases , 255Maxam–Gi lbert method of sequencing, 380MCAD deficiency, 87McArdles disease, 180, 312Meckel–Gruber Syndrome, 12, 182Medium-chain acyl -CoA dehydrogenase (MCAD), 87Medium-chain fatty acids , 83Megaloblastic anemias , 163Melanocyte-s timulating hormone, 293Membrane, 93–94

channels , 98functions , 97s ignal ing, 99–102structure, 94–96

Membrane attack complex (MAC), 232Membrane channels , 98, 98bMembrano-prol i ferative glomerulonephri ti s , 266tMembrane l ipid, 24

bas ic components , 24fatty acids , 24glycerol , 24head group, 25, 26f

Membrane receptors , 93Membrane s ignal ing receptors , 100tMembrane transport proteins , schematic presentation, 97fMenke’s “kinky ha i r” disease, 57

ATPase, Cu2+ transporting, a lpha polypeptide (ATP7A), 57excess copper accumulation, 57

Menstrua l cycle, 303, 304f, 316express ion of reproductive hormones during, 299ffol l i cular/prol i ferative phase (days 5–13), 303lutea l/secretory phase (days 15–28), 303menstruation (days 1–4), 303

Mepol izumab, 259Mesangia l cel l s , 265Messenger RNA (mRNA) molecules , 42Metabol ic fate

of chylomicrons , 244fof very-low-dens i ty l ipoprotein (VLDL), 245f

Metabol ic systems, 137fMetabol i sm, 141

epinephrine effects , 141tglucagon effects , 140thormonal control , 136–140insul in effects , 139t

Metachromatic leukodystrophy (MLD), 355tMethamphetamine, 152Methionine, 4, 6tMethionine malabsorption syndrome (Smith–Strang disease/Oasthouse urine disease), 341t

Meth mouth, 152Methotrexate, 40, 51, 260Methylmalonic acidemia (MMA), 278t, 337tMetoprolol , 239Metronidazole, 166Mevalonate, 92Microca lci fi cation, 246Microtubule-based moti l i ty, 180, 181f, 182Microtubule motors , 181fMicrotubule polymerization and cancer therapy, 180Microtubules , 11Migl i tol , 76Minera l i zation in bone matrix, 195fMinera locorticoids , 34, 52Minera ls , 130, 143, 145t–147tMini -chromosome maintenance complex, 106Minimal change disease, 265Minimal change glomerulonephri ti s , 266tMinor enzymatic pathway, 56Misoprostol , 154Mitochondria , 236Mitochondria l DNA (mtDNA), 42Mitochondria l Tri functional Protein (MTP), 84Mitogen-activated protein kinase (MAPK), 255Modified carbohydrates , 76Molecular markers , 375Molecular “shock absorbers ,” 76Molybdenum, 147tMonocytes , 227

and macrophages , 227Monomeric enzymes

al losteric regulation of, 61Monomers” or “subunits”, 9Monosaccharides , 16

in human biology, 16fMontelukast, 258Moti l in, 153tMothers carrying a fetus with LCHAD deficiency, 84“Motor” proteins , 11–12Mouth, 150–152, 151t, 152tmRNA, tRNA, and rRNA, characteris tics of, 43tMTP deficiency, 84Mucins , 152Mucopolysaccharides , 152tMucus , 152, 152tMul lerian ducts , 297, 299Mul lerian inhibi ting factor, 298Multiple Acyl -CoA Dehydrogenase Deficiency (MADD), 87Multiple enzymes , 55Multiple protein subunits , 9Muscle, types of, 168fMuscles , 253

fatigabi l i ty, 174Muscle contraction, 11Muscular dystrophies , 9Muscles and moti l i ty. See also Skeleta l muscle.

