جــــامـــعة...

SSecond year students Second semester

بسم هللا الرحمن الرحیمالصيدلة جامعة املنوفيةرؤية كلية

جامعة املنوفية رائدة على املستوى احمللي و اإلقليمي يف جودة -أن تكون كلية الصيدلة.التعليم الصيديل و البحث العلمي و خدمة اتمع

رسالة كلية الصيدلة جامعة املنوفية

تقديم برامج دراسية متطورة تضمن ختريج صيادلة متميزين مهنياٌ و خلقيا قادرين علاملنافسة يف الداخل و اخلارج مع اإلرتقاء بالبحث العلمي و تطوير صناعة الدواء مبا يؤدي إىل

حتسني مستوى اخلدمات الصحية.

األهداف اإلسرتاتيجية لصيدلة املنوفية

رفع كفاءة العملية التعليمية مع التحديث املستمر للمناهج الدراسية مبا يتوافق مع سوق العمل. أ.توجيه البحث العلمي خلدمة اتمع احمللي و اإلقليمي. ب.

توثيق التعاون مع اتمع و زيادة الوعي باملشاركة اتمعية. ت.رفع كفاءة العاملني بالكلية من أعضاء هيئة التدريس و معاونيهم و اجلهاز اإلداري و الفين. ث.

تنمية املوارد الذاتية من الوحدات اخلاصة و الربامج التعليمية. ج.حتقيق معايري ضمان جودة التعليم بكلية الصيدلة جامعة املنوفية. ح.

Course Specifications

B-Professional Information

1-Overall aims of the course:

Demonstrate metabolism of different nutrients, its component pathways, and somemetabolic disorders.

Illustrate different techniques for analysis of biological fluids constituents.

2-Intended learning outcomes (ILO’s):

a-Knowledge and Understanding:By the end of this course, the student should be able to:

a1-Know the different metabolic pathways of different nutrients.

a2-Know different metabolic abnormalities and its management.

a3-Understand regulation of different metabolic pathways.

b-Intellectual Skills:By the end of this course, the student should be able to:

b1- Discriminate different metabolic pathways and its regulation.

b2- Design full scheme for identification and quantification of different constitue of urine.

A-Basic Information

Course code: PB

Course name: Biochemistry 2

Credit hours of the course: 3 Lecture: 2

Practical: 1

Total: 3

Pre-requisite of the course: Biochemistry 1

Department teaching the course: Biochemistry Department

Course Co-ordinator: Dr. Mohamed Badr

Dr. Nada Osama

c-Professional and Practical Skills:By the end of this course, the student should be able to:

c1- Differentiate the different metabolic pathways of a given nutrient material.

c2- Apply recent chemical methods to analyze urine samples.

c3-Select the most suitable method for urine analysis.

c4-Estimate and interpret the results of urine analysis to different disease.

d-General Skills:By the end of this course, the student should be able to:

d1-Communicate effectively with his teacher and colleagues.

d2-Cooperate in a team, and independently on solving problems.

d3- Usng internet and e- learning.

d4-Exchange ideas, principles and information by oral, written and visual means

3-Teaching and Learning Methods (lectures, open discussion, role plays, ..etc):

- Lectures using data show - Laboratory work- Open discussion - Diagrams - videos

4- Student Assessment:

a-Assessment Methods and Weighing:- Class participation: 10 %- Practical exam: 20 %- Oral exam: 20%- Final exam: 50%

b-Assessment Schedule: - Class participation: assessment each laboratory- Practical exam: Week 9- Oral exam: According to semester timetable - Final exam: According to semester timetable

Course Notes Hands out written by the instructor

Required Books ---------------

Recommended Books Lippincott ( Biochemistry)

Oraby's illustrated review ofBiochemistry

Periodicals ----------------

Web Sites -----------------

Matrix of knowledge and skills of the Biochemistry 2 course

General and transferable

skills

Professional and

practical skills

Intellectual skills

Knowledge and understanding

skills

Week of

study

Topics

d1,d2,d3,d4c1b1a1,a2,a31,21. Carbohydrate metabolism

(Glycolysis & Kreb’s cycle) d1,d2,d3,d4c1b1a611. Introduction about blood (Practical)

d1,d2,d3,d4c1b1a1,a2,a33,42. Carbohydrate metabolism(HMP shunt, Uronic acid pathway, Glycogen

metabolism, Fructose and galactose metabolism, Gluconeogenesis,

d1,d2,d3,d4 c2,c3,c4b2a62-82. Determination of different parametersin blood (Practical)

d1,d2,d3,d4c2b1a1,a2,a34,5,63. Lipid metabolism(Digestion & absorption, Storage & mobilization of lipids, Biosynthesis of TAG, Lipolysis, Fatty acid oxidation)Biosynthesis of fatty acids Metabolism of conjugated lipids, Ketone bodies, Cholesterol metabolism, Plasma lipid & plasma, lipoproteins, Fatty liver.)

d1,d2,d3,d4c2,c3,c4b2a693. Revision + Quiz Theo. Practical(Practical)

d1,d2,d3,d4c2b1a1,a2,a36,7,84. Protein metabolism(Digestion & absorption, General catabolic pathways of amino acids, Ammonia, Urea cycle, Metabolism of different amino acids)

d1,d2,d3,d4c2,c3,c4b2a6104. Final practical

d1,d2,d3,d4c2b1a1,a2,a310, 11

5. Integration of metabolism & Revision

CARBOHYDRATE METABOLISM

1

DIGESTION OF CARBOHYDRATES

- More than 60% of our foods are carbohydrates. Starch, glycogen, sucrose, lactose and cellulose are the chief carbohydrates in our food. - Digestion of carbohydrates occurs in mouth and small intestine. The digestion of carbohydrates involves their hydrolysis into monosaccharides by a family of glycosidases. They catalyze hydrolysis of glucosidic bonds.

Digestion of Carbohydrates: 1- In mouth:- Digestion of carbohydrates starts in the mouth by salivary α-amylase enzyme.

- This enzyme is produced by the salivary gland. Its optimum pH is 6.7 and it is activated by chloride ion.

- During mastication, salivary α-amylase acts on dietary starch and glycogen breaking some α-(1-4) bonds to form a mixture of maltose and smaller branched oligosaccharide molecules called starch dextrins(amylodextrin, erythrodextrin and achrodextrin).

- Because both starch and glycogen also contain α-(1-6) bonds, the resulting digest contains isomaltose.

- Because food remains for short time in the mouth, digestion of starch and glycogen may be incomplete and gives a partial digestion. The digestion product in the mouth gives maltose, isomaltose and starch dextrins.

2- In stomach:- No digestion in the stomach for carbohydrate because of the high acidity inactivates salivary α-amylase.


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Sucrose Glucose + Fructose

Maltose Glucose + Glucose

Lactose Glucose + Galactose

Sucrase

Lactase

Maltase

3- In small intestine: - When the acidic contents of stomach reach the small intestine, they are neutralized by bicarbonate secreted by pancreas, and the pancreatic α-amylase continues the process of starch digestion into maltose, isomaltose, sucrose and lactose. - The optimum pH for pancreatic α-amylase is 7.1- The final digestive process occurs at the small intestine and includes the action of several disaccharidases (maltase, isomaltose, sucrase and lactase) which can hydrolyze disaccharides into their corresponding monosaccharides.

Digestion of Cellulose:- The human body cannot produce β-(1-4) amylase so that they are unable to digest cellulose β-(1-4) glucosidic linkage. So cellulose passes as such in stool. - Cellulose helps water retention during the passage of food along the intestine producing larger and softer feces, so preventing constipation.

Absorption of monosaccharides by intestinal mucosal cells: - The end products of carbohydrate digestion are monosaccharides: glucose, galactose and fructose. They are absorbed from jejunum to portal vein to the liver where galactose and fructose are transformed into glucose. - There are two mechanisms for absorption of monosaccharides:

1- Active transport (against concentration gradient). 2- Passive transport (facilitated diffusion).

1- Active transport: - Monosaccharides absorbed by active transport are glucose and galactose. - Active transport occurs when the hexose sugars (glucose and galactose) have a hydroxyl group at C2 at the right side. Fructose, which does not contain -OHgroup to the right at position 2 is absorbed by passive diffusion. - Active transport requires energy and specific carrier protein.

- Mechanism of active transport:- In the cell membrane of the intestinal cells, there is a mobile carrier proteincalled sodium-dependent glucose transporter (SGLT-1). It transports glucose to


3

G

Carrier protein

G

Na+ GK+

K+Na+

ATPaseSodiumpump

ATP

ADP+Pi

G

To capillaries

Na+

Na+

Cell

mem

bran

eCy

topl

asm

Mechanism of active transport of glucose

inside the cell using energy. The energy is derived from sodium-potassium pump.

- The transporter has 2 separate sites, one for sodium and the other for glucose. - It transports them from the intestinal lumen across cell membrane to the cytoplasm, allowing the carrier to return for more transport of glucose and sodium.

- Sodium is transported from high to low concentration (with concentration gradient) and at the same time causes the carrier to transport glucose against its concentration gradient.

- Sodium is expelled outside the cell by sodium pump. It needs ATP as a source of energy. The reaction is catalyzed by an enzyme called "Adenosine triphosphatase (ATPase)". Sodium is exchanged with potassium.

- Insulin increases the number of glucose transporters in tissues containing insulin receptors e.g. muscle and adipose tissue.

Inhibitors of active transport:A- Ouabain (cardiac glycosides): inhibits ATPase enzyme necessary for hydrolysis of ATP that produces energy of sodium pump. B- Phlorhizin: inhibits the binding of sodium to the carrier protein.

2- Passive transport (facilitated diffusion): - Sugars pass with concentration gradient i.e. from high to low concentration. It needs no energy. - It occurs by means of a sodium-independent facilitated transporter (GLUT-5). - Fructose and pentoses are absorbed by this mechanism. - Glucose and galactose can also use the same transporter if the concentration gradient is favorable. - There is also sodium-independent transporter (GLUT-2) that is facilitates transport of sugars out of the cell i.e. to circulation.


4

Disorders of carbohydrate digestion: 1- Lactose intolerance:- This is a deficiency of lactase enzyme that hydrolyzes lactose into glucose and galactose. - Lactase deficiency may be congenital which develops soon after birth or acquired which occurs later on life. - The deficiency of lactase enzyme leads to accumulation of lactose in smallintestine which increased osmotic pressure, so water will be drawn from the mucosa into the large intestine causing osmotic diarrhea. Moreover, bacterial fermentation action on lactose leads to formation of carbon dioxide which causes flatulence and abdominal cramps. - Treatment simply removes lactose from diet.

2- Sucrase deficiency:- This is an inherited deficiency of the sucrase. - Symptoms are the same as those described in lactase deficiency.

Fate of absorbed sugars: - Monosaccharides (glucose, galactose and fructose) resulting from digestion of carbohydrates are absorbed and can undergo the following: 1- Uptake by the tissue mainly by the liver: - After absorption, the liver takes up sugars, where galactose and fructose are converted into glucose.

2- Utilization by the tissues: - Glucose may undergo one of the following fates: 1- Oxidative pathway: through glycolytic pathway which metabolizes glucose into pyruvate or lactate for production of energy.

2- Pentose phosphate pathway (Hexose monophosphate shunt, HMP): for production of ribose and deoxyribose and NADPH+H+.

3- Storage as glycogen in liver and muscle (glycogenesis).

4- Storage as fat: lipogenesis. 5- Conversion: to substances of biological importance: a- Pyruvate and intermediates of citric acid cycle provide the carbon skeleton for the synthesis of amino acids. b- Ribose and deoxyribose enter in the biosynthesis of RNA and DNA.


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c- Lactose enters in the biosynthesis of milk. d- Fructose which is the sugar of semen. e- Glucosamine, galactosamine and glucuronic acid enter in the biosynthesis of mucopolysaccharides. f- Acetyl CoA which is the building unit for long chain fatty acid and cholesterol synthesis.

Outline Of Glucose Metabolism

GlucoseKinase Glucose-6-P

ATPADP

Fructose-1,6-di-P

Glycolysis HMP-Shunt

PentoseNADPH+H+

2 -Triose-P

2-Phosphoenolpyruvate

2-Pyruvate Lactate

2-Acetyl CoA

Anaerobic

Aerobic

Krebs Cycle (TCA)

Lipogenesis

B-OxidationFatty acids

ATP (energy)H2OCO2

Glucogenic A.A

Ketogenic A.A

GlycogenGlycogenesis

Glycogenolysis


6

Glucose-6-P

Fructose-6-P

GlucoseATP

ADP

GlucokinaseHexokinase Mg2+

Phosphohexoseisomerase

Phosphofructo-kinase

ATP

ADP

Mg2+

Fructose-1,6-bisphosphate

2 Glyceraldehyde -3-P Dihydroxyacetone-P (DHAP)

Aldolase

Glyceraldehyde-3P-Dehydrogenase

NAD+

NADH+H+

Pi

(2) 1,3 Bisphosphoglycerate

PhosphoglycerateKinase

ADP

ATPMg2+

(2) 2 PhosphoglycerateMutase

EnolaseMg2+

H2O

(2) PhosphoenolpyruvatePyruvate kinase

ADPATP

Mg2+(2) Enol Pyruvate

Spontaneous

(2) Keto Pyruvate

(-1 ATP)

(-1 ATP)

2x (+3 ATP)

Inhibited byFluoride

Inhibited byiodoacetate

(2) 3 Phosphoglycerate

2x (+1 ATP)

2x (+1 ATP)

PhosphotrioseIsomerase

Lactate Dehydrogenase

NADH+H+ NAD+

(2) Lactate

GLYCOLYSIS

- Glycolysis means oxidation of glucose to give pyruvate (in the presence of oxygen) or lactate (in the absence of oxygen). - Site: All glycolytic reactions occur in the cytosol. - Steps:


7

Fructose-6-P

ATP

ADP

GlucokinaseHexokinase Mg2+

Phosphohexoseisomerase

Phosphofructo-kinase

ATP

ADP

Mg2+


2 Glyceraldehyde -3-P

Dihydroxyacetone-P (DHAP) Aldolase

Glyceraldehyde-3P-Dehydrogenase

NAD+

NADH+H+

Pi

(2) 1,3 Bisphosphoglycerate


ADP

ATPMg2+


Mutase

H2O

(2) Phosphoenolpyruvate

Pyruvate kinase

ADPATP

Mg2+(2) Enol Pyruvate

Spontaneous

(2) Keto Pyruvate

(-1 ATP)

(-1 ATP)

2x (+3 ATP)Inhibited byiodoacetate


2x (+1 ATP)

2x (+1ATP)

OH

OH

H

OHH

OHH

OH

CH2OH

H

OH

OH

H

OHH

OHH

OH

CH2-O-P

H OH

CH2OH

HOH H

H OHO

CH2-O-P

OHHOH H

H OHO

CH2-O-P

CHOC OHHCH2-O-P

CH2- O- P

C OCH2OH

COO - PC OHHCH2-O-P

COOHC OHHCH2-O-P

COOHC O-PHCH2OH

Inhibited byFluoride

COOHC O - PCH2

COOHC OHCH2

COOHC = OCH3

Glucose

Glucose -6-P

COOHC - OHCH3


NADH+H+ NAD+

H

(2) Lactate

PhosphotrioseIsomerase

CH2-O-P

EnolaseMg2+


8

Reactions of Glycolysis:1- Phosphorylation of glucose: - The first reaction in glucose metabolism (phosphorylation of glucose) is irreversible reaction and catalyzed by hexokinase or glucokinase.

- Hexokinase and Glucokinase:

a) Hexokinase: - It is present in most tissues. - It has broad specificity and able to phosphorylate several hexoses in addition to glucose. - It is inhibited by reaction products, glucose-6-phosphate (feed back inhibition).- It has a low Km and therefore has a high affinity for glucose. - It has low Vmax for glucose and therefore cannot phosphorylate large quantities of glucose. - Hexokinase is one of three regulatory enzymes of glycolysis.

b) Glucokinase: - It is present in liver and β-cells of pancreas. [- It is the predominant enzyme for glucose phosphorylation. - It is not inhibited by reaction products, glucose-6-phosphate (feed back inhibition).- It has a high Km and therefore has a low affinity for glucose. - It has high Vmax for glucose, allowing the liver to remove the blood glucose from portal blood. This prevents large amount of glucose entering the systemic circulation following a carbohydrate-rich meal, and thus minimizes hyperglycemia during the absorptive period. - Glucokinase level increased by a carbohydrate-rich diet and by insulin.