cardiac muscle, 175–176, 176fcomponents of muscle, 168, 168f

actin, 168, 169factin-binding proteins (ABP), 171myos in, 170myos in l ight cha ins , 171tropomyos in, 170, 170f

energy production and use in muscles , 178, 179f, 180exci tation–contraction coupl ing, 171–173, 172fintermediate fi laments , 182–183, 182f, 183tmicrotubule-based moti l i ty, 180, 181f, 182nonmuscle cel l s , 183, 183foverview of, 167skeleta l muscle

muscle types , 174, 175fstructure and overview of, 173–174, 173f

smooth muscle, 176–178, 177f, 178f, 179tMutations , 113, 113f

types , 113f

Myasthenia gravis (MG), 174Mycomplasma, 287Myel in, 253, 283Myel in sheaths , 253, 306Myocardia l infarction, 319Myocardia l infarction, aspi rin in treatment of, 248fMyoglobin, 174Myos in, 12, 170Myos in I , 12Myos in II molecules , 12Myos in force generation, 171Myos in l ight cha in kinase (MLCK), 171, 178f, 237f, 240Myos in l ight cha in phosphatase (MLCP), 171, 178fMyotonic dystrophy (DM), 362tMyotubularin, 173Myris tylated a lanine-rich C kinase, 152

NN-acetyl -ga lactosamine (a .k.a . Ga lNAc), 27, 28fN-acetylglutamic acid, 63N-Acetyl glutamate synthase, 278tN-Acetylglutamate synthase (NAGS) deficiency, 342tN-acetylneuraminic (NANA), 29, 29fN-acetylmuramoyl -L-a lanine amidase, 151t

Na +–Cl – channel , 270

Na +–Cl – cotransporter, 267

Na +–K+-ATPase pumps, 270, 285f

Na +–K+–2Cl - transport, 267Nasal cavi ty, 252Natura l ki l ler (NK) cel l s , 227NE/epinephrine, 288–290Nephrin, 265Nephri tic syndrome, 266Nephrogenic diabetes ins ipidus , 50, 315Nephrogenic (kidney-associated) diabetes ins ipidus , 276Nephron, 267–268

diuretic medications , 267overview, 268ftask of, 268

Nephronopthis i s , 182Nephrotic syndrome, 266Nervous systemand anesthes ia , 294–295autonomic nervous system (ANS), 286–287, 287tbiochemistry of vi s ion, 294, 294fcomponents of, 281–283nerve impulse conductionnerve impulse, 284, 285f, 286fneuron at rest, 283–284repolarization, 284synaptic cleft, 286fneurotransmittersacetylchol ine (Ach), 290, 295catecholamines , 290–292, 291fdopamine, 287–288γ-aminobutyric acid (GABA), 292, 293f, 295glutamic acid, 292glycine, 292neuropeptides , 293–294norepinephrine (NE)/epinephrine, 288–290serotonin (5-HT), 290overview, 281parasympathetic, 287somatic nervous system, 286sympathetic, 286Neura l tube defects , 313Neurofi laments , 183tNeurogenic (bra in-associated) diabetes ins ipidus , 276Neurohormone, 299Neuropeptides , 293–294Neurotransmitters , 236Neutra l proteases , 257tNeutrophi l elas tase, 255

Neutrophi l extracel lular traps (NET), 227Neutrophi l s , 227Nexin, 181, 181f, 183tNiacin, 246Nickel , 147tNicotinamide adenine dinucleotide (NADH), 57Nicotinamide adenine dinucleotide phosphate (NADPH), 57, 73, 271Nicotinic–acetylchol ine receptor, 174Niemann–Pick C disease, 315Niemann–Pick disease, 355tNi fedipine, 238f, 238tNitric oxide synthetase (NOS), 271Nitrogen (N), 396, 396tNitroglycerin, 242f, 248, 319Nitrous oxide, mechanism of action of, 242fNon-essentia l amino acids , 4, 61Non-Hodgkin’s lymphoma, 114bNoninfective diseases , of respi ratory systemacute respiratory dis tress syndrome (ARDS), 259–260asthma, 256biochemica l processes leading to, 256–259bronchiti s , 255–256emphysema, 255intersti tia l lung diseases (ILDs), 261pneumoconios is diseases , 261resul t of an occupation/environmenta l condition, 261Nonmoti le ci l ia , 11Nonmuscle cel l s , 183, 183fNonspeci fic inters ti tia l pneumonia , 261tNonsteroida l anti -inflammatory drugs (NSAID), 214, 267Nose, 252Nuclear factor-kappa b (NF-k b), 255Nuclear export sequence, 104Nuclear lamins , 183tNuclear matrix, 105, 106Nuclear matrix/scaffold, 104–106Nuclear membrane, 104Nuclear pores , 104Nucleolus , 106Nucleoporins , 104Nucleos ides and/or nucleotidesanalogues as chemotherapy agents and antibiotics , 40bas ic s tructure of, 38fbreakdown of purines and pyramidines , 40–41components of carbohydrate, 39ni trogenous base, 38Nucleotide polymers , 50Nucleotides , 38ni trogenous base, 38Nucleus , 104Nurse cel l s , 309Nutrients , absorption of, 163f

OO2 binding. See under BloodO2 del ivery, phys iologic response to inadequate, 209Obes i ty, 161Odd number fatty acid degradation, 86fOkazaki fragments , 106Ol igomer protein, 9Ol igosaccharide, 18, 76Omal izumab, 259Omega-3 (Ω-3) fatty acids , 25, 246Omega-6 (Ω-6) fatty acids , 25Oncotic pressure, 202Oogenes is , 300Opiates , 295Ops in, 294Opsonization, 227Oral contraceptive pi l l s , 305Ornithine transcarbamylase (OTC), 278tdeficiency, 342tOrosensory phase of eating, 150Orotic aciduria (type I), 358t

Oseltamivi r / Tamiflu, 76Osmotic pressure gradient, 267Ostei ti s deformans , 198Osteoarthri ti s (OA), 76, 193Osteoblasts , 187, 193f, 197Osteocalcin, 194Osteochondrogenic cel l s , 186Osteoclasts , 194, 195Osteocytes , 193f, 196Osteogenes is imperfecta (OI), 10, 344tOsteoid, 194Osteoid minera l i zation, 196Osteomalacia/rickets , 196Osteomyel i ti s , 211Osteopontin, 194Osteoporos is , 197Osteoprogenitor cel l s , 187Osteoprotegerin (OPG), 195Oxaloacetate, 68, 68f

concentration, 88Oxidative phosphorylation, 66, 69–71, 69f, 174

complex I, 70complex II , 70complex II I , 70complex IV, 70cytochrome c molecules , 70, 69fmitochondria l enzyme, 71mitochondria l matrix, 70Q protein, 70respiratory cha in portion, 70“super” complex, 70

Oxygen–hemoglobin dissociation curve, 207fOxygen (O2), 396, 396tOxygen (O2)–hemoglobin (Hgb) dissociation curve, 207fOxygen (O2) transportation in body, 253Oxytocin, 306, 307t

PPaget’s disease, 198Palmitate (16-carbon), 24f, 81Palmitoyl -CoA, 80Panacinar emphysema, 255Pancreas , 161, 161f, 162t

impact of O2–CO2 exchange, 254impairment of, 315

Pancreatic acini , 161Pancreatic amylase, 162tPancreatic carboxypeptidases , 162tPancreatic enzymes , exocrine, 162fPancreatic hormones , endocrine, 162tPancreatic l ipase, 162tParathyroid hormone (PTH), 196, 197, 199, 199t, 200, 200f, 279Paresthes ia , 163Parieta l (oxyntic) cel l s , 153–154

bicarbonate ion, 154gastric acid, 153–154intrins ic factor, 154

Parkinson’s disease, 94, 182Pass ive di ffus ion exchange mechanism, 253Pass ive immunity, 222Pathways , 55Patients with CPT I deficiency, 83Patients with MADD, 87Patients with OI, 10Patients with PKU, 56Patients with Refsum disease, 87PCR technique, 383, 384fPDH deficiency, 351tP-dimethylaminobenzaldehyde, 155Pentose phosphate pathway, 65, 73–74