2- Isomerization of glucose-6-phosphate:- The Isomerization of glucose-6-phosphate (aldose) to fructose-6-phosphate (ketose) is catalyzed by phosphohexose isomerase. This reaction is reversible.

3- Phosphorylation of fructose-6-P: - Phosphorylation of fructose-6-P into Fructose-1,6-biphosphate is irreversible reaction catalyzed by phosphofructokinase-1 (PFK-1) which is the rate limiting step in glycolysis.


9

- PFK-1 is inhibited allosterically by elevated level of ATP (indicating an energy rich signal); conversely, PFK-1 is activated allosterically by AMP (asignal indicating that the cell's energy stores are depleted). - Citrate also inhibits PFK-1.

Comparison between glucokinase and hexokinase

Points of comparison Glucokinase Hexokinase

Tissue distribution Liver and β-cells of pancreas Most tissues

Substrate specificity Acts on glucose only Acts on glucose, fructose and galactose.

Km and Vmax Have high Km and high Vmax(low affinity for glucose)

Have low Km and low Vmax (high affinity for glucose)

Inhibition by glucose-6-phosphate

Not inhibited by reaction product.

Inhibited by reaction product.

Carbohydrate diet on activity

Affected by fasting or by high carbohydrate diet

Not affected by fasting or by high carbohydrate diet

Diabetes mellitus Decreased in diabetes M. No change in diabetes M.

Insulin hormone Its synthesis is induced by insulin

Not induced by insulin.

Function Acts in liver after meals. It removes glucose coming in portal circulation, converting it into glucose-6-phosphate

It phosphorylates glucose inside the body cells. This makes glucose concentration more in blood than inside the cells. This leads to continuous supply of glucose for tissues even in the presence of low blood glucose level

4- Cleavage of fructose 1,6-bisphosphate:- Aldolase A cleaves fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glycerladehyde 3-phosphate.

5- Isomerization of dihydroxyacetone phosphate:- Interconversion of dihydroxyacetone phosphate and glycerladehyde 3-phosphate is catalyzed by triose phosphate isomerase. The isomerization results in the production of two molecules of glycerladehyde 3-phosphate.

6- Oxidation of glycerladehyde 3-phosphate:- The conversion of glycerladehyde 3-phosphate to 1,3 bisphosphoglycerate by glycerladehyde 3-phosphate dehydrogenase is the first oxidation reduction


10

reaction of glycolysis. (Note: The NADH formed must be reoxidized to NAD+

for glycolysis to continue).

7- Formation of ATP from 1,3-bisphosphoglycerate and ADP (Substrat- level phosphorylation): - The high energy phosphate group in 1,3-bisphosphoglycerate is used to synthesize ATP from ADP in a reaction catalyzed by phosphoglycerate kinase.- Note that this is only reaction of kinases which is reversible. - This is an example for substrate level phosphorylation in which the production of high energy phosphate is coupled directly to the oxidation of the substrate instead of oxidative phosphorylation via electron transport chain.

8- Shift of phosphate group from carbon 3 to carbon 2: - This reaction is catalyzed by phosphoglycerate mutase.

9- Dehydration of 2-phosphoglycerate: - The dehydration reaction by enolase redistributes the energy within the molecule, resulting in the formation of phosphoenolpyruvate (PEP). The reaction is reversible.

10- Formation of Pyruvate and ATP (Substrat- level phosphorylation): - Another example for substrate level phosphorylation is the conversion of phosphoenol pyruvate into pyruvate by pyruvate kinase enzyme which is thethird irreversible reaction of glycolysis.

11- Reduction of pyruvate to lactate:- Lactate formed by lactate dehydrogenase, is the final product of anaerobic glycolysis. The formation of lactate is the major fate for pyruvate in red blood cells, lens and cornea of the eye, kidney and leukocytes.

Alternate fate of pyruvate: 1- Oxidative decarboxylation of pyruvate: - Oxidative decarboxylation of pyruvate by pyruvate dehydrogenase complex isan important pathway in tissues with a high oxidative capacity, e.g. cardiac muscle. Pyruvate dehydrogenase complex irreversibly convert pyruvate into

COOHC = OCH3

COOHC - OHCH3


NADH+H+ NAD+

H

Pyruvate Lactate


11

COOHC = OCH3

Pyruvate carboxylase

CO2

Biotin

C - COO-

CH2 - COO-

=O

OxaloacetatePyruvate

active acetate (Acetyl CoA) which is major fuel for citric acid cycle (Krebscycle) and the building block for fatty acid and cholesterol biosynthesis.

2- Carboxylation of pyruvate to oxaloacetate:- Another pathway of fate of pyruvate is its carboxylation to oxaloacetate by pyruvate carboxylase which is biotin dependent reaction.

Energy yield of glycolysis: - ATP energy Yield = ATP produced – ATP utilized.

Reaction ATP (+ or -)

1- Hexokinase / Glucokinase - one ATP

2- PFK-1 - one ATP

3-Glyceraldehyde-3-P dehydrogenase (aerobic) + 2 NADH+H+ × 3 = + 6 ATP

4- Phosphoglycerate kinase + 2 × 1 = + 2 ATP

5- Pyryvate Kinase + 2 × 1 = + 2 ATP

Total aerobic glycolysis + 8 ATP

Total anaerobic glycolysis + 2 ATP

Regulation of Glycolysis:- Glycolysis is primarily regulated in response to the cell's requirement for ATP as follows: 1- Phosphofructokinase: the rate-limiting enzyme of the pathway. - It is allosterically inhibited by ATP and stimulated by AMP. It is also inhibited by citrate.

COOHC = OCH3

Pyruvate Dehydrogenase Complex

TPP, CoASH, Lipoic acid, FAD & NAD+ CH3 - C O ~CoA

Acetyl CoAPyruvate


12

2- Pyruvate kinase: is inhibited by ATP and stimulated by fructose 1,6 biphosphate.

3- Hexokinase: is inhibited by its product, glucose-6-phosphate.

4- Fructose 2,6 biphosphate: it mediates the effects of insulin and glucagon onthe glycolytic pathway. The concentration of fructose 2,6 biphosphate is increased under conditions which increase the insulin/glucagon ratio (after meal rich in carbohydrate). This compound acts as allosteric activator of phosphofructokinase.

5- Hormonal regulation:- Insulin stimulates the three irreversible reactions of glycolysis while glucagon inhibits these reactions.

In vitro inhibition of glycolysis: 1- Iodoacetate: by inhibiting the enzyme glyceraldehyde-3-p dehydrogenase. 2- Fluoride: inhibits enolase enzyme. Clinical laboratories use fluoride to inhibit glycolysis by adding it to the blood before measuring blood glucose.3- Arsnate: inhibits substrate level phosphorylation reactions.

Glycolysis in RBCs.: - RBCs. contain no mitochondria, thus: a- They depend only upon glycolysis for energy production (= 2ATP). b- Even under aerobic condition, glycolysis terminates in lactate due to the enzymatic machinery of pyruvate oxidation is not present in RBCs. (due to absence of mitochondria). - RBCs have the ability to form 2,3 bisphosphoglycerate (2,3 BPG) which lower the affinity of hemoglobin to oxygen leading to good oxygenation of tissues in case of hypoxia.

2,3-Bisphosphoglycerate Shuttle: - 1,3-bisphosphoglycerate is converted into 2,3-bisphosphoglycerate (2,3-BPG) by the action of bisphosphoglycerate mutase in RBCs which is then hydrolyzed into 3-phosphoglycerate by phosphatase enzyme without production of high energy phosphate (ATP). Then 3-phosphoglycerate complete glycolysis.- 2,3-BPG is an allosteric effector of hemoglobin. 2,3-BPG is present in RBCs and promotes the release of oxygen from hemoglobin through its reduction of hemoglobin affinity to oxygen, thus allow oxygen delivery to tissues in case of hypoxia.


13

2)1,3-Bisphosphoglycerate


ADP

ATPMg2+

(2)3-Phosphoglycerate

COO - PC OHHCH2-O-P

COOHC OHHCH2-O-P

COOHC O - PHCH2-O-P

2) 2,3-Bisphosphoglycerate

BisphosphoglycerateMutase

Phosphatase

Pi2,3-Bisphosphoglycerate Shuttle

SH

SHL

S

SL

- Pyruvate kinase deficiency: - Genetic deficiency of pyruvate kinase in RBCs leads to hemolytic anemia (excessive destruction of RBCs) due to reduced rate of glycolysis.

MITOHONDRIAL PATHWAY FOR GLUCOSE OXIDATION

- Complete oxidation of glucose occurs in both cytoplasm (glycolysis) and mitochondria (Krebs' cycle). In the presence of O2, pyruvate (the product of glycolysis) passes by special pyruvate transporter into mitochondria which proceed as follows: 1- Oxidative decarboxylation of pyruvate into acetyl CoA. 2- Acetyl CoA is then oxidized completely into CO2, H2O through Krebs' cycle.

Oxidative decarboxylation of pyruvate: - Pyruvate dehydrogenase complex (PDH) is a multienzyme complex located in the mitochondrial matrix. It converts pyruvate into acetyl CoA. This pathway is irreversible and explains why glucose cannot be formed from acetyl CoA (derived from fatty oxidation) in gluconeogenesis. - This enzyme needs 5 coenzymes: 1- Vitamin B1 = TPP = Thiamine diphosphate.

2- Lipoic acid, (reduced), (oxidized).

3- Coenzyme A = CoASH. 4- Flavin adenine dinucleotide = FAD. 5- Nicotinamide adenine dinucleotide = NAD+.


14

COOHC = OCH3

Pyruvate Dehydrogenase Complex

TPP, CoASH, Lipoic acid, FAD & NAD+ CH3 - C O ~CoA

Acetyl CoAPyruvate

COOHC = OCH3

CO2

TPP

CH3 - CH - TPP

OHL

SS

CH3 - C ~S- L - SH

=O

CoA-SHCH3 - C O ~CoA

LSH

SH

FAD

FADH2 NAD+

NADH+H+

The overall reaction of pyruvate dehydrogenase complex

Acetyl CoA

Pyruvate

Pyruvyl TPP

Acetyl Lipoic A

Energy production: (3 ATP): - This pathway produces one molecule of NADH+H+ which produces 3 ATP molecules through respiratory chain phosphorylation.

Regulation of oxidative decarboxylation: a- Factors stimulating PDH: 1- Pyruvate 2- CoASH. 3- NAD+. 4- Insulin hormone.

b- Factors inhibiting PDH: 1- NADH+H+. 2- ATP.3- Acetyl CoA. 4- Calcium ions.

c- Mechanism of regulation: - PDH exists in two forms: 1- Active form (dephosphorylated). 2- Inactive form (phosphorylated). - Thus, protein kinase inhibits PDH and phosphatase stimulates it.- PDH kinase is stimulated by excess acetyl CoA, NADH+H+ and ATP while phosphatase is stimulated by insulin.


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CH3 - CO~S - CoA Acetyl CoA

OxaloacetateCitrate Synthase

CoASH

Citrate

Aconitase

H2O

Inhibited byFluoroacetate

Cis-aconitate

Aconitase

Fe2+

Fe2+

H2O

Isocitrate

Isoc

itrat

eDe

hydr

ogen

aseNAD

+

NADH+H+

OxalosuccinateIsocitrate

Dehydrogenase

CO2

Mn2+

Alpha-ketoglutarate

Alpha-ketoglutarate

dehydrogenase complex

NAD+

NADH+H+

CoASH

CO2

Succin

yl-CoA

Succ inatet hiok ina se/ M

g2 +

ADP + Pi

ATP

CoASH

Succinate

Succinatedehydrogenase

FAD

FADH2

Fumarate

Fumarase

H2O

L-Malate

MalateDehydrogenase

NAD+ NADH+H+

Inhibited by Arsenate

Inhibited bymalonate Krebs Cycle

CITRIC ACID CYCLE (Krebs' Cycle) (Tricarboxylic Acid Cycle)

- Tricarboxylic acid cycle (TCA) is a series of reactions in which acetyl CoA is oxidized into CO2, H2O and energy. Mitochondrial matrix is the location of the enzymes of TCA.

- The cycle is started by acetyl CoA (2 carbons) and oxaloacetate (4 carbons). It ends by oxaloacetate (4 carbons). The difference between the starting compound (6 carbons) and the ending compound (4 carbons) is 2 carbons that are removed in the form of 2 CO2. These 2 carbons are derived from acetyl CoA. For this reason acetyl CoA is completely catabolized and never gives glucose.


16

CH3 - CO~S - CoA Acetyl CoA

C - COOHCH2 - COO-

=O

OxaloacetateCitrate Synthase

CoASH

C - COOHCH2 - COOH

CH2 - COOHHO

CitrateAconitase

C - COOHCH - COOH

CH2 - COOHH2O

Inhibited byFluoroacetate

Cis-aconitate

Aconitase

Fe2+

Fe2+

H2O

CH - COOHCH - COOH

CH2 - COOH

HO

IsocitrateIs

ocitr

ate

Deh

ydro

gena

se

NAD

+NA

DH+H

+

CH - COO-

C - COO-

CH2 - COO-

O

OxalosuccinateIsocitrate

Dehydrogenase

CO2

Mn2+CH2C - COO-

CH2 - COO-

O

Alpha-ketoglutarate

Alpha-ketoglutaratedehydrogenase complex

NAD+

NADH+H+

CoASHCO2

CH2C ~S - CoA

CH2 - COOH

O

Succinyl-CoA

Succi na t ethiok inas e/M

g2+

ADP + Pi

ATP

CoASH

CH2 - COOHCH2 - COOH

Succinate

Succ

inat

ede

hydr

ogen

ase

FAD

FADH2

CHCH - COOH

COOH

CH - COO-

Fumarate

Fumarase

H2O

HOCH2 - COO-

L-Malate

MalateDehydrogenase

NAD+NADH+H+

Inhibited by Arsenate

Inhibited by malonate

Krebs Cycle


17

Regulation of Krebs cycle (TCA): The key enzymes are citrate synthase and isocitrate dehydrogenase and α-ketoglutarate dehydrogenase:

a) Citrate synthase: 1) Stimulated by acetyl CoA, oxaloacetate, ADP and NAD+. 2) Inhidited by long chain acyl CoA, citrate, succinyl CoA, ATP and NADH+H+.

b) Isocitrate dehydrogenase and a-ketoglutarate dehydrogenase: 1) Stimulated by NAD+, ADP. 2) Inhibited by NADH+H+ and ATP.

c) Availability of Oxygen: - Citric acid cycle needs oxygen to proceed (i.e. aerobic pathway). This is because in absence of oxygen respiratory chain is inhibited leading to increase NADH+H+/NAD. NADH+H+ will inhibit TCA cycle.

- In vitro inhibition of TCA cycle: a- Flouroacetate: inhibits aconitase enzyme. b- Arsenate: inhibits: α-ketoglutarate enzyme. c- Malonic acid: inhibits succinate dehydrogenase enzyme.

Oxaloacetate

NADH+H+ / NAD+

ATP / ADP

Acetyl CoA

Citrate

Isocitrate

Oxalosuccinate

-Ketoglutrate

Citrate synthase

Isocitrate DH

-Ketoglutrate DH

Succinyl CoA

Long chainacyl CoA

Regulation of citric acid cycle


18

Energy yield: - Oxidation of one NADH+H+ by electron transport chain yield 3 ATP while oxidation of one FADH2 yields 2 ATP molecules.

Energy yield from oxidation of one molecule of acetyl CoA in TCA cycle:

Reaction enzyme ATP produced

1- Isocitrate dehydrogenase (NADH+H+). 3 ATP

2- α-Ketoglutarate dehydrogenase (NADH+H+). 3 ATP

3- Succinate thiokinase (substrate level). 1 ATP

4- Succinate dehydrogenase (FADH2). 2 ATP

5- Malate dehydrogenase (NADH+H+). 3 ATP

Total 12 ATP

- Complete oxidation of one molecule of glucose under aerobic condition produce 38 ATP molecules as follow: 1- Aerobic glycolysis = 8 ATP 2- Oxidative decarboxylation of 2 molecule of pyruvate= (2 × 3 ATP = 6 ATP). 3- Oxidation of one molecule of acetyl CoA in TCA cycle = 12 ATP (2 × 12 = 24 ATP). - Energy produced from complete oxidation of one molecule of glucose =

8 + 6 + 24 = 38 ATP molecules.

- On the other hand complete oxidation of one molecule of glucose under anaerobic condition yield only 2 ATP molecules.

Functions (significance) of TCA: 1- The cycle is the major source of energy for cell containing mitochondria. 2- The cycle is amphibolic i.e. it has catabolic (breakdown) and anabolic (formation) functions. a) Catabolic function: TCA is the final common pathway for oxidation ofcarbohydrates, fats and proteins (amino acids).

b) Anabolic function: Formation of:1) Amino acids:- α-Ketoglutarate Transamination Glutamate.


19

- Oxaloacetate Transamination Aspratate. - Pyruvate Transamination Alanine.

2) Glucose: e.g. - α-Ketoglutarate Transamination Glucose.

3) Heme synthesis: - Succinyl CoA → Heme.

4) Fatty acid and cholesterol: - Citrate (diffuse to cytoplasm) → Oxaloacetate + Acetyl CoA → Fatty acid and cholesterol.

5) CO2 produced is used in the following (CO2 fixation) reactions: - Pyruvate + CO2 → Oxaloacetate Gluconeogenesis Glucose. - Acetyl COA + CO2 → Malonyl CoA → Fatty acids.- Ammonia + ATP + CO2 → Carbamoyl phosphate → Urea and pyrimidine.- Propionyl CoA + CO2 → Methyl malonyl CoA → Odd number fatty acid.- Synthesis of H2CO3 / HCO3 buffer.

Cytoplasm

Mitochondria Pyruvate

Glucose

Pyruvate

Acetyl CoA

Citrate

Oxaloacetate Acetyl CoA

CoASH ATP citrate lyase

FA SynthesisCholesterol

TCACitrate

oxalo.A


20

OH

OH

H

OHH

OHH

OH

CH2OP

H

Glucose-6-P

NADP+ NADPH+H+

Mg2+

Glucose -6-PDehydrogenase

OH

OHH

OHH

OH

CH2OP

H = O

6-Phosphogluconolactone

H2O/Mg2+

COOHC OHHC HHOC OHHC OHHCH2O-P

6-Phosphogluconic acid

6-Phosphoglucono-lactonase

6-PhosphogluconateD

ehydrogenase

NADP+

NADPH+H+

CO2

Mg2+

CH2 OHC = OC OHHC OHHCH2O-P

Ribulose-5-P

Epimerase

CH2 OHC = OC HHOC OHHCH2O-P

Xululose-5-P

IsomeraseCH2 OHC - OHC OHHC OHHCH2O-P

H

Ribose -5-P

HEXOSE MONOPHOSPHATE PATHWAY (HMP SHUNT) (Pentose phosphate pathway)

- HMP pathway is an alternative route for the oxidation of glucose. - The enzymes of this pathway are present in cytosol as glycolysis. - HMP shunt does not generate ATP but has 2 major functions:

1- The generation of NADPH+H+ for reductive synthesis such as fatty acids and steroids biosynthesis.

2- The production of pentoses (ribose) for nucleotide and nucleic acid biosynthesis.

- HMP shunt is divided into 2 phases oxidative and non-oxida tive phase.

1- Oxidative phase:- Glucose-6-P undergoes dehydrogenation and decarboxylation to give a pentose (ribulose-5-P), CO2 and 2 NADPH+H+


21

HEXOSE MONOPHOSPHATE PATHWAY (HMP SHUNT) (Pentose phosphate pathway)

- HMP pathway is an alternative route for the oxidation of glucose. - The enzymes of this pathway are present in cytosol as glycolysis. - HMP shunt does not generate ATP but has 2 major functions:

1- The generation of NADPH+H+ for reductive synthesis such as fatty acids and steroids biosynthesis.

2- The production of pentoses (ribose) for nucleotide and nucleic acid biosynthesis.

- HMP shunt is divided into 2 phases oxidative and non-oxida tive phase.

1- Oxidative phase:- Glucose-6-P undergoes dehydrogenation and decarboxylation to give a pentose (ribulose-5-P), CO2 and 2 NADPH+H+


22


Xululose-5-P

CH2 OHC - OHC OHHC OHHCH2O-P

H

Ribose -5-P

Tran

sket

ola s

eTP

P

CH2 OHC = O

C - HC - OHC OHHC OHHCH2O-P

HHO

Sedoheptulose-5-P

CHOC - OHCH2 -O- P

H

Glyceraldehyde-3-P

Tra n

sald

olas

eCH2-OHC = OC HHOC OHHC OHHCH2OH

Fructose-6-P

CHOC OHHC OHHCH2-O-P

Erythrose-4-P

Tran

sket

olas

eTP

P


Xululose-5-P

CHOC - OHCH2 -O- P

H

Glyceraldehyde-3-P

Non-Oxidative (Reversible) reactions of HMP shunt

Oxidative (Irreversible) reactions of HMP Shunt

2- Non-oxidative phase:- Ribulose-5-P is converted back to glucose-6-P (through intermediates of gluconeogenesis) by a series of reactions involving mainly 2 enzymes, transketolase and transaldolase.

- The non-oxidative reaction catalyzes the interconversion of 3 to 7 carbon sugars. In this reaction, ribulose-5-phosphate (produced in the oxidative phase) can be converted to either ribose-5-phosphate, or to intermediates of glycolysis such as fructose-6- phosphate and glycerladehyde-3- phosphate. The only coenzyme required in the non-oxidative reaction is TPP (thiamine pyrophosphate) in the transketolase reactions.

The difference between HMP shunt and glycolysis: 1- Oxidation in HMP pathway occurs in the first reaction utilizing NADP+

rather than NAD+. 2- CO2, which is not produced at all in glycolysis, is a characteristic product in HMP pathway. 3- ATP is not generated in HMP, whereas it is a major function of glycolysis.4- Pentose-P is generated in HMP pathway but not in glycolysis.

:Regulation of HMP shunt- The pentose phosphate pathway is regulated primarily at the G6PD

reaction. - NADPH is a potent competitive inhibitor of the enzyme: - The ratio of NADPH/NADP+ is sufficiently high; it inhibits enzyme

activity.


23

H2O2

2 H2O

2 GSH

GSSG

Glutathioneperoxidase

NADPH+H+

NADP+

Role of NADPH+H+ in H2O2 detoxification

GlutathioneReductase

- The ratio of NADPH/NADP+ decreases and so the cycle increases in response to the enhanced activity of G6PD.

- Insulin induce enzyme via gene expression

The Metabolic significance of HMP shunt: 1- HMP is active in liver, adipose tissues, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary gland. All of these tissues use NADPH+H+ inreductive synthesis. e.g.: synthesis of fatty acids, steroids or reduced glutathione (in RBCs).

2- HMP provides ribose for nucleotides and nucleic acids biosynthesis. The role of NADPH+H+ in reduction and detoxification of H2O2:- H2O2 is one of a family of reactive oxygen species (ROS) that are formed from partial reduction of molecular oxygen.

- H2O2 can cause damage to DNA, proteins and unsaturated lipids through its ability to generate a more reactive and damaging hydroxyl radical which attack these molecules causing DNA strand break and base hydroxylation, protein oxidation and lipid peroxidation.

- H2O2 removed from the body by the aid of glutathione peroxidase enzyme using reduced glutathione (GSH) as electron donating substrate and converted into oxidized glutathione (GSSG). GSH is regenerated from GSSG by the aid of glutathione reductase enzyme which need NADPH+H+ as coenzyme as a source of reducing electrons.

Role of NADPH+H+ in synthesis of steroid Hormones: -. Mitochondrial Cytochrome P-450, NADPH dependent, monooxygenase system: for steroid hydroxylation, for e.g. In the steroid hormone producing tissues, placenta, ovary and testes, it used this system to hydroxylate the intermediates of cholesterol to steroid hormones. Role of NADPH+H+ in detoxification of drugs- Microsomal Cytochrome P-450, NADPH dependent monooxygenase system: for detoxification of drug & xenobiotic metabolism and hydroxylate these


24

materials by using NADPH as a source of reducing equivalent, lead to more soluble form and non-toxic product.

Glucose-6-P Dehydrogenase (G-6PD) deficiency: - It is an inherited disease characterized by hemolytic anemia caused by the inability to detoxify oxidizing agents (such as H2O2).

- Deficiency in G-6PD activity impairs the ability to form NADPH+H+ by HMP shunt which is essential for detoxification of oxidants such as H2O2 and other free radicals formed within the cells.

Favism (deficiency in G-6PD enzyme): - It is a hemolytic anemia (excessive destruction of RBCs) especially after ingestion of fava beans and some other compounds such as: - Antibiotics (as sulfamethoxazole and streptomycin) and antimalarial (as primaquine).


25

URONIC ACID PATHWAY

- This is a minor pathway for the conversion of glucose to glucuronic acid. - Location of the pathway: a- Intracellular location: cytoplasm. b- Organ location: mainly liver.

- Uronic acid pathway is an alternative pathway for glucose oxidation that does not provide ATP, but synthesizes D-Glucuronic acid.

- Uses of Glucuronic acid:1 It is an essential component of glycosaminoglycans. 2 It is required in detoxification reactions of a number of insoluble

compounds, such as bilirubin, steroids and several drugs, including morphine.

3 It serves as a precursor of ascorbic acid (vitamin C) in plants and mammals (other than guinea pigs and primates, including humans).


26

GLYCOGEN METABOLISM

- A constant supply of blood glucose is an absolute requirement for human life. Blood glucose can be obtained from three sources: 1- Dietary intake of glucose and glucose precursors (starch, fructose, sucrose, etc…).2- Gluconeogenesis can provide sustained synthesis of glucose, but tends to be slow in responding to a falling blood glucose level. 3- The body has developed mechanism for storing a supply of glucose in a rapidly mobilizable form i.e. glycogen. In the absence of dietary glucose, glucose is rapidly released from liver glycogen.

Function of glycogen: - The main stores of glycogen in the body are found in skeletal muscle and liver. 1- The function of muscle glycogen is to serve as a source of energy within the muscle itself during muscle contractions.

2- The function of liver glycogen is to maintain the blood glucose level, particularly during the early stages of fasting. After 12-18 hours fasting, liver glycogen is depleted.

Location of glycogen: - Glycogen is present mainly in cytoplasm of liver and muscle. - In liver: About 120 grams represents up to 10 % of the liver weight. - In muscle: About 350 grams represents up to 1-2 % of the total muscle weight.

Structure of glycogen: - Glycogen is a branched-chain homopolysaccharide made from α-D-glucose. - The primary glycosidic bond is α-1,4 linkage. After every 8-10 glucose units (glucosyl residues), there is branch containing α-1,6 linkages.


27

Glucose Glucose-6-P Glucose-1-P

UDP-Glucose

UDP-glucosepyrophosphorylase

PPi

Glukinase Phosphoglucomutase

UTPATP ADP

O

HO

HO

OH CH2OH

O

OHO

OH CH2OH

O

OHO

OH CH2OH

O

HO

HO

OH CH2O

O

1

2

3

45

6

1

2

3

4

5

6

1

2

3

4

5

6

1

2

3

45

6

alpha-1,4-bond

alpha-1,6 bond(at branching)

alpha-1,4-bondO

alpha-1,

Structure of glycogen

GLYCOGENESIS

- It is the formation of glycogen in liver and muscles. - Glycogen is synthesized from molecules of α-D-glucose. The process occurs in cytosol, and requires energy supplied by ATP for the phosphorylation of glucose and uridine triphosphate (UTP) for activation of glucose. 1- UDP-glucose is the source of all glucosyl residues that are added to the growing glycogen molecules. UDP-glucose is synthesized as follow.

2- Glycogen synthase is responsible for making the α-1,4 linkage in the glycogen. This enzyme cannot initiate chain synthesis using free glucose as acceptor of a molecule of glucose from UDP-glucose. Instead, it can only elongate already existing chain of glucose. Therefore, a fragment of glycogencan serve as a primer in cells whose glycogen stores are not totally depleted.

- In absence of glycogen fragment, a specific protein called glycogenin, can serve as an acceptor of glucose residues. The OH group of a specific tyrosine side chain serves as the site at which the initial glycosyl unit is attached.


28

GlycogeninHOUDP-glucose

UDP

O

Glycogen initiator synthase

UDP

UDP

O

Alpha-1,4- bonds

Non reducing endBranching enzyme

O

Further elongation and branching

Glycogen

Tyrosineside chain

Alpha-1,6- bond

GLYCOGENESIS

Transfer of the first molecule of glucose from UDP-glucose to glycogenin is catalyzed by glycogen initiator synthase.

3- Elongation of glycogen chains involves the transfer of glucose from UDP-glucose to non-reducing end of the growing chain, forming a new glycosidic bond between the OH of carbon 1 of activated glucose and carbon 4 of accepting glycosyl residue. The enzyme responsible for elongation of glycogen chain called glycogen synthase.

4- Branching enzyme transfers a chain of 5-8 glycosyl residues from the non-reducing end of glycogen chain to another residues of the chain and attaches it by α-1,6 linkage.

5- The new branches are elongated by the glycogen synthase and the process is repeated.


29

Phosphorylase

8 Pi

8 Glucose 1-P

2

6

Transferase

(Alpha-1,6 glucosidase)

1 Glucose

Phosphorylase

14 Pi

14 Glucose-1-Phosphate

Phosphoglucomutase

In liver & KidneyPhosphatase

Pi

Glucose

Glycogen core

Debranching Enzyme

Glycogen core

Glycogen core

Glycogen core

GLYCOGENOLYSIS

Glucose-6-phosphate

Degradation of glycogen (Glycogenolysis) - It is the breakdown of glycogen into glucose (in liver) and lactic acid (in muscle). Steps: 1- Phosphorylase (the key enzyme of glycogenolysis) acts on α-1,4 bonds, breaking it down by phosphorolysis (i.e breaking down by addition of inorganic phosphate "Pi"). So, it removes glucose units in the form of glucose-1-phosphate. 2- Phosphorylase enzyme acts on the branch containing more than 4 glucosyl units.3-When the branch contains 4 glucose units, 3 of them are transferred to a next branch by transferase enzyme, leaving the last one. 4- The last glucose unit that is attached to the original branch by α-1,6 bond is removed by debranching enzyme by hydrolysis (i.e braking the bond by addition of H2O). 5- Glucose-1-phosphate molecules are converted to glucose-6-phosphate, by mutase enzyme.


30

Glycogen synthase Protein kinase PhosphorylatedGlycogen synthase

ATP ADP

Phosphorylase PhosphorylatedPhosphorylase

Inactive

Active

Protein kinase

ATP ADP

6- Fate of Glucose-6-phosphate:a) In liver: glucose-6-phosphate is converted to glucose by glucose-6-phosphatase. b) In muscles: There is no glucose-6-phosphatase, so glucose-6-phosphate enters glycolysis to give lactate. Regulation of glycogenesis and glycogenolysis: - A highly regulated process, involving reciprocal control of glycogen phosphorylase and glycogen synthase - Glycogen phosphorylase is allosterically activated by low energy and inhibited by ATP, glucose-6-P.- Glycogen Synthase is stimulated by glucose-6-P- Differences in Liver Glycogen Phosphorylase

Liver glycogen phosphorylase is very similar to, but not identical to the one found in muscle as it is allosterically inhibited by the accumulation of glucose. This does not happen in muscle and is an important control in the liver, allowing it to shut down when glucose accumulates faster than it is needed. - Both enzymes are regulated by covalent modification – phosphorylation- During fasting, glycogenolysis is stimulated and glycogenesis is inhibited. This provides blood glucose. - After meal, part of absorbed blood glucose goes to portal circulation to be utilized. The remaining is converted into glycogen in liver. Therefore, after meal, glycogenesis is stimulated and glycogenolysis is inhibited - The principles enzymes controlling glycogen metabolism are glycogen synthase and phosphorylase. These are regulated as follow: 1- During Fasting:a) Blood glucose level tends to be decreased. This stimulates secretion of epinephrine, nor-epinephrine and glucagon hormones. [

b) These hormones stimulate adenylate cyclase enzyme, which converts ATP into cyclic AMP (cAMP).

c) cAMP stimulates protein kinase enzyme causing phosphorylation of both glycogene synthase and glycogen phosphorylase.