nonoxidative phase, 73, 74foxidative phase, 73, 74f

Pentose ribose, 16

Peps in, 154Peptides , 227, 269tPeptide bond, 109Peptidoglycan cel l wal l , syneths is of, 58Perforin, 224Periphera l proteins , 96Pernicious anemia, 163Peroxin proteins , 87Peroxisomes , 87

diseases of, 87Pertuss is toxin, 262PGD2, 31tPGE1, 31tPGE2, 31tPGG2, 31tPGH2, 31tPGI2, 31tPGIs , 29Phagocytos is , 224Phas ic contraction, 177Phenothiazines , 293Phenyla lanine, 5t, 50, 56Phenyla lkylamines , 238f, 238tPhenylketonuria (PKU), 50–51, 56, 338tPhosphate, 269tPhosphate group (PO3 and PO4), 397, 397tPhosphate (P), 396, 396tPhosphatidic acid, 88Phosphodiester bond, 41

found in RNA and DNA, 41fPhosphodiesterase inhibi tor, 272Phosphatidylglycerol , 253Phosphatidyl ethanolamine from phosphatidyl serine,formation, 89fPhosphatidylchol ine (PC), 94Phosphatidylethanolamine (PE), 94Phosphatidyl inos i tol , 94Phosphatidylserine (PS), 94Phosphatidylserine or inos i tol , formation of, 893-Phosphoglycerate dehydrogenase deficiency, 338tPhosphoenolpyruvate carboxykinase, 71Phosphofructokinase deficiency, 66Phosphofructokinase-1, 66Phosphol ipase, 162tPhosphol ipase C, 273Phosphoglycerate kinase, 66Phosphoglyceride synthes is , 88Phosphoglycerides , 88, 89f–90fPhosphol ipid components and formation, 26fPhosphol ipid molecules , 25Phosphol ipids , 24, 25, 30f, 94Phosphorous , 146tPhosphoserine aminotransferase deficiency, 338tPhyloquinone, menaquinone, 145tPink puffers , 255Pinocytos is , 222Pitui tary hormone, 314Placenta l vasculature, 209Plaque, 150Plasma, 202Plasmin

activation of, 219fand clot dissolution, 218–219, 219f

Plasminogen, 218Platelet

aggregation, prevention of, 214clot formation, 217–218plug formation, 213–214, 215f

Platelet-activating factor, 257tPlatelet plug, formation of, 215fPneumoconios is diseases , 261, 261tPodocin, 265Podocytes , 264

Podosome, 195Point mutation, 113

class i fi cation, 113Polyacrylamide gel , 374Polyacrylamide gel electrophores is (PAGE) technique, 373–375

bas is , 374fsodium dodecyl sul fate (SDS), 375ftwo-dimens ional (2D), 375, 375f

Polyadenylation, 107Polycystic kidney disease (PCKD), 264Polymerization reaction, 50Polyol pathway, 140bPolymyos i ti s , 261tPolypeptide, 7

hormones (Gs receptor), 100tPorphyrias , 206tPosterior polymorphous dystrophy, 190tPostinfectious glomerulonephri ti s , 266tPost-trans lational modi fication, 110Potass ium, 145t, 269tPotass ium-sparing diuretics , 275Precursor or proenzymes , 58–59Pregnancy testing and hCG, 303Prekal l ikrein, 215Preprocol lagen, 188fPrimary Ci l iary Dyskines ia , 182Prinzmeta l ’s angina, 248Prions , 111bProgesterone, 35, 301–302, 307t

impact of increased levels , 301influence on endometrium, 302

Progesterone antagonis ts , 302Progestogens , 301Programmed cel l death (apoptos is ), 278Prolactin, 306, 307t, 318Prolactinoma, 306Prol ine, 4, 6f, 6tPromoter sequence, 107Propionic acidemia, 278tPropionyl -CoA, 81Propofol , 295Propranolol , 239Prosecretin, 164tProstacycl ins (PGIs ), 29Prostaglandins (PGs), 29Prostaglandin D2, 257tProstaglandin E2, 154, 155fProstaglandin precursor (PGH2), 29Prostaglandin thromboxane, 214Prostanoids , 29Protamines , 104Protease-antiprotease theory, 255Protease enzymes , 257tProtein and deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) precipi tation, 380Protein “backbone”, 7Protein C, 218Protein folding, 9Protein kinase C (PKC), 101, 273Protein kinase G, 271Protein s tructure, levels of, 7–9

primary (1°) s tructure, 7quaternary (4°) s tructure, 9, 10, 10fsecondary (2°) s tructure, 8, 8ftertiary (3°) s tructure, 8, 9f

Protein synthes is , 108–109elongation (EF) factor, 108ini tiation factor (IF), 108

Protein trafficking, 110Protein tyros ine kinase (PTK), 255Proteins , 96

categories of, 9–13class i fi cation, 96

Proteoglycans , 20

Proteolys is , 216Proteosome, 226Prothrombin, 216Proton pump inhibi tors , 98b

Protons (H+), 269tProtoporphyrin, 203, 205Proximal convoluted tubule, 268Proximal his tidine, 203Pseudohypera ldosteronism, 275Pseudomembranous col i ti s , 166PTK phosphorylations , 255P-type fimbriae of Escherichia coli, 267Pudendal nerve, 266Pulmonary arteries , 252–253, 252fPulmonary compl iance, 253Pulmonary edema, 208, 272Pulmonary hypertens ion, 272Pulmonary surfactant, 251

l ipid component, 253and lung development, 253and premature bi rth, 254protein component, 253

Pulmonary veins , 252–253, 252fPurine ring, consti tuents of, 39Purines , 38Pyelonephri ti s , 267Pyridostigmine, 174Pyrimidine ni trogenous bases , 39Pyrimidine uraci l ring, 40fPyrimidine-derived nucleos ides cytidine, 40fPyrimidine-derived nucleos ides uridine, 40fPyrimidines , 38Pyruvate, 69, 135, 178

carboxylase, 71, 135, 312dehydrogenase, 69dehydrogenase kinase, 135kinase, 66kinase deficiency, 346t

QQuinone, 151t

RRadia l spokes , 182Radiation, 260Random coi l , 8Rapidly progress ive (crescentic) glomerulonephri ti s , 266tRapidly progress ive glomerular nephri ti s , 264Ras GTPase protein, 299Reabsorption process , 268Reactive oxygen species , 140bReactive phase of bone heal ing, 198Receptor activator of nuclear factor kappa B (RANK), 194Red blood cel l s (RBC), 69, 96

breakdown of, 155components of, 203ffunctions , 203, 203f, 204f, 205f

Refractory period of repolarization, 284Refsum disease, 87, 356tRegulatory myos in l ight cha ins , 171Regulatory RNA, 42Rejection, transplant, 234Relaxed hemoglobin (Hgb), 205, 205fRemiki ren, 270Renal corpuscle, 265Renal l i thias is , 359tRenin, 265, 316Renin inhibi tors , 270Renin–angiotens in system, 318Renin–angiotens in–aldosterone system (RAAS), 263, 270–276, 271f

a ldosterone, 275–276angiotens inogen/angiotens in I and II , 273atria l natriuretic peptide (ANP), 276, 276fmacula densa and blood flow/osmolari ty, 270–272

renin and blood pressure, 270vasopress in, 276

Rennin, 154Repeated peptide bonds , 7Reperfus ion injury, 248Reproductive system

bas ic anatomy and development, 297–299female

breast ti s sue development and lactation, 306–307, 307testrogens , 301FSH, 299–300GnRH, 299hCG, 302–303LH, 300menstrua l cycle, 303, 304fprocess of ferti l i zation, 305, 305fprogesterone, 301–302