31

Glycogen synthase a (Active) Glycogen synthase b

(Inactive)

P

phosphorylase

H2OPi

ATP ADP

Protein Kinase

cAMP

Activate (+)

ATPAdenylate cyclase

PPi

Epinephrine, Nor-epinephrine,Glucagon

Activate (+)

Phosphodiesterase5/-AMP

Inhibit gylcogenesis

REGULATION OF GLYCOGENESIS

Phosphatase

Insulin

Activate (+)

P

d) As results, glycogenolysis will proceed causing increase of blood glucose. At the same time, glycogenesis will be inhibited.

e) Epinephrin and nor-epinephrin stimulate mobilization of calcium ions from mitochondria to cytosol. Calcium ions then combine with a protein called calmodulin causing conformational changes in it and activating it. The active calmodulin causes phosphorylation of both phosphorylase and glycogen synthase (like protein kinase). This leads to stimulation of glycogenolysis. This pathway is very important in muscle which uses the ion to trigger muscular contraction thus the same ion that stimulates muscular contraction also activates phosphorylase kinase which release glucose 1-P from glycogen which can be used to produce ATP to support muscular contraction.

2- After meal: a) Blood glucose level tends to be increased. This stimulates secretion of insulin hormone. b) Insulin causes the followings: i- Stimulation of phosphodiesterase enzyme, which converts, cAMP into AMP i.e. abolishes the stimulatory effect of cAMP. ii- Stimulation of phosphatase enzyme, which remove phosphate from phosphorylase (inhibiting it) and glycogen synthase (stimulating it). As result, glycogenesis will proceed and glycogenolysis will be inhibited.


32

H2OPi

ATP ADP

Protein Kinase

cAMP

Activate (+)

Adenylate cyclase

PPi

EpinephrineNor-EpinephrineGlucagon

Activate (+)

Phosphodiesterase5/-AMPATP

Glycogenphosphorylase b (Inactive)

Glycogenphosphorylase a (Active)

P

REGULATION OF GLYCOGENOLYSIS

Phosphatase

phosphorylase

Insulin

Activate (+)

Stimulate Glycogenolysis


33

GLUCONEOGENESIS

Gluconeogenesis is the formation of glucose from non-carbohydrate sources. These sources include: 1- Pyruvate. 2- Lactate. 2- Glucogenic amino acids. 3- Glycerol (the product of metabolism of fats in adipose tissue).

Site of gluconeogenesis:- This pathway occurs in both the cytosol and mitochondria. - Liver (90%) and kidneys (10%) are the major tissues involved in gluconeogenesis, since they contain all the necessary enzymes. Importance of gluconeogenesis:1- Gluconeogenesis meets the needs of the body for glucose when carbohydrate is not available in sufficient amount from diet.

2- A continual supply of glucose is necessary as a source of energy especially in the brain and erythrocytes. Severe hypoglycemia can leads to coma and death.

3- Gluconeogenetic mechanisms are used to clear the products of the metabolism from the blood such as lactate and glycerol.

Metabolic pathways involved in gluconeogenesis: - The steps of gluconeogenesis is mainly the reversal of glycolysis except for the three irreversible kinases reactions which are replaced by the reactions described below: The irreversible reactions (Energy barriers that obstruct a simple reversal of glycolysis): 1- Between pyruvate and phosphoenolpyruvate. 2- Between fructose 1,6-biphosphate and fructose -6-phosphate. 3- Between glucose-6-phosphate and glucose.


34

1- Phosphoenolpyruvate Pyruvate Lactate LDH

ADP ATPMg2+

Cytosol

Pyruvate Carboxylase (1)

CO2

Oxaloacetate

In mitochondria

In mitochondria

ATPBiotin

OxaloacetateIn Cytosol

PEP

car b

oxyk

inas

e( 2

)

CO2

Pyruvate kinase

2- Fructose-6-P Fructose-1,6-bisphosphatePhosphofructokinase 1

Fructose-1,6-bisphosphatase

(3)

3- Glucose Glucose -6-P Hexokinase

Glucose-6-phosphatase

(4)

The solutions to overcome the irreversible reactions of glycolysis : - Both fructose-1,6-bisphosphatase and glucose-6-phosphatase enzymes arepresent in liver and kidney. - Glucose-6-phosphatase is absent in the muscle and adipose tissues. - Dephosphorylation of fructose 1,6-bisphosphate, (by F-1,6-bisphosphatase-1bypasses the irreversible phosphofructokinase-1 and so formation of fructose 6-phosphate. This reaction is an important regulatory site for gluconeogenesis:


35

Glucose-6-P

Isomerase

Fructose-6-P

Phosphofructose kinase

Aldolase

Triose-phosphate

Phosphoenol pyruvate

Pyruvate kinase

PyruvateLDH

Transamination

CarboxylaseOxaloacetate

Aspartic acid

Transamination

phosphoenol pyruvatecarboxykinase

Fr-1,6-bisphosphatase

GlucoseHexokinase

(1)

(2)

(3)


Lactate

AlanineCystineSerine

(4) Gl-6-phosphatase

GlycerolGlycerokinase

Glutamine

Alpha-KG

Transamination

1- Fructose 1,6 bisphatase is inhibited by elevated levels of AMP. Conversely, high levels of ATP and low concentrations of AMP stimulate gluconeogenesis. 2- Fructose 1,6 biphatase is inhibited by fructose 2,6-bisphosphate, an allosteric modifier whose concentration is decreased by the level of circulating glucagon.


36

Glycerol as a substrate for gluconeogenesis: - Glycerol is released from triacylglycerols in adipose tissue to blood and transported to liver.

- Adipocytes lack glycerol kinase & cannot phosphorylate glycerol.Cori Cycle: - Lactate is released into the blood by cells that lack mitochondria, such as RBCs and by exercising skeletal muscles. In Cori cycle, glucose is converted by exercising muscles to lactate, which diffuse into the blood. This lactate is taken up by the liver and converted into glucose by gluconeogenesis which is released back into the circulation.

Regulation of Gluconeogenesis:

I- Allosteric Regulation: - Gluconeogenesis is controlled at the first step of the pathway, catalyzed by pyruvate carboxylase, which carboxylates pyruvate to oxaloacetate. It is allosterically activated by acetyl CoA which increased during starvation by increased fatty acid β-oxidation.- The second control point is the reaction catalyzed by fructose-1,6-bisphosphatase. This enzyme is activated by ATP and inhibited by AMP and fructose 2,6-bisphosphate.

II- Hormonal regulation: 1- Insulin: it inhibits gluconeogenesis. It acts by inhibiting the biosynthesis of the enzymes in this pathway (pyruvate carboxylase, PEP carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase). 2- Anti-insulin hormones: (Cortisol, Epinephrine and Glucagon): they stimulate gluconeogenesis by initiating the biosynthesis of the enzymes in this pathway. III- Gluconeogenic substrate availability: 1- Glucogenic amino acids: markedly increase the rate of gluconeogenesis. 2- Glycerol: This elevated in the blood during fasting, diabetes, or stress due to increase in lipolysis in adipose tissues, favors the rate of gluconeogenesis. 3- Lactate: which is increased in the blood during muscular exercise and by the RBCs, can be converted to glucose in the liver (Cori cycle).


37

Fructose Fructose-1-P

GlyceraldehydeDHAP

Glyceraldehyde-3-P

Fructokinase

Aldolase B

Kinase

Isomerase

ATP ADP

ATP

ADP

FRUCTOSE METABOLISM

- The major source of fructose is the disaccharides sucrose, which when cleaved release equimolar amounts of fructose and glucose.

- Fructose is also found as free monosaccharide in fruits and vegetables and in honey.

- The entry of fructose into cells is not insulin dependent.

- Importance of Fructose: - Energy production: 15% of daily energy is derived from fructose. - Fructose is the major energy source for spermatozoa in the seminal vesicle. Deficiency of fructose in semen correlates with male infertility.

Phosphorylation of fructose: -Fructose is firstly phosphorylated by hexokinase or fructokinase to fructose-1-phosphate using ATP as the phosphate donor. - Fructose-1-phosphate is then cleaved by aldolase B into dihydroxyacetone phosphate (DHAP) and glyceraldehyde (note that aldolase A cleaves fructose-1,6-bisphosphate). - DHAP can directly enter glycolysis or gluconeogenesis pathways, whereas glyceraldehyde was phosphorylated to glyceraldehydes-3-phosphate by triose kinase, which is then enter glycolysis or gluconeogenesis.


38

Conversion of glucose into fructose via sorbitol formation: - Aldose reductase enzyme reduces glucose into sorbitol in lens, retina, peripheral nerves, kidneys, placenta, RBCs, ovaries, and seminal vesicles. - In liver, ovaries sperm and seminal vesicles cells, there is another enzyme called sorbitol dehydrogenase which oxidizes sorbitol into fructose. Complications of diabetes: Large amounts of glucose may enter these cells during times of hyperglycemia (uncontrolled diabetes). Elevated intracellular glucose concentrations and an adequate supply of NADPH cause aldose reductase to produce a significant increase in the amount of sorbitol, which cannot pass efficiently through cell membranes and, therefore, remains trapped inside the cell This is exacerbated when sorbitol dehydrogenase is low or absent, for e.g, in retina, lens, kidney, and nerve cells. As a result, sorbitol accumulates in these cells causing strong osmotic effects and therefore cell swelling as a result of water retention leading to cataract, neuropathy & retinopathy.


39

Non-classicalgalactosemia

Classical galactosemia

In liverIn other tissues Glycolysis

Glucose

Galactose Galactose-1-P

Glucose-1-PGlucose-6-P

Kinase

ATPADP

UDP-glucose

UDP-galactose

UDP-galactose4-epimerase

UDP-glucosegalact-1-Puridyl transferase

Mutase

GALACTOSE METABOLISM

- The major dietary source of galactose is lactose that obtained from milk and milk products. Like fructose, galactose entry into cells is not insulin dependent.

Metabolism:


40

Inborn error of galactose metabolism:

- Galactose, along with glucose, is a constituent of milk disaccharides lacose. - Deficiency in UDP-glucose-galactose-1-P uridyl transferase enzyme and/or galactokinase can leads to inborn error of galactose metabolism.

1- Classical galactosemia: - It is genetic disease due to deficiency of UDP-glucose-galactose-1-P uridyl transferase enzyme that converts galactose1-phosphate to UDP-galactose. - Ingested galactose accumulates in blood causing galactosemia and galactosuria. - The accumulated galactose is reduced by aldose reductase in the eye, nerve tissue and liver to polyol (galactitol), causing cataract, liver damage and severe mental retardation. - Treatment includes removal of galactose as well as lactose from diet. The body needs for galactose can be provided by glucose by the action of the epimerase.

2- Non-classical galactosemia: (Galactokinase deficiency): Leads to galactosemia and galactosuria in addition to increased galactitol production (via aldose reductase enzyme) which leads to cataract.

PROTEIN METABOLISM

- ٢٤ -| P a g e

Pepsinogen (Inactive) Pepsin (Active)

Pepsinogen (Inactive) Pepsin (Active)

HCl

PROTEIN METABOLISM

Digestion of protein: - Proteins are too large to be absorbed by the intestine. Therefore they must be hydrolyzed at first to give amino acids, which can be easily absorbed. - Three organs produce the enzymes needed for protein digestion: stomach, pancreas and small intestine.

I- In Stomach:- The following enzymes start the digestion of proteins: pepsin, rennin and gelatinase.

1- Pepsin (optimum pH: 1-2): - It is secreted by the body chief cells of the stomach as an inactive proenzyme: pepsinogen. - Pepsinogen is activated at first by gastric HCl, then by autocatalytic action of the formed pepsin that activate the remaining pepsinogen.

- Action of pepsin: a) Pepsin is an endopeptidase (acts on amino acids in the middle of polypeptide chain). b) It hydrolyses peptide the bonds formed by aromatic amino acids. c) It hydrolyses protein into large polypeptides and few free amino acids.

2- Rennin: - This enzyme present only in infant stomach and its optimum pH is 4 (infantile pH of stomach) and cause coagulation of milk. Milk clot prevent the rapid passage of the milk from stomach and so give the baby the sense of fullness.

Action of rennin: a) Rennin acts on casein (main milk protein). b) In the presence of calcium ion, rennin converts casein into insoluble calcium para-caseinate (milk clot). c) The digestion of calcium para-caseinate is completely by pepsin.

3- Gelatinase: - The enzyme that digest gelatin.

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II- In intestine: - Two different organs, pancreas and intestine produce enzymes act on protein in the intestine.

A- Pancreatic Enzymes: - These include trypsin, chymotrypsin, carboxypeptidase, elastase and collagenase.

1- Trypsin (optimum pH: 8): - Trypsin is secreted as inactive proenzyme: trypsinogen. - Trypsinogen is activated to trypsin at first by enterokinase enzyme (produced by intestinal mucosa), then by autocatalytic action of the formed trypsin that activate the remaining trypsinogen.

Action of trypsin:a) Trypsin is an endopeptidase enzyme hydrolyzing the peptide bonds formed by basic amino acids e.g. lysine and arginine. b) Trypsin also acts as activator for all other inactive pancreatic enzymes.

2- Chymotrypsin: - Chymotrypsin is secreted as inactive proenzyme: chymotrypsinogen which is activated by trypsin enzyme.

Action of chymotrypsin: a) It is Endopeptidase hydrolyzing the peptide bonds formed by aromatic amino acids (its action is similar to that of pepsin).

3- Carboxypeptidase: - It is secreted as inactive procarboxypeptidase and activated by trypsin enzyme.

Action of carboxypeptidase: a) It is exopeptidase i.e acts on peripheral polypeptide chains. b) It hydrolyzes peptide bonds adjacent to free -COOH group of the polypeptide chain releasing each time a single free amino acid.

4- Elastase: - It is secreted as an inactive proenzyme: proelastase, and activated also by trypsin enzyme.

PROTEIN METABOLISM

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Trypsinogen (Inactive) Enterokinase Trypsin (Active)

Trypsinogen

Chymotrypsinogen

Proelastases

Pro-carboxypeptidase(Inactive)

Trypsin

Chymotrypsin

Elastase

Carboxypeptidase(Active)

Action of elastase: a) It is endopeptidase. b) It hydrolyzes peptide bonds formed by small amino acids e.g. glycine, alanine and serine.

5- Collagenase: - An enzyme that catalyzes the hydrolysis of collage.

B- Intestinal Enzymes:

1- Aminopeptidase:- It is an exopeptidase, hydrolyzes the peptide bonds adjacent to the free –NH2group of the polypeptide chain releasing each time a single amino acid.

2- Dipeptidases: - It completes the digestion of dipeptides.

Gatrointestinal hormones that help protein digestion:

A- Gastrin: It initiates pepsinogen secretion.

B- Cholecystokinin: It stimulates the release of several inactive zymogens from pancreas.

C- Secretin: It stimulates the release of pancreatic juice rich in bicarbonate to neutralize gastric secretion.

Absorption of Amino Acids: - Under normal conditions, the dietary proteins are almost completely digested into amino acids. - These amino acids are then rapidly absorbed from small intestine into the portal circulation. - In few cases protein may absorbed as such as in:

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Sources and Fate of Amino Acid Pool

(1) Dietary Proteins (2) Body Proteins (3) Synthesis of non-essential A.As

Amino Acid Pool

Synthesis of SpecializedProducts e.g. epinephrineNor-epinephrine, T3, T4,Serotonin……etc.

-ketoacids Ammonia Synthesis of body proteins

Urea

Glucose CO2+H2O+ATP Ketone bodies and F.A.

1- Normally in infants: where gamma globulins present in colostrum are absorbed as such which give immunity to the baby. (colostrum is the milk secreted from mammary gland in the first few days after labour).