malefol l i cle-s timulating hormone (FSH), 308f, 309luteinizing hormone (LH), 308f, 309testosterone, 307

overview, 297Reproductive tract, 179tReserpine, 293Resorption, 200Respiratory acidos is , 254–255Respiratory a lka los is , 254–255Respiratory bacteria , 255Respiratory bronchiol i ti s -associated inters ti tia l lung disease, 261tRespiratory dis tress syndrome, 312Respiratory muscle fatigue, 254Respiratory system

bas ic anatomy and development, 252–254role in body, 251–252, 252

Respiratory tract, 179tResusci tation, 314Reticular fiber, 186Reticuloendothel ia l cel l s , 212Retina l i somerase, 294Retin-opathy, 143bRetrovi ra l therapy, 107, 107bRetrovi ra ls , 107Reverse transcriptase, 107b

inhibi tors , 107, 107bR-Group class i fi cations , 5t–6tR-group, 3

characteris tcs of, 4Rhabdo, 174Rhabdomyolys is , 174Rheumatoid arthri ti s , 261t, 262Rho kinase, 171Rhodops in, 294Rhodops in kinase, 294RIA, 376–377, 376fRibonucleic acid (RNA), 38Ribosomal RNA (rRNA), 42, 106Ribosome synthes is , 106Ribozyme, 107Rickets , 196RNA molecules , 42RNA polymerase, 107Rotavi ruse, 110bRough endoplasmic reticulum (RER), 188fRoxi thromycin, 110br-proteins , 106RU-486, 302Ryanodine receptor, 176, 240SSal iva , compos i tion of, 151t–152tSa l ivary acid phosphatases , 151tSandhoff disease, 356tSarcoidos is , 261t, 262Sarcolemma, 168Sarcoma homology domains , 223

Sarcomere, 168Sarcopenia , 175Sarcoplasmic reticulum (SR), 168, 240Saturated fatty acids , 24

melting points , 25Saturated l ipid ta i l s , 94fSchmid-type metaphyseal chondrodysplas ia (SMCD), 190tScurvy, 194Secretin, 153t, 154Secretion, 268Selective progesterone receptor modulators (SPRMs), 302Selenium, 147tSel f-antigens , 222Sel f-hugging, 61Senior–Loken syndrome, 12, 182Sequence recognition particles (SRPs), 110Serine kinase activi ty, 193Serine, 5tSerotonin (5-HT), 290Sertol i cel l s , 309

aromatase, 309Serum, 202Severe combined immunodeficiency disease (SCID), 359tSex-determining region, on the Y chromosome (SRY), 298

hormonal involvement, in sexual di fferentiation, 298fSexual infanti l i sm, 299Short gut syndrome, 163Short-chain acyl -CoA dehydrogenase (SCAD), 87Sia l ic acid molecules , 29Sickle cel l disease (SCD), 209–211, 210f, 317Side chains , 51Sideros is , 261tSigmoid, 205Signal sequences , 110Signal transducer, 102f

and activator of transcription (STAT), 209, 224, 306Si ldenafi l , 272Si l i cos ideros is , 261tSi l i cos is , 261tSimple one and two substrate enzyme reaction, 56f

molecular shape, 56fSingle subunit (“monomeric”) enzyme, 59Single-s tranded binding proteins , 106Sinoatria l and trioventricular nodes , 236, 237Sinus i ti s , 262Skeleta l muscle, 11, 168f, 173f. See also under Muscles and moti l i ty

contraction and relaxation, cycle of, 316fiber types , 175f

Skin, 179tSl i t diaphragms, 265Slow-twitch fibers , 174Smal l intestine, 161, 163, 163f

functions of, 164t–165tinfectious/inflammatory diseases of, 163

Smal l s igna l ing molecules , 100tSmith-Magenis syndrome (SMS), 339tSmooth muscle, 168f, 176–178, 177f, 178f, 179t

contraction, 177fmyos in force generation, regulation of, 178f

Smooth muscle cel l s , 179tca lcium activation of, 237f

Sodium, 145t, 269tSodium fluoride, 197

Sodium–potass ium ATPase (Na +–K+-ATPase) pump, 98Soluble receptor-associated tyros ine kinases , 101Somatostatin, 153tSorbi tol–a ldolase reductase pathway, 140bSpecia l R-groups , 4Sphingol ipid, 27, 28fSphingomyel in, 27Sphingos ine, 27, 90Spinobulbar muscular atrophy (SBMA) (Kennedy’s disease), 363tSpondylometaphyseal dysplas ia (SMD), 190t

Squamous cel l carcinoma, 113bSrc kinase, 306SRP docking protein, 110Starch, 18Statins , 92Stearate (18-carbon), 24fSteroid hormone receptor, activation, 99f.Steroid hormones , 34, 99, 111Steroid production, during pregnancy, 302fSteroid response elements , 111Stomach. See under Digestive systemStreptococcus pyogenes, 266tStretch-activated channel , 270Structura l proteins , 11Struvi te s tones , 279“Stuck” fatty acid degradation, 87Subl ingual ni troglycerin, 240Substance P, 293Substrate concentration, 56Substrate-binding s i tes /active s i te, 56Substrates , 56Succinate dehydrogenase, 70Sugar ribose, 39Sul fa drugs , 73Sul fatide, 27, 28fSul fur, 147, 147tSul fur atom-conta ining sul fate (SO4–2) group, 27Sul fur (S), 396, 396tSul fur–sul fur bonds (–S–S–), 397, 397tSuperoxide dismutase, 151tSurfactants , 306Syncoi l in, 183tSynemin (Desmus l in), 183tSystemic lupus erythematous (SLE), 261tSystemic scleros is , 261t

TT lymphocytes , 223–227, 225f, 226fTadalafi l , 272Tangier disease, 357tTartrate res is tant acid phosphatase (TRAP), 195TATA box, 111Taxol , 114bTay–Sachs disease, 91, 357tT-cel l receptor recognition of antigen, 225fTemperature and pH on hemoglobin binding of oxygen, 207fTendon xanthoma, 245Tense hemoglobin (Hgb), 205, 205fTestosterone, 307, 309Tetramethylethylenediamine (TMED), 374Thalassemia, 206t, 312Thiamine-deficient cel l , 317Thiazide diuretics , 267Thick fi lament, 170f, 171

compos i tion of, 169fThin basa l lamina disorder, 266tThin fi lament, 170fThin layer (paper) chromatography (TLC), 377, 378fThiocyanate, 152tThreonine, 5tThrombin, 216Thrombolys is , 248Thrombopoietin, 159tThromboxanes (TXs), 29Thrombus , 246Thymidine deoxyriboncleotide (dTMP), 40

synthes is of, 40fThymidine dimer repair, 113bThymine (T), 39Thyroid-s timulating hormone (TSH), 299Tocopherols/tocotrienols , 145tTonic contraction, 177Tota l ca lcium, 200Toxic oxygen “radica ls”, 73

Trabecular bone, 198Trachea, 251, 252Tracking dye, 374Trans fatty acids , 25Transcription (JAK–STAT) s igna l ing, activator of, 102fTranscription factor, 107–108, 111Transducin, 294Transfection, 385Transfer RNA (tRNA) molecules , 42Transferrin, 111, 211, 212f

in i ron transportation, 212fTrans i tion mutation, 113Transplant rejection, 234Transport

channel proteins , 12–13of fatty acids us ing CPT I and CPT II , 83fof food from mouth to rectum/anus , 150f