2- Abnormally in adults: as in certain diseases. This may lead to allergic signs and symptoms.

Site of absorption: - Small intestine, in jejunum & ileum

Mechanism of absorption: - L-amino acids are absorbed by active transport mechanism need specific carrier protein and ATP, while D-amino acids are absorbed by passive transport mechanism.

Fate of absorbed amino acids:-

1- Anabolic pathways: a) Synthesis of proteins: tissue proteins, enzymes and hormones.

b) Synthesis of specialized products e.g.: creatine, choline, formation of nitrogen bases of neucleic acids.

c) Synthesis of small peptides e.g. glutathione.

2- Catabolic pathways: a) Removal of amino group (NH2) from amino acids by Transamination and deamination processes. b) The resulting products are ammonia, urea and α-keto acids. c) α-keto acids are further metabolized to be completely oxidized into CO2, H2Oor may be converted into glucose or fatty acids and ketones.

PROTEIN METABOLISM

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Amino acid + -Keto acid -Keto acid (new) + Amino acid (new)Transaminases

H2N CHCCH3

OHO

Alanine

+ O C CCH2

OHO

CH2COH

O

-Ketoglutarate

ALT/GPTPLP

O C CCH3

OHO

+ H2N CHCCH2

OHO

CH2COH

OPyruvic acid

Glutamic acid

General catabolic pathways of amino acids: - In human, the end products of proteins and amino acids catabolism are ammonia and urea. They are produced through the following catabolic pathways: a) Transamination.

b) Deamination: Oxiadative – Non oxidative – Hydrolytic.

c) Trandeamination: Transamination followed by deamination.

d) Decarboxylation.

a) Tranamination: - It is the transfer of amino group from α-amino acid to α-keto acid to form a new α-amino acid and a new α-keto acid.

- Enzymes catalyze transamination reaction called transaminases (or amino transferases).

- All transaminases utilize pyridoxal phosphate (PLP) (active form vitamin B6)as the coenzyme in the transamination reactions. - All transamination reactions are reversible.

- The transaminases enzymes are named for the amino acid that donate the amino group, for example, alanine transaminase, aspartate transaminase, and leucine transaminase. - All transaminases are present either in cytoplasm or in both cytoplasm and mitochondria of most tissues. - Among all tranaminases, 2 are present in most mammalian tissues and they are of clinical importance. These are: ALT and AST.

1- Alanine transaminase (ALT):a) ALT also called glutamate pyruvate transaminase (GPT). b) ALT is present in the cytosol of the cells. c) ALT is an enzyme that catalyzes the transfer of amino group from Alanine toα-ketoglutarate to form glutamate and pyruvate. The reaction is reversible.

PROTEIN METABOLISM

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+ O C CCH2

OHO

CH2COH

O

-Ketoglutarate

AST/GOTPLP

+ H2N CHCCH2

OHO

CH2COH

O

Glutamic acid

H2N CHCCH2

OHO

COH

O

O C CCH2

OHO

COH

O

OxaloacetateAspartate

2- Asparatate transaminase (AST):a) AST also called glutamate oxaloacetate transaminase (GOT). b) AST is present in the cytosol and mitochondria of the cells. c) AST is an enzyme that catalyzes the transfer of amino group from Aspartateto α-ketoglutarate to form glutamate and oxaloacetate. The reaction is reversible.

Diagnostic importance of transaminases (ALT and AST): a) Transaminases are normally intracellular enzymes.

b) The presence of elevated levels of transaminases in the blood indicates damage to cells producing these enzymes. This occurs in certain diseases:

1- Elevated levels of both ALT and AST indicate possible damage of liver cells with subsequent escape of hepatic enzymes into blood. 2- An elevated level of AST only suggests damage to heart muscle (myocardial infarction), skeletal muscle or kidney.

b) Deamination:- It is the removal of amino group from amino acids in the form of ammonia (NH3).- Deamination mostly occurs in liver and kidney. There are three types of deamination:

1- Oxidative deamination:- By oxidative deamination reaction, both oxidation (removal of hydrogen) and deamination (removal of ammonia) occur together. Several enzymes are involved, e.g.:

1- Oxidative deamination by L-amino acvid oxidase: a) This emzyme is present in liver and kidney. b) Its coenzyme is FAD. c) It deaminates most naturally occurring L-amino acids.

PROTEIN METABOLISM

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H2N CHCCH2

OHO

OH

Serine dehydrataseH2O

H3C C CNH

OHO NH3

H3C C C OHO

Pyruvic Acid

Serine

PLP

H2O

-Imino propionateCO

L-A.A.OXIDASE

FAD

Catalase

R CHCNH2

OHO

R C CNH

OHO H2O

R C C OHO

-KetoacidFADH2

O2H2O2

H2O

NH3

CO

O C CCH2

OHO

CH2COH

O

-Ketoglutarate

H2N CHCCH2

OHO

CH2COH

O

L-Glutamate

L-Glutamate dehydrogenase

NAD (P)+ NAD (P)++H+

HN C CCH2

OHO

CH2COH

O

-Iminoglutaric Acid

H2O NH3

d) H2O2 is produced as a by product. It is highly toxic to the cells. It is hydrolyzed rapidly by catalase into H2O and O2.

2- Oxidative deamination by L-glutamate dehydrogenase:a) It is highly active enzyme catalyzes the deamination of L-glutamate. b) This enzyme is present in the cytosol and mitochondria of most tissues. c) Its coenzyme is either NAD+ or NADP+.

2- Non-Oxidative deamination:a) This occurs for hydroxy containing amino acids e.g. serine and threonine without removal of hydrogen (non-oxidative). b) Its coenzyme is pyridoxal phosphate.

3- Hydrolytic deamination:a) This occurs for glutamine and asparagine. Both are hydrolytically deaminated by glutaminase and asparaginase respectively.

PROTEIN METABOLISM

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H2N - CH - COOH

CH2

CH2 - CO - NH2

GlutamineH2O NH3

Ammonia

GlutaminaseH2N - CH - COOH

CH2

CH2 - COOHGlutamate

b) Glutaminase enzyme is present in kidney. It produces ammonia, which is used in regulation of acid base balance by kidney.

c) Trandeamination:- It is transamination of most amino acids with α-keto glutarate to form glutamate. Then glutamate is deaminated to give ammonia. - It is the main pathway by which amino group (NH2) of most amino acids is released in the form of ammonia (NH3).

d) Decarboxylation: - Decarboxylation (removal of CO2) of amino acids produces its corresponding amines. - Some amines have physiological importance such as: 1- Histamine (from histidine) is vasodilator. 2- γ-amino butyric acid (from glutamate) is neurotransmitter. - The resulting amines are further oxidized, after carrying out their functions, by amine oxidase enzymes in the presence of pyridoxal phosphate as coenzyme.

AMMONIA

- Ammonia is toxic substance especially for CNS. - Any ammonia formed in the peripheral tissue must be moved to the liver to be converted into urea. This maintains ammonia at low level in the circulating blood.- Blood contain trace amounts of ammonia (10-110 ug/dl).

Sources and fate of ammonia:Sources:1- From amino acids by deamination or transdeamination. 2- From glutamine in the kidneys by glutaminase enzyme. 3- In intestine, ammonia is produced by action of intestinal bacterial enzymes on urea secreted into the intestine and dietary amino acids. 4- Purines and pyrimidines metabolism.

PROTEIN METABOLISM

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- Ketoglutarate + Ammonia GlutamateGlutamate Dehydrogenase

Transamination Non-essential amino acids

H2N CHCCH2

OHO

CH2COH

O

+Glutamine synthetase

ATP ADP+PiH2N CHC

CH2

OHO

CH2CNH2

O

Glutamic acid Ammonia Glutamine

NH3

Fate of Ammonia: 1- Formation of non-essential amino acids: through transdeamination reactions.

2- Formation of urea: It is the main pathway by which the body can get rid of ammonia.

3- Excretion in urine.

4- Formation of Glutamine: - Glutamine synthetase is a mitochondrial enzyme present in many tissues as kidney and brain. - Glutamine has the following functions: a) Regulation of acid-base balance. b) Removes the toxic effect of ammonia in the brain. c) Glutamine is the source of N3 and N9 of purine bases. d) Glutamine is used in detoxication of phenyl acetic acid (a toxic substance).

UREA

-Urea (H2N-CO-NH2) is the main end product of protein (amino acids) metabolism. - Urea formation is the pathway by which liver can convert toxic ammonia into non-toxic urea.

Site of urea formation: - Liver is the only site for urea formation. - Then urea is transported in the blood to the kidney to be excreted in urine (urine urea is v20-40 g/day).

PROTEIN METABOLISM

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CO2 + NH3Carbamoyl Phosphate synthetase I

2ATP 2ADP+ Pi

Biotin

Mg++N-acetylglutamate

Carbamoylphosphate

(+)(+)(+)

Ornithine CitrullineOrnithinetranscarbamoylase

Citrulline

Aspartate

Arginosuccinatesynthetase

ArginosuccinateLyase

Fumarate

Arginine

UreaOrnithine

MITOCHONDRIACYTOSOL

Mn++

Reactions of Urea Biosynthesis

H2O

Arginosuccinase

Arginase

Urea formation: (Urea cycle): - It is also called Kreb's Henselit cycle - Site: Liver. The first two reactions occur in mitochondria where other reactions occur in the cytoplasm. - Five amino acids share in urea cycle: N-acetylglutamate, ornithine, citrulline, aspartate, and arginine.

Regulation of Urea cycle: - The key enzyme of urea cycle is carbamoyl phosphate synthetase I. It is activated allosterically by N-acetylglutamate, Mn++ and Mg++ ions. - N-acetylglutamate synthesis is stimulated by high protein diet and amino acids specially arginine.

Relation between tricarboxylic acid cycle and Urea cycle: - CO2 and ATP needed for urea cycle produced from TCA cycle. - Fumarate produced from urea cycle can be oxidized in TCA cycle. - Aspartate can give oxaloacetate and vice versa (transamination).

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Ammonia Intoxication

- Excess ammonia is toxic to the central nervous system. This condition is called ammonia intoxication or hyperammonemia. - Blood contain traces of ammonia: 10-110 μg/dl.

Symptoms: Include: - Flapping tremors, blurring of vision and vomiting. High concentration of ammonia may cause coma and death.

Types and causes of hyperammonemia:

1- Acquired Hyperammonemia:a) Liver cirrhosis by bilharziasis, alcoholism, hepatitis or biliary obstruction. b) Liver cell failure. c) Renal failure.

2- Inherited Hyperammonemia: - Result from genetic deficiency of one or more of enzymes involved in urea cycle. It includes:

a) Hyperammonemia Type I - Deficincy of carbamoyl phosphate synthetase (reaction I).

b) Hyperammonemia Type II - Deficiency of ornithine transcarbamoylase (reaction II).

c) Citrullinemia: - Deficiency of arginosuccinate synthetase (reaction III).

d) Argininosuccinic Aciduria - Deficiency of argininosuccinase (reaction IV).

e) Argininemia: - This is due to deficiency of arginase (reactions V).

mechanism of ammonia intoxication:

- At normal blood ammonia level, any ammonia reaches the brain is incorporated into glutamine formation by glutamine synthetase enzyme. - In cases of hyperammonemia, ammonia reacts not only with glutamate, but also with α-ketoglutarate by glutamate dehydrogenase enzyme.

PROTEIN METABOLISM

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-ketoglutarate + NH3 GlutamateGlutamate Dehydrogenase

NADH+H+ NAD+

- This depletes α-ketoglutarate, which is an essential intermediate of citric acid cycle. - This results in a decrease in ATP and energy production. The brain and central nervous system require large amounts of energy and therefore leading to the symptoms of ammonia intoxication and even coma. - Significant improvement is noted on a low-protein diet, and much of the brain damage may thus be prevented. Food intake should be in frequent small meals to avoid sudden increases in blood ammonia levels.

Nitrogen Balance: - Nitrogen balance means that nitrogen intake (from diet) is equal to nitrogen loss (output).

A- Nitrogen intake: Nitrogen is taken in the form of dietary proteins. Every 100 gm proteins contain 16 gm nitrogen.

B- Nitrogen loss (output): The nitrogen output may be in the form of: 1- Urinary non-protein nitrogenous compounds as urea, creatinine, creatine, uric acid, ammonia and hippuric acid. 2- In milk and menstrual fluids in female. 3- Undigested protein in stool. 4- Traces of urea and uric acid in sweat.

Positive Nitrogen Balance: The nitrogen intake exceeds the nitrogen output. It occurs in conditions associated with increased formation of tissue proteins e.g. growing children, muscle training and pregnancy.

Negative Nitrogen Balance: The nitrogen loss exceeds the nitrogen intake. It occurs in conditions associated with increased breakdown of tissue proteins as in tuberculosis, diabetes mellitus, starvation, cancer and prolonged fever.

Equilibrium Nitrogen Balance: Nitrogen intake = nitrogen output.

PROTEIN METABOLISM

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H3CCH CH COOH

NH2

H3C

Valine

H3CCH CH COOH

NH2

H3C

Leucine

CH2

H3C

CH CH COOH

NH2

H3C

Isoleucine

CH2

CH2

VALINE ISOLEUCINE LEUCINE

R - CH - COOHNH2

Branched chain amino acids

Transamination Transaminase

-Ketoacid

-Amino acid

OR - C - COOH

Corresponding -Ketoacid

TPP CoASHOxidative

decarboxylation

2H

-Ketoaciddecardoxylase

CO2

R - C - SCoANext lower acyl CoA

Several steps

Propionyl CoA Acetyl CoA

Succinyl CoA Acetoacetyl CoA

O

The

sam

est

eps a

s is o

leuc

ine

The

s am

es t

eps a

s iso

l euc

i ne

Metabolism of Individual Amino Acids

Valine, Leucine and Isoleucine

- Valine, leucine and isoleucine are essential amino acids. -Valine is glycogenic, leucine is ketogenic and isoleucine is glycogenic and ketogenic. - Catabolism of them is discussed as a group.

PROTEIN METABOLISM

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H2N CHCCH2

OHO

Phenylalanine hydroxylase

THBP DHBP

NADPH+HNADP+

DHBPReductase H2N CHC

CH2

OHO

OH

Phenylalanine Tyrosine

O2 H2O

Maple syrup urine disease (Branched-chain ketoaciduria): - Maple syrup urine disease is caused by a deficiency of the enzyme α-ketoacid decarboxylase.

Features: - Accumulation of α-ketoacids of branched chain amino acids and their excretion in urine. - Urine smell like maple syrup or burnt sugar odour. - This leads to metabolic acidosis and mental retardation.

Treatment and/or management: - Dietary restriction of branched chain amino acids. Infants who are suffering from this disease rarely survive beyond the first year of life.

Phenylalanine

- Phenylalanine is a ketogenic and glycogenic essential amino acid. - Phenylalanine is the precursor for: Body protein and tyrosine. - Phenylalanine can be converted to tyrosine mainly in liver as follows:

- This reaction needs phenylalanine hydroxylase enzyme and tetrahydrobiopterin as coenzyme. This results in the formation of dihydrobiopterin (DHB) which must be regenerated by dihydrobiopterinreductase enzyme with NADPH+H as coenzyme.

- Deficiency of either phenylalanine hydroxylase or dihydrobiopterin reductaseresults in disease called phenylketonuria.

H2N CHCCH2

OHO

PROTEIN METABOLISM

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H2N CHCCH2

OHO

Phenylalanine hydroxylase

THBP DHBP

NADPH+HNADP+

DHBPReductase H2N CHC

CH2

OHO

OH

Phenylalanine Tyrosine

O2 H2O

Tyrosine

- Tyrosine is a ketogenic and glycogenic non-essential amino acid. - Tyrosine becomes essential in cases of deficiency of phenylalanine hydroxylase enzyme. - Synthesis:

Functions: Tyrosine is the precursor of: 1- Catecholamines (Dopamine, Epinephrine and Norepinephrine). 2- Melanine pigments. 3- Thyroid hormones. 4- Tyramine, phenol and cresol. These are putrefactive substances that produced by the action of bacteria present in large intestine on tyrosine.

1- Catecholamines:- These are dopamine, epinephrine and norepinephrine.