Transporter associated with antigen process ing (TAP transporters ), 226Transverse tubules (T tubules ), 168Triacylglycerol , 24, 25f, 29, 81, 88, 242

synthes is , 88synthase enzyme complex, 88

Triacylglycerol -rich l ipoprotein, 241Tricarboxyl ic acid cycle, 69Triglyceride, 141t, 159, 160, 174Triose glycera ldehydes , 16Trioventricular nodes , 236, 237Triple hel ix, 50Tropocol lagen, 194Tropomyos in, 170, 170fTroponin, 170, 247Tryps in, 162tTryps inogen, 162tTryptophan, 5tTuberculos is , 262Tubule system, 268Tubul in, 11, 180Tuftel in, 150Tumor (swel l ing), 30Tumor necros is factor (TNF), 231t, 255, 257tTXA2, 31tType I col lagen mutation, 315Type I hypersens i tivi ty response, 256Type I tyros inemia (tyros inos is ), 339tType II a lveolar cel l s , 253, 260Type II tyros inemia, 339tType II I hypersens i tivi ty, 262Type II I tyros inemia, 339tType IV col lagen, 264Tyros ine, 5tTyros ine kinases , 273

UUloric (febuxostat), 41Unesteri fied cholesterol molecule, 32fUnsaturated fatty acids , 24, 24f

with carbon–carbon double bonds , degradation of, 86, 86fUnsaturated l ipid ta i l s , 94fUraci l (U), 38Urea, 62, 63f, 266, 269tUrea cycle, 62–63, 63f, 157, 157t

regulation of, 62Ureters , 266Urethra , 266Uric acid, 279Uridine monophosphate (UMP), 39Urinary system

bas ic anatomy and phys iology, 263–264components , 263, 266inul in/creatinine clearance, 270nephron, 267–268renal corpuscle, 264–266, 265f

Urinary tract, 179tUrinary Tract Infections (UTIs ), 267

Urobi l inogen, 154Uterine leiomyoma, 302

VVagal nervectomy, 154Vagus nerve, 154Val ine, 5tVancomycin, 166Vardenafi l , 272Vasoconstriction, 240Vasodi lation process , 240Vasopress in, 276Vector, 384Vein, 241fVeno-occlus ive cri ses , 211Verapami l , 238tVery-long-chain fatty acids (VLCFA), 87Very-low-dens i ty l ipoprotein (VLDL), 88, 159, 160, 241, 242, 243, 244f

metabol ic fate of, 245fVimentin, 183Vincris tine, 114bVirions , 111bVis ion, biochemistry of, 294, 294fVi tamin A (retinol es ters ), 50, 294

deficiency, 51Vitamin B12 absorption, 163Vitamin C (ascorbic acid), 50, 194

deficiency, 313Vitamin D, 35, 50, 198

from cholesterol , production of, 35fVi tamin D3, 279Vitamin E, 50Vitamin K, 50, 218Vitamins , 130, 143, 144t–145tVi tel l ine layer of egg, 305VLDL. See Very-low-dens i ty l ipoprotein (VLDL)Voltage-dependent ca lcium channels (VDCC), 239Von Wi l lebrand’s Disease (vWD), 213, 217, 365t

WWarfarin, 218Warts , 229“Water hating” R-groups , 7Wegener’s granulomatos is , 262, 266tWernicke–Korsakoff syndrome, 130Western blotting, 383White blood cel l s , 225fWhooping cough, 262Wi lm’s tumor, 114bWi lson’s disease, 57

ATPase, Cu2+ transporting, beta polypeptide (ATP7B), 57Wolffian ducts , 297, 299Wolff–Parkinson–White syndrome, 317Woman’s bra in ti s sue, a fter hunger s trike, 314

XXanthanuria , 359tXanthine, 40Xanthomas, 245, 312Xeroderma pigmentosum (XP), 113b, 363tX-l inked disease, 94. See also Barth syndrome

ZZafi rlukast, 258Zanamivi r/ Relenza, 76Zel lweger spectrum, 87Zel lweger syndrome, 87

Lorenzo’s oi l , 87treatment for, 87

Zeta-chain-associated protein kinase-70 (ZAP-70), 223Zidovudine, 107, 107bZi leuton, 258Zinc, 146tZinc finger, 112t

Zona pel lucida, 305ZP3, 305