Function of Catecholamines: a) Neurotransmitter b) Regulation of metabolism (stimulate glycogenolysis and lipolysis). c) Increase cardiac output and blood pressure. d) Relaxation of smooth muscle of bronchi and intestine.

Synthesis:- It is synthesized from tyrosine at storage sites in adrenergic neurons, sympathetic ganglia and chromaffrin cells of adrenal medulla.

PROTEIN METABOLISM

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CH2 - NHCH3CH-OH

OHOH

CH2 - NHCH3CH-OH

OHOCH3 CHO

CH-OH

OHOCH3

COMT

MAOEpinephrine Metanephrine

3-methoxy-4-hydroxymandelic aldehyde

COOHCH-OH

OHOCH3

3-methoxy-4-hydroxymandelic Acid (VMA)

CH2 - NH3CH-OH

OHOH

CH2 - NH3CH-OH

OHOCH3

COMT

Norepinephrine Normetanephrine

MAO

H2N CHCCH2

OHO

OH

Tyrosine hydroxylase

THBP DHBP

NADPH+HNADP+

DHBPReductase

H2N CHCCH2

OHO

OHOH

Tyrosine3,4-DihydroxyPhenylalanine (DOPA)

DOPA Decarboxylase

H2N CH2CH2

OHOH

Dopamine

Dopamine B-Oxidase

AscorbicAcid + O2

Dehydroascorbate+ H2O

H2N CH2CH-OH

OHOH

Norepinephrine

PhenylethanolamineN-methyltransferase

S Adenosyl methionine S Adenosyl homocysteine

N CH2CH-OH

OHOH

CH3

Epinephrine

O2 H2O

CO2

H

Catabolism of Catecholamines: - Enzymes of catecholamines catabolism include 2 enzymes: a) Monoamine oxidase (MAO): It is present mainly in the mitochondria of adrenergic nerve endings. b) Catechol-ortho-methyl transferase (COMT): This is present in all tissues.

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H2N CHCCH2

OHO

OH

H2N CHCCH2

OHO

OHOH

Tyrosine 3,4-Dihydroxyphenylalanine(DOPA)

TyrosinaseTyrosinaseMelanin

Dopaquinone

H2N CHCCH2

OHO

O

O

Intermediate steps

- Vanillylmandelic acid (VMA) is used as indicator in diagnosis of adrenal tumors (pheochromocytoma) that produce huge amount of catecholamines.

2- Melanin pigments:- Melanins are pigments present in many tissues particularly in the eye, iris, hair and skin. - Melanins are synthesized to protect underlying cells from the harmful effects of sun lights.

Synthesis: In the skin, melanins are synthesized in melanosomes. These are particles bound to the membrane of melanocytes (pigment forming cells).

3- Thyroid hormones:- The two major hormones produced by the thyroid gland are triiodothyronine(T3) and thyroxine (T4).

Functions: a) They increase heat production and oxygen consumption in most tissues through stimulation of ATPase activity.

b) They produce many metabolic changes (increase lipolysis and glycogenolysis).

c) They act together with growth hormone as major anabolic agent during growth.

Synthesis: - Synthesis and release of T3 and T4 are stimulated by thyroid stimulating hormone (TSH) released from pituitary gland. - TSH secretion is under control of thyrotropin releasing hormone (TRH) which is tripeptide hormone produced in the hypothalamus.

PROTEIN METABOLISM

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I- I- OxidatonH2O2

I+ Iodination T T

T TT

TT T

TG

CH2 - CH - COOH

I

OH

NH2

Monoiodotyrosine (MTI)CH3 - CH - COOH

NH2Alanine

TG

TG

T3

TG

TBG + Free T3

TBG T3

TG

CH2 - CH - COOH

I

OH

NH2

Diiodotyrosine (DIT)

I

TG

T4

TBG + Free T4

TBG T4

(DIT)

TG ThyroidFolliclePlasma

Biosynthesis of thyroid hormones

T=TyrosineTG=ThyroglobulinTBG=Thyroxin binding globulin

Steps of synthesis:1- The thyroid gland contains many follicles, each composed of a shell of single layered cells surrounding a central space filled with glycoprotein called: thyroglbulin (TG).2- Thyroglbulin contains about 150 tyrosine residues. 3- Iodide ions (I-) can be taken up by thyroid cells and oxidized into higher value state (positive ions, I+). This needs H2O2 and thyroid peroxidase enzyme. 4- I+ is then incorporated into the tyrosine residue of TG. 5- The resulting monoiodotyrosine and diiodotyrosine residues react together to give T3 or T4. 6- Enzymes hydolysis of thyroglbulin releases T3 and T4 into the plasma.

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Inborn errors of phenylalanine and tyrosine metabolism:

1- Phenylketonuria (PKU): - It is inherited deficiency of phenylalanine hydroxylase enzyme. - Atypical phenylketonuria: It results from deficiency of dihydrobiopetrin reductase enzyme.- Phenylalanine hydroxylase catalyses the hydroxylation of phenylalanine to tyrosine. The enzyme deficiency leads to an increased plasma phenylalanine level, and impaired tyrosine production. In PKU phenylalanine accumulates in the plasma and tissues and is converted into the phenylketones (phenylpyruvate, and phenyllactate), which are not normally produced in significant amounts.

Effects: - High levels of phenylalanine may impair brain development leading to mental retardation. - Failure to walk and talk. - Tremors and skin lesion.

Frequency: - The frequency of phenylketonuria is 1 in 10.000 live births.

Treatment and/or management: - Restriction of dietary phenylalanine, however phenylalanine is an essential amino acid, therefore too much dietary restriction can also cause poor growth. - This regimen of diet is terminated at 6 years of age when a high concentration of phenylalanine has no longer effect on brain cells.

NB: Tyrosine cannot be synthesized in patients with PKU and becomes an essential amino acid.

2- Tyrosinemia: - It is inability to metabolize tyrosine and p-hydroxy phenylpuruvate. - It is due to deficiency of enzyme activity as tyrosine α-ketoglutarate transaminas and p-hydroxy phenylpuruvate oxidase. - Two forms of tyrosinemia a) Acute: Characterized by diarrhea, vomiting and failure to grow. Death from liver failure occurs within 7 months. b)Chronic: Occurs latter in life, liver cirrhosis and hepatocellular carcinoma arecommon.

PROTEIN METABOLISM

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H2N CHCCH2

OHO

H2N CHCCH2

OHO

OH

Phenylketoneuria Albinism

Phenylalanine hydroxylase Tyrosinase

Transamination Transamination

Melanine

Tyrosinemia

PhenylalanineTyrosine

COCCH2

OHO COC

CH2

OHO

OHPhenylpyruvate

CHCCH2

OHOHO

CH2

OH

OH

COOH

Tyrosinemia

AlkaptonureaHomogentesic acid Oxidase

P-hydroxy Phenylpyruvate

Phenyllactate Homogentisic acid

P-hydroxy Phenylpyruvate Oxidase

Maleyl acetoacetateUrine

Fumarate + Acetoacetate Fumaryl acetoacetate HydrolysisIsomerase

Treatment: - Feeding of affected infant and children diet containing very low level of tyrosine and phenylalanine.

Catabolism of Phenylalanine and Tyrosine

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3-Alkaptonuria:- Alkaptonuria is caused by a deficiency of the enzyme homogentisic acid oxidase that normally involved in the breakdown of tyrosine to fumarate. - Homogentisic acid oxidase deficiency leads to an accumulation of homogentisate, which is deposited in cartilage and other connective tissue causing generalized pigmentation (ochronosis).

Alkaptonuria symptoms include: 1- Joint damage and arthritis. 2- Homogentisate is excreted in the urine that is oxidized in the air giving the black urine due to the formation of alkapton.

Treatment and/or management: - No specific treatment.

4- Albinism: - It is a hereditary deficiency of the tyrosinase enzyme in melanocytes. This results in defective synthesis of melanin pigments. Eye, skin and hair are affected. - Many types are present, according to the site affected: a) Eye: ocular albinism.b) Skin: cutaneous albinism.c) Eye and skin: oculo-cutaneous albinism.

Treatment and/or management: - High sun protection.

Tryptophan

- It is glycogenic and ketogenic essential amino acid.

Functions: Tryptophan is the precursor of: 1- Serotonin. 2- Melatonin. 3- Niacin (nicotinic acid). 4- Indol and skatole.

NH

CH2 - CH - COOHNH2

PROTEIN METABOLISM

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NH

CH2 - CH - COOH

NH2

NH

CH2 - CH - COOHHO

NH

HOCH2 - COOH

NH

CH2 - CH2 - NH2HO

Tryptophan 5-Hydroxy tryptophan

5-Hydroxy indol aceticacid (5-HIAA)

5-Hydroxy Tryptamine (5-HT )(Serotonin)

MAO

5-Hydroxy tryptophanDecarboxylase

NH3 O2

Tryptophan hydroxylase

THBPDHBP

NADPH+HNADP+

DHBPReductase

O2 H2O

NH2

NH

HOCH2 - CH2 - NH2

SerotoninNH

HO

N-Acetyl serotonin

NH

CH3OCH2 - CH2 - NH - C - CH3

O

N-Acetyl-5-methoxy serotonin (Melatonin)

CH3-CO~SCOA

N-acetyltransferase

CoASH

Transmethylation

CH2 - CH2 - NH - C - CH3

O

Acetyl CoA

~CH3

1- Serotonin: (also called 5-hydroxy tryptamine) - Seretonin is synthesized and stored in hypothalamus, brain steam, pineal gland and platelets

Synthesis of serotonin:

Functions of serotonin: - Neurotransmitter. - Vasoconstriction - Contraction of smooth muscle fibers.

2- Melatonin:- Melatonin is synthesized by pineal gland.

Synthesis:

Functions of melatonin: a) It inhibits gonadal functionsb) It has sleep inducing effect.

PROTEIN METABOLISM

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NH

CH2 - CH - COOHNH2

Tryptophan

TryptophanOxygenase

PLP

C - CH2 - CH - COOHCHO

NH

O

Formyl Kynurenine

C - CH2 - CH - COOH

NH2

OC - CH2 - CH - COOH

NH2

O

OH

COOH

NH2

COOH

O2

NH2

NH2

H2O

H.COOH

Kynurenine (O)

NH2

3-Hydroxy Kynurenine

3-Hydroxy anthranilic acidOH

NNicotinic Acid (Niacin)

H2O

Alanine

Acetoacetyl CoA

Intermediatesteps

c) It plays a role in circadian rhythm regulation. d) It inhibits synthesis and secretion of other neurotransmitters such as dopamine and GABA.

3- Niacin (nicotinic acid):- Niacin is a member of vitamin B-complex, being synthesized in liver.Functions: - Essential for synthesis of NAD and NADP coenzymes. - Niacin has been used therapeutically for lowering plasma cholesterol. This is because it inhibts the flow of free fatty acids (FFA) from adipose tissue, which provides acetyl CoA essential for cholesterol synthesis.

Synthesis: - It is synthesized during tryptophan catabolism. - Every 60 mg tryptophan is converted into 1 mg niacin.

PROTEIN METABOLISM

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CH3 - CH - CH - COOH

OH NH2

CH2 - CH2 - CH2 - CH2 - CH - COOH

NH2 NH2

4- Indole and skatol: - These are putrefactive products of tryptophan produced by bacteria in large intestine. - Indole and skatol give the characteristic odour of stool. - Indole and skatol may be absorbed and go to the liver to be hydroxylated and conjugated with sulphate and excreted in urine as salt form (indican).

Hartnup's disease: - It is a hereditary abnormality in tryptophan metabolism where the intestinal absorption and renal tubular reabsorption of this amino acid are impaired. - It is characterized by pellagra skin rashes and mental retardation. - There is excess excretion of tryptophan together with lysine and histidine in urine (aminoaciduria).

Threoninie

- It is Essential, glycogenic amino acid.- Threonine enters in the synthesis of phosphoproteins and glycine.

Lysine

- Lysine is Essential and glycogenic amino acid. -Lysine is important for: a) Hydroxylysine synthesis: which enter in synthesis of collagen. b) Carnitine synthesis: It is β-hydroxy γ-trimethyl amino butyric acid. Carnitine acts as carrier, transporting long chain acyl CoA across inner mitochondrial membrane. This is essential for fatty acid oxidation.

Arginine

- It is glycogenic and semi-essential amino acid i.e. formed in amount not sufficient for body especially in children and pregnant women.

Functions of arginine: 1- Enter in urea formation. 2- Creatine formation. 3- Nitric oxide synthesis: L-Arginine serves as a precursor of nitric oxide (NO). NO is an intracellular signaling molecule and serves as neurotransmitter, smooth muscle relaxant and vasodilator.

H2N CH C

CH2

OH

O

CH2

CH2

NH

C

NH2

NH

PROTEIN METABOLISM

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Creatine: (methyl-guanidoacetate): - Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase enzyme using ATP as phosphate donor. This occurs in muscles. - Creatine phosphate acts as a store of high energy phosphate in muscles and used during muscle exercise as it can give phosphate to ADP to form ATP.

Creatine biosynthesis: - Creatine synthesized from 3 amino acid, glycine, arginine and methionine. This occurs in liver and kidney.

1- In kidney: The first reaction is transamidation by arginoglycine transamidase i.e. transfer of guanido group (H2N-[C=NH]-NH2) from arginine to glycine to form guanidoacetate.

2- In Liver: The second reaction is tranmethylation by guanidoacetate methyl transferase i.e. transfer of methyl group from S-adinosyl-methionine to form methyl guanidoacetate (creatine).

3- In muscle: Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase enzyme using ATP as phosphate donor. This occurs in muscles.

Degradation of creatine: - Creatine and creatine phosphate lose either water or phosphate molecule,respectively, to form a substance called creatinine (anhydrous creatine). - Creatinine is the end product of creatine metabolism and is normally rapidly removed from the blood and excreted by the kidney in urine.

Creatine Kinase enzyme (CK): Also called creatine phosphokinase (CPK). - This enzyme catalyzes the formation of creatine phosphate. - It is present in 3 forms (isoenzymes) in serum.

1) CK-MM: derived mainly from skeletal muscle and its serum level is elevated in muscle disease (muscle atrophy).

2) CK-MB: derived mainly from heart muscle and its serum level is elevated in recent myocardial infarction.

3) CK-BB: derived from brain and its serum level is elevated in brain damage.

PROTEIN METABOLISM

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H2N CHCCH2

OHO

CH2CH2NHCNH2

NH H2N CHCH

OHO

Arginine Glycine

+

Arginoglycinetransamidase

Ornithine COOHCH2NHCNH2

NH

Guanidoacetate

COOHCH2N-CH3CNH2

NH

Guanidoacetatemethyl transferase

COCH2N-CH3C NH

NH

Creatine

Creatinine

CO-PCH2N-CH3CNH2

NH

Creatine phosphate

ATPADP + H+

S-Adenosylmethionine

S-Adenosylhomocysteine

Creatine kinase

H2O

Pi

H2N CHCH2

COOH

NNH

H2N CH2CH2

NNH

Histidine decarboxylase

CO2PLP

Histidine Histamine

H2N CHCH2

COOH

NNH

Histidine

Histidine

- It is essential, glycogenic amino acid. - It is used for the synthesis of histamine.

Histamine: - Histamine is derived from histidine by Decarboxylation catalyzed by histidine decarboxykase.

- Functions of histamine: a) Vasodilator. b) Contraction of smooth muscle of bronchi. c) Stimulation of gastric secretion. d) Neurotransmitter.

PROTEIN METABOLISM

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CH2 - CH2 - CH - COOH

NH2S-CH3

CH2 - COOH

NH2

CH2 - CH - COOH CH2 - COOH

OH NH2 NH2

Serine hydroxymethyl transferase

Serine Glycine

Methionine

- Methionine is glycogenic, essential amino acid.

Functions: It enters in the synthesis of:1- S-adenosylmethionine (SAM): The main methyl donor used in transmethylation reactions.

2- Cysteine synthesis: - Through formation of homocysteine, this reacts with serine to give cysteine.

3- Lipotropic factor: - Methionine is one of lipotropic factors, which prevent fatty liver.

Glycine

- Glycine is non-essential glycogenic amino acid. Synthesis: - Glycine can be synthesized from serine by the enzyme serine hydroxymethyl transferase.

Functions: Glycine is the precursor of:

1- Purine bases: Carbon atoms No.4, 5 and nitrogen atom No. 7 are derived frm glycine.

2- Heme:-Heme is the pigment, which combines with globin protein to form hemoglobin. - Glycine reacts with succinyl CoA to form porphobilinogen in the presence of pyridoxal phosphate. Porphobilinogen is finally converted to heme.

3- Glutathione: (Glutamyl-Cysteinyl Glycine) - Glutathione is tripeptide formed of three amino acids: glutamate, cysteine and glycine. - It is synthesized in two steps catalyzed by glutamyl cysteine synthetase to form glutamyl cysteine then by glutathione synthetase forming glutathione.

Purine Nucleus

N

NNH

N 1

2

34

567

8

9

PROTEIN METABOLISM

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H2O2

2 H2O

2 GSH

GSSG

Glutathioneperoxidase

NADPH+H+

NADP+

GlutathioneReductase

CH2 - CH - COOH CH2 - COOH

OH NH2 NH2

Serine hydroxymethyl transferase

Serine Glycine

- Two forms are present: reduced (G-SH) and oxidized (G-S). The –SH group indicates the sulfhydryl group of the cysteine and it is the most active part of the molecule.

Functions of glutathione: a) Glutathione is an important defense mechanism against certain compounds, as some drugs and carcinogens, through conjugation with them to produce non-toxic compounds. b) Glutathione acts as antioxidant and breaks down the toxic hydrogen peroxide (H2O2).c) Glutathione acts as an activator for some enzymes. d) Glutathione is essential for maintaining red cell structure and keeping hemoglobin in ferrous state. e) Glutathione protects β-cell of pancreas against degenerative effect of alloxan(diabetogenic agent).

4- Creatine: (methylguanido acetic acid): See arginine.

5- Bile Salts: - These are sodium and potassium salts of glyocholic acid. - Glycine conjugates with cholic acid to form glyocholic acid.

6- Serine: (Reversible reaction)

7- Hippuric acid: - Glycine conjugates with the toxic benzoate to form the non-toxic hippuric acid. This occurs in liver

PROTEIN METABOLISM

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H2N CHCCH3

OHO

Alanine

+ O C CCH2

OHO

CH2COH

O

-Ketoglutarate

ALT/GPTPLP

O C CCH3

OHO

+ H2N CHCCH2

OHO

CH2COH

OPyruvic acid

Glutamic acid

COASH H2O

CO~SCOA

Glycine COASHATP ADP+Pi

CO-NH-CH2-COOHCOOH

Hippuric acid(Non-Toxic)

Benzoate(Toxic)

Benzoyl COA

Alanine

H2N CHCCH3

OHO

-Alanine

CH2 CCH2 NH2

OHO

CH2 CCH2 NH2

OHO

-Alanine

H2N CHCCH2

OHO

COH

O

Aspartate

Aspartate decarboxylase

CO2

CH2 - CH - COOH

OH NH2

Serine

Alanine and β-Alanine - It is non-essential glycogenic amino acid. Synthesis:

- Β-Alanine is synthesized from aspartic acid by decarboxylation.

Importance: - β-Alanine enter in synthesis of COASH and pantothenic acid. - Alanine is a major component of bacterial cell wall.

Serine- It is non-essential glycogenic amino acid.

Synthesis: - From glycine by hydroxymethyl transferase enzyme.

Importance: Serine enters in synthesis of: 1) Phosphoprotein by esterification. 2) Sphingosine base: Serine reacts with palmityl CoA to form sphingosine base. It enters in the structure of sphingomyelin.

PROTEIN METABOLISM

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+ O C CCH2

OHO

CH2COH

O

-Ketoglutarate

AST/GOTPLP

+ H2N CHCCH2

OHO

CH2COH

O

Glutamic acid

H2N CHCCH2

OHO

COH

O

O C CCH2

OHO

COH

O


H2N CHCCH2

OHO

OH

CH2-NH2CH2OH

Decarboxylase

CO2

EthanolamineSerine

Choline Acetyl CholineAcetylationTramsmethylation

H2N CHCCH2

OHO

OH

H2N CHCCH2

OHO

CH2HS

+ H2N CHCCH2

OHO

S CH2

CHNH2

CH2

COOH

CystathionineSynthetase

Vit.B6H2OSerine Homocysteine

Cystathionine

CystathionaseCH3CH2CCOOH

O+

H2N CHCCH2

OHO

SH

-ketobutyricacid

Cysteine

3) Purine base: β-carbon of serine enters in the formation of C2 and C8 of purine bases. 4) Ethanolamine and Choline synthesis:

5) Cysteine synthesis:

Aspartic acid

- It is non-essntial glycogenic amino acid.Sunthesis:

Functions:

1) Aspartate acts as neurotransmitter. 2) Aspartate shares in the synthesis of purine and pyrimidine. 3) Aspartate reacts with citrulline to form arginosuccinate in urea cycle. 4) Asparagine is formed from aspartate by asparagines synthetase which enters in synthesis of some hormones as oxytocin.

H2N CHCCH2

OHO

COH

O

PROTEIN METABOLISM

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+ O C CCH2

OHO

CH2COH

O

-Ketoglutarate

AST/GOTPLP

+ H2N CHCCH2

OHO

CH2COH

O

Glutamic acid

H2N CHCCH2

OHO

COH

O

O C CCH2

OHO

COH

O


H2N CHCCH2

OHO

CH2COH

O

+Glutamine synthetase

ATP ADP+PiH2N CHC

CH2

OHO

CH2CNH2

O

Glutamic acid Ammonia Glutamine

NH3

H2N CHCCH2

OHO

CH2COH

O

Glutamic acid

H2N CHCCH2

OHO

CH2COH

O

CH2CH2COH

O

CH2-NH2

Glutamate Gamma-aminobutyric acid (GABA)

Glutamate decarboxylasePLP

CO2

Glutamate

- It is non-essential amino acid glycogenic amino acid.

Synthesis:

Functions: 1) Acts as enzyme activator: N-acetylglutamate activates carbamoyl phosphate synthetase 1 enzyme (see urea biosynthesis). 2) Glutamate enters in synthesis of glutathione. 3) Glutamate acts as neurotransmitter. 4) Glutamate enters in the synthesis of glutamine by glutamine synthetase.

5) Glutamate enters in the biosynthesis of foilc acid. 6) Glutamate enters in synthesis of Gamma-aminobutyric acid (GABA), which is inhibitory transmitter in brain and spinal cord.

PROTEIN METABOLISM

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H2N CHCCH2

OHO

SH

Cysteine

NH

COOHNH

COOHHO

Proline Hydroxyproline

Cysteine- It is non-essential glycogenic amino acid.

Synthesis: See serine metabolism.

Functions: 1) Cysteine enters in synthesis of glutathione.2) Cysteine enters in synthesis of taurine, which combines with cholic acid toform taurcholic acid. Its sodium salt is one of bile salts.3) Cysteine enters in synthesis of thioethanolamine by Decarboxylation.Thioethanolamine enters in synthesis of COA and acyl-carrier protein (component of fatty acid synthetase enzyme). 4) Cysteine is very important amino acid in keratins formation. Keratins aresimple proteins that are present in hair, nail and skin. 5) Cysteine is important for the detoxification of some aromatic amino acidse.g. bromobenzene.

Proline and Hydroxyproline

- These are non-essential glycogenic amino acids.Synthesis: - From glutamate by oxidation to glutamate semialdehyde then to proline. Functions: - Proline and hydroxyproline enter in collagen synthesis.

PROTEIN METABOLISM

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Role of liver in protein metabolism:

1- Transamination, deamination and transdeamination occur in liver. 2- Synthesis of urea from ammonia. 3- Synthesis of amino acid derivatives as creatine, taurine. 4- Synthesis of plasma protein and coagulation factors. 5- Synthesis of non-essential amino acids. 6- Catabolism of carbon skeleton of amino acids and formation of glucose from glucogenic amino acids and ketone bodies from ketogenic ones. 7- Detoxification of many toxic compounds by conjugation with some amino acids as glycine and glutamine.

Role of Pyridoxal phosphate (PLP) in protein metabolism:

1- Play an important role in absorption of amino acids. 2- Transamination reactions:

Glutamate + Pyruvate → α-Ketoglutrate + Alanine. 3- Non-oxidative deamination:

Serine → Pyruvate. 4- Decarboxylation:

Glutamate → GABA. 5- Trans-sulfration:

Homocysteine + Serine → Homoserine + Cysteine. 6- Heme synthesis: (PLP acts as coenzyme for ALA synthase). 7- Nicotinic acid synthesis: (PLP acts as coenzyme for tryptophan oxygenase).

Hormonal regulation of protein metabolism:

1- Anabolic Hormones: Insulin, growth hormones and androgens are anabolic hormones stimulate protein biosynthesis.

2- Catabolic Hormones: Glucocorticoids are catabolic hormones inhibit protein biosynthesis and stimulate Gluconeogenesis.

3- Thyroid Hormones are anabolic in physiological levels and catabolic in elevated levels.

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LIPID METABOLISM - On average an adult human eats 100-150 grams lipids per day. The main lipids in diet are triglycerides (99%). Diet also contains some phospholipids, cholesterol and fat soluble vitamins.

Importance of lipids in diet: 1) Lipids are one of the main sources of energy in the body. 2) Lipids supply the body with essential fatty acids. 3) Lipids supply the body with fat soluble vitamins. 4) Lipids make the diet palatable.

Digestion of lipids

- Fats are essentially insoluble in the aqueous intestinal environment. - Solubilization (emulsification) of dietary lipid is accomplished via bile salts (to increase surface area of lipid droplet) that are synthesized in the liver and secreted from the gallbladder.

Digestion of Triglycerides:

- In infants: Triacylglycerols containing short or medium chain fatty acids (found in milk) can be degraded by gastric lipase. This enzyme acts only at neutral pH and is therefore of little function in adult stomach. - Triacylglycerols are digested by a group of enzymes. These are lingual, gastric, pancreatic and intestinal lipase enzymes. 1) Lingual lipase: a- Secreted by Ebner's glands on the dorsal surface of the tongue. b- Because foods remain for a short time in the mouth, digestion of triacylglycerols by lingual lipase is minimal. 2) Gastric lipase: a- Optimum pH for gastric lipase is 7. Thus it cannot act in adult stomach (pH 1-2). b- Gastric lipase may be of value in infants stomach (pH: 5). It acts on mother milk fat that contains triacylglycerols consisting of short or medium chain fatty acids can be degraded by gastric lipase. 3) Pancreatic lipase: a- It is the most important lipase in digestion of triacylglycerols.

DR. MOHAMED BADR

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b- It digests the primary ester bonds (position 1 and 3), hydrolyzing them into fatty acids and 2-monoacylglycerols. c- The resulting 2-monoacylglycerols will undergo: i- 72% are absorbed as such. ii- 28% are converted into 1-monoacylglycerols by isomerase enzyme which are then:

1- Absorbed as 1-monoacylglycerols (6%). 2- Hydrolyzed by intestinal lipase into glycerol and fatty acids (22%)

which are then absorbed. d- The presence of emulsifying agent as bile salts and phospholipids is important for action of pancreatic lipase. Emulsification means breakdown of large globules into small ones. This increase the surface area of lipids exposed to lipase enzyme.

4) Intestinal lipase: - It acts on 1-monoacylglycerols converting them into glycerol and free fatty acids. - Thus the end products of triacylglycerols are: a) 72%: 2-monoacylglycerols. b) 6%: 1-monoacylglycerols. c) 22%: Glycerol and fatty acids. Digestion of cholesterol:

- Cholesterol itself undergoes no digestion and absorbed as such. - Cholesteryl esters are digested by cholesterol esterase enzyme into cholesterol and fatty acids.

- Digestion of Phospholipids: - Phospholipids may be absorbed as such or digested by phospholipase enzymes (A1, A2 (B), C and D). - They act on phospholipids hydrolyzing them into fatty acids, glycerol, phosphate and nitrogenous base. - Phospholipase A2 (B) acts on the secondary ester linkage (position 2) releasing unsaturated fatty acid leaving lysophosholipids. - Phospholipase A1 acts on the primary ester linkage (position 1) releasing saturated fatty acid leaving glyceryl phosphoryl base. - Phospholipase C hydrolyzes the glycerol part leaving phosphorylated nitrogenous base.

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Intestinal Wall

Liver GlycerolPortal circulation

Short chain FA

Lumen

Glycerol

Short chain FA

Triacylglycerols

Chylomicrones

Protein

SystimicCirculation

Thoracic D

uct

Bile salts

Monoacylglycerol

Long chain FA

Cholesterol

Phospholipids

Micelles

+

+

+

+

Bile salts

Monoacylglycerol

Long chain FA

Cholesterol

Phospholipids

- Phospholipase D separates the phosphate part from the base.

Absorption of lipids

- The end products of lipid metabolism are monoacylglycerols, fatty acids [short chains (C4-C10) and long chains (C12-C18)], glycerol, phospholipids and cholesterol. They are absorbed from the jejunum and ileum. - Short chain fatty acids (less than 12 carbons) and glycerol are water-soluble and pass via portal circulation to the liver. - Other lipids are water insoluble. They combine with bile salts to form water-soluble complex called micelles, which enter the mucosal cell. - Bile salts are reabsorbed to the liver again (enterohepatic circulation). - Long chain fatty acids are activated in the mucosal cells and combine with mono- and diacylglycerols to form triacylglycerols again. - Triacylglycerols, phospholipids and cholesterol are bound to a protein called apolipoprotein B to form chylomicrons which enter the circulation through lymphatic vessels. - In the blood; chylomicrons are bound with other 2 proteins (apolipoprotein E and C).

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Errors of lipids digestion and absorption:

1) Steatorrhoea: - It is a condition in which the fat content of the stool is abnormally increased. Normally it is less than 5 grams per day.

- Causes: Steatorrhoea results from deficiency of any factor essential for digestion or absorption of lipids as pancreatic lipase, bile salts or healthy intestinal mucosa.

2) Chyluria (milky urine): - It is the presence of fat (chylomicrons) in urine after fatty meals.

- Cause: It is due to abnormal connection between the lymphatic drainage of the intestine and the urinary system.

Fate of absorbed lipids:- After fatty meal, plasma shows a milky appearance. This due to venous blood contains excess chylomicrons after absorption. - Excess chylomicrons stimulate mast cells to produce heparin that stimulate the lining epithelium of blood vessels to produce an enzyme called lipoprotein lipase (plasma clearing factor). - Lipoprotein lipase enzyme will act on triacylglycerols of chylomicrons, converting them into glycerol and free fatty acids.

Triacylglycerol Glycerol + Fatty acidsLipoprotein lipase

- Glycerol and fatty acids are taken up by different tissues for the following functions:

1) Formation of depot fat: a- It is formed mainly of triacylglycerols. b- It is present in fat cells of adipose tissue.

2) Oxidation for production of energy: a- Fatty acids: Converted into acetyl CoA that is oxidized in citric acid cycle. b- Glycerol: Converted into glycerol-3-phosphate (by glycerol kinase) that converted into dihydroxyacetone phosphate. The latter is undergoing oxidation in glycolysis.

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3) Glucose formation by gluconeogenesis: a- Glycerol → Glucose. b- Odd number fatty acid oxidation → propionyl CoA → Glucose

4) Synthesis of biologically active compounds:e.g. different steroids and eicosanoids (prostaglandins, prostacyclin, thromboxanes and leuckotriens).

5) Synthesis of tissue fats (structural fats): - They enter in the cellular structure. They include phospholipids and Cholesteryl ester.

Storage and mobilization of lipids

I- Types of body lipids: - Body lipids are of 2 types: A) Tissue lipids. B) Adipose tissue (depot fat).

A) Tissue lipids: 1- These lipids enter in the structure of body cells as cell membrane and mitochondria. 2- Tissue lipids never oxidized to give energy.

B) Adipose tissue (depot fat): It is of 2 types white and brown.

1- White adipose tissue: A- Composition: - Triacylglycerols: The main content. They contain saturated and unsaturated fatty acids. - Little phospholipids and cholesterol.

B- Site: 1) Under skin and breast. 2) Around important organs e.g. kidney.

C- Sources: 1) Absorbed fat.

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R-COOHFatty acid

Thiokinase

ATP +CoASH

AMP + PPiH2O

OR-C-CoAAcyl CoA

CH2OHHO - C - H

CH2 - O - P - O-

O-

O

Glycerol phosphate

CoA - C - R1

O

CoA

Acyltransfearase

CH2 - O - P - O-HO - C - H

CH2 - O - C R1

O-

O

Lysophosphatidis acid

O

CoA - C - R2

O

CoA

Acyltransfearase

CH2 - O - P - O-R2 - C - O - C - H

CH2 - O - C R1

O-

O

OO

Phosphatidic acid

H2O

Pi

Phosphatase

CH2OHR2 - C - O - C - H

CH2 - O - C R1

O

O

Diacylglycerol

CoA - C - R3

O

CoA

Acyltransfearase

R2 - C - O - C - HCH2 - O - C R1

O

O

TriacylglycerolCH2 - O - C - R3

O

Synthesis of triacylglycerol

2) Carbohydrate (by lipogenesis).

D- Functions: Depot fat is important for:

1) Energy production: During fasting, the triacylglycerols stored in depot fat provide the body with free fatty acids which oxidized in tricarboxylic acid cycle and give energy.

2) Fixation of some organs e.g. kidney.

3) Heat insulator around the body.

4) Production of vitamin D3: Exposure of skin to ultraviolet rays of sun where 7-dehydrocholesterol, present in depot fat, converted into vitamin D3.

2- Brown adipose tissue: a- Certain areas of adipose tissue appear brown in color as they contain high content of mitochondria, cytochromes and well developed blood supply.

b- The brown fat may have a special function in the production of heat.

- Two processes control the amount of triacylglycerols in depot fat: Lipogenesis and lipolysis.

I- Lipogenesis

A- Definition:- Lipogenesis is the synthesis of triacylglycerol from fatty acids (acyl CoA) and glycerol (glycerol-3-phosphate).

B- Steps: 1- Activation of fatty acids into acyl CoA:

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Glycerol + ATP Glycerokinase Glycerol phosphate + ADP

2- Synthesis of glycerol-3- phosphate: a- In liver, kidney, intestine, and lactating mammary glands: Glycerol-3-phosphate is formed from glycerol by glycerokinase or from glucose through glycolysis.

b- In muscles and adipose tissue, glycerokinase is deficient. In these tissues glycerol-3-phosphate is formed from glucose (through glycolysis) as follows:

Glucose → Dihydroxyacetone Phosphate → Glycerol phosphate

C- Regulation of lipogenesis:

1- After meal, lipogenesis is stimulated: - Insulin is secreted which stimulates glycolysis. Glycolysis supplies dihydroxyacetone phosphate that converted into glycerol-3-phosphate in adipose tissue, so lipogenesis is stimulated.

2- During fasting lipogenesis is inhibited: - Anti-insulin hormones are secreted. These inhibit lipogenesis and stimulate lipolysis.

II- Lipolysis

A- Definition:- Lipolysis is the hydrolysis of triacylglycerols in adipose tissue into glycerol and fatty acids.

B- Steps:- Lipolysis is carried out by a number of lipase enzymes, which are present in adipose tissue. These are: 1. Hormone sensitive triacylglycerol lipase. 2. Diacylglycerol lipase. 3. Monoacylglycerol lipase.

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Adipose Tissue

Lipolysis Estrification

TG

Glucerol Acyl CoA Glycerol-3-p

ATP

ADP

Glycerokinase

Glycerol-3-p

Dihydroxyacetone-p

Glycolsis

Glucose

PlasmaFFA

AlbuminLiverKidney

Tissues

FFA

Triacylglycerol

Hormone-sensitivetriacylglycerol lipase

diacylglycerol + free fatty acids

Diacylglycerol lipase

Glycerol +free fatty acids

Monoacylglycerollipase monoacylglycerols + free fatty acids

C- Fate of products of lipolysis:

1- Fate of fatty acids: a- Oxidation by tissues to give of energy.

b- Fatty acids may remain in adipose tissue to be re-esterified into triacylglycerols again.

2- Fate of glycerol: - Glycerol may diffuse to blood and then taken up by the liver to give: a- Glucose by gluconeogenesis. b- Pyruvate by glycolysis. c- Triacylglycerols by lipogenesis.

- Note: In adipose tissue, glycerokinase enzyme is deficient, which is essential to convert glycerol into glycerol phosphate. Thus glycerol in adipose tissue cannot be used in re-esterification of fatty acids to form triacylglycerols.

DR. MOHAMED BADR

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D- Regulation of lipolysis:- The key enzyme controlling lipolysis is the hormone sensitive triacylglycerol lipase enzyme. It exists in 2 forms: active (phosphorylated) and inactive (dephosphoryalted).

1. During fasting:

a- Many hormones (epinephrine, norepinephrine, glucagon, ACTH and TSH) are secreted. They stimulate adenylate cyclase enzyme →formation of cAMP → activation of protein kinase enzyme →phosphorylation of hormone sensitive triacylglycerol lipase →stimulation of lipolysis.

b- Prostaglandin E and nicotinic acid inhibit adenylate cyclase →inhibition of lipolysis.

H2OPi

ATP ADP

Protein Kinase

cAMP

(+)

Adenylate cyclase

PPi

EpinephrineNor-EpinephrineGlucagonThyroid H.Glucocorticoids H.

(+)

Phosphodiesterase5/-AMPATP

HS Lipase (b)(Inactive) HS Lipase (a)

(Active)

P

Rregulation of Lipolysis

Lipase phosphatase

phosphorylase

Insulin(+)

Stimulate Lipolysis

(-)Prostaglandin ENicotinic acid

(-)Caffeine

Insulin

(+)

DR. MOHAMED BADR

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2. After meal: a- Insulin is secreted → stimulation of both phosphodiesterse enzyme and lipase phosphatase enzyme → dephosphorylation and inactivation of hormone sensitive triacylglycerol lipase enzyme → inhibition of lipolysis.

b- Caffeine is a substance present in coffee and tea. It inhibits phosphodiesterase enzyme → stimulation of lipolysis.

E. Causes of excessive lipolysis:- In conditions where the need for energy is increased e.g.: 1- Starvation. 2- Diabetes mellitus. 3- Low carbohydrate diet.

Fatty acid oxidation

- Among the different foodstuffs, lipids give the maximum amount of energy.

I) β-Oxidation: - It is the major catabolic pathway of fatty acids in mitochondria in which 2-carbon fragments are successively removed from the carboxyl end of the fatty acyl-CoA, producing acetyl-CoA. - This process of fatty acid oxidation is called β-oxidation because all the reactions involve the β-carbon.

A- Site: 1- Intracellular location: Mitochondria. 2- Organ location: a- Liver, kidney and heart. b- β-Oxidation never occur in brain.

B- Activation and transport of fatty acids into the mitochondria: 1- Fatty acids must be activated in the cytoplasm before being oxidized in the mitochondria. Activation is catalyzed by acyl CoA synthetase (Fatty acid thiokinase). 2- The net result of this activation process is the consumption of 2 molar equivalents of ATP.

Fatty acid + ATP Acyl CoA + AMP + PPiAcyl CoA synthetase

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3- Because the β-oxidation occurs in the mitochondrial matrix, the acyl CoA must be transported across the mitochondrial inner membrane, which is impermeable to acyl CoA. Therefore, a specialized carrier present in the inner mitochondrial membrane called carnitine transports the acyl CoA from cytosol into the mitochondrial matrix. The transport process in is called carnitine shuttle.

4- Carnitine shuttle: a- Structure of carnitine: - Carnitine is β-hydroxy-γ-trimethyl ammonium butyric acid.

b- Function of carnitine: - It transports long chain acyl CoA inside the mitochondrial matrix where enzymes for β-oxidation are present.

c- Steps of shuttle: Three enzymes are involved:

1) Carnitine acyl transferase I: (i) For palmitic and, it is called carnitine palmitoyl transferase I. (ii) It is present in the outer mitochondrial membrane. (iii) It transfers the acyl group from acyl CoA to carnitine to form acyl carnitine. 2) Carnitine acylcarnitine translocase:(i) It is present in the inner mitochondrial membrane. (ii) It transports acyl carnitine across inner mitochondrial membrane (in exchange with carnitine).

3) Carnitine acyl transferase II: (i) For palmitic acid, it is called carnitine palmitoyl transferase II. (ii) It is present in the inner mitochondrial membrane. (iii) It transfers the acyl group from acyl carnitine to from acyl CoA again.

CH2-CH-CH2-COOHN+ OH

(CH3) Carnitine

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d- For short chain fatty acids e.g. acetyl CoA, another enzyme called carnitine acetyl transferase is used. C- Steps of β-Oxidation: 1- Activation of fatty acid:

a- Catalyzed by acyl CoA synthetase enzyme. b- 2 high energy bonds (~P) are utilized: ATP → AMP + PPi.2- Unsaturation of fatty acids (First oxidation):

a- Catalyzed by acyl CoA dehydrogenase enzyme. b- This enzyme is one of flavoproteins and its coenzyme is FADH2 which gives 2 ATP when oxidized in the respiratory chain. 3- Hydration:

a- Catalyzed by enoyl CoA hydratase enzyme. b- It helps the addition of water to saturate double bonds. 4- Oxidation (Second oxidation):

a- Catalyzed by β-hydroxy acyl CoA dehydrogenase enzyme.

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R - CH2 - CH2 - C - OHFatty acid

CoA.SH ATP

O

Acyl CoA synthetase

H2O AMP + PPi

1Activation

R - CH2 - CH2 - C ~ S - CoAAcyl - CoA (Active fatty acid)

O

Inner mitochodrial membrane Carnitine transporter

R - CH2 - CH2 - C ~ S - CoAAcyl - CoA

O

Acyl CoAdehydrogenasee

2Unsaturation

FP

FPH2Respiratory chain 2ATP

R - CH = CH - C ~ S - CoAUnsaturated acyl CoA

O

Enoyl CoAhydratase

H2O 3Hydration

R - CH - CH2 - C ~ S - CoA-Hydroxy acyl ~ CoA

OOH

-Hydroxyacyl -CoA Dehydrogenase

NADH + H+

NAD+4

-Oxidation

Respiratory chain 3ATP

R - C - CH2 - C ~ S - CoA-Ketoacyl ~ CoA

OO

CoA. SHThiolase5

Splitting

R - C ~ S - CoA + CH3 - C ~ S - CoAO O

Acyl - CoA Acetyl - CoA

Citricacidcycle CO2

Rep

eat t

he st

eps o

f oxi

datio

n be

gini

ng fr

om re

actio

n (2

) los

ing

each

tim

2 ca

rbon

ato

ms (

in th

e for

m o

f ace

tyl C

oA)

b- Its coenzyme is NADH+H+ which gives 3 ATP when oxidized in the respiratory chain.

5- Splitting (Cleavage) step:

a- Catalyzed by thiolase enzyme. b- It splits acyl CoA into acetyl CoA and acyl CoA (shorter than the original one by 2 carbon atoms) which repeat the cycle again and again until complete oxidation of acyl CoA into acetyl CoA. c- The acetyl CoA, the end product of each round of β-oxidation enters the TCA cycle to produce energy and CO2.

DR. MOHAMED BADR

87CH3 - C - CH2 - C ~ S - CoA Acetoacetyl CoA

O O

D- Energy production of β-Oxidation:

1- Calculation of energy production of oxidation of palmitic acid (16 carbons): a- β-Oxidation of palmitic acid will be repeated 7 times (turns) to produce 8 acetyl CoA.

b- In each turn, one molecule of reduced FADH2 and one molecule of reduced NADH+H+ are produced. They are oxidized in respiratory chain to give 5 ATP.

- FADH2 → 2 ATP- NADH+H+ → 3 ATP

.˙. 7 turns x 5 ATP → 35 ATP.

c- Oxidation of one molecule of acetyl CoA in citric acid cycle gives 12 ATP.

.˙. 8 Acetyl CoA x 12 ATP = 96 ATP.

d- Tow high energy phosphate bonds are utilized in the first reaction (catalyzed by acyl CoA synthetase) which occurs for one time only.

e- .˙. Net energy gain = Energy produced – Energy utilized = (35 ATP + 96 ATP) – 2 ATP = 131 ATP – 2 ATP = 129 ATP

2- Calculation of energy production of oxidation of any fatty acid:

= {(N/2 – 1) X 5 ATP} + {N/2 X 12 ATP} – 2 ATP

Where N= Number of carbons of fatty acid e.g. palmitic acid = 16 carbons, so energy production = = {(16/2 – 1) x 5 ATP} + {16/2 X 12 ATP} – 2 ATP= {(8 – 1} X 5 ATP} + {8 X 12 ATP} – 2 ATP = 129 ATPE- Importance (functions) of β-Oxidation: 1- Energy production e.g. palmitic acid produces 129 ATP. 2- Production of acetyl CoA which enter in many pathways. 3- Ketone bodies formation: Acetoacetyl CoA is the last 4 carbons product in the course of β-Oxidation of any fatty acid. It may be converted into acetoacetate; one of ketone bodies.

DR. MOHAMED BADR

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F- β-Oxidation of odd number fatty acids:1- They are oxidized by β-Oxidation until a propionyl CoA (3 carbons) is produced. Then propionyl CoA is converted to succinyl CoA as shown in the figure.

2- Sources and fate of succinyl CoA:a- Sources: 1) Oxidation of odd number fatty acids. 2) Citric acid cycle. 3) Catabolism of some amino acids e.g. leucine, valine and methionine.

b- Fates: 1) Glucose synthesis (gluconeogenesis). 2) Heme synthesis. 3) Oxidation in citric acid cycle. 4) Activation of ketone bodies. 5) Detoxication.

Sources and fate of active acetate (Acetyl CoA) I- Sources:

A- Lipids: Oxidation of fatty acids.

B- Carbohydrate: Glucose oxidation → Pyruvate → Acetyl CoA.

C- Proteins: 1- Ketogenic amino acids: Give directly active acetate or acetoacetate → active acetate. 2- Glucogenic amino acids → Pyruvate → Active acetate.II- Fate: A- Oxidation: Through tricarboxylic acid cycle.B- Lipogenesis: Formation of fatty acids.

FA with odd number carbon atoms

Acetyl CoA(s)

CH3 - CH2 - C ~ S - CoA

O

Propionyl CoAATP

ADP + Pi

Propionyl CoA Carboxylase* CO2

Biotin

* COOH O

CH3 - CH - C ~ S - CoAMethyl malonyl CoA

Isomerase

HOOC - CH2 - CH2 - C ~ S - CoA

O

Succinyl CoA

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Protein

Glucogenic Ketogenica.a.

Pyruvate

Acetoacetate

Glucose Fatty acid

Pyruvate

Acetyl CoA

Sources:

Fate:

Oxidation Synthesis ofFA.

Synthesis ofKetone bodies

Synthesis ofcholesterol

Synthesis ofacetyl choline

Krebscycle

Bile acids Vit. D3 Steroid hormones

Site of formatin:

Mitochondria.

Note: Acetyl CoA used for fatty acid synthesis always derived from glucose and never from fatty acids. This because insulin hormone secreted after meal stimulates both glucose oxidation (→ acetyl CoA) and lipogenesis (= Fatty acid synthesis) and inhibits lipolysis (→ Fatty acid oxidation → Acetyl CoA).

C- Ketogenesis: Synthesis of ketone bodies.

D- Acetylcholine synthesis.

E- Cholesterol synthesis: which is the precursor of: 1- Bile acids. 2- Vitamin D3. 3- Steroid hormones: glucocorticoids, mineralocorticoids, male sex hormones (testosterone) and female sax hormone (estrogens and progesterone).

DR. MOHAMED BADR

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Lipogenesis

- Fatty acids synthesis: - A considerable number of fatty acids are derived from diet. Living organisms have the capacity to synthesize fatty acids from acetyl CoA. Any substance can give acetyl CoA (e.g. glucose) is called lipogenic.- There are 3 mechanisms for fatty acids synthesis: Cytoplasmic, mitochondrial and microsomal.

I- Cytoplasmic system for fatty acid synthesis (de novo synthesis of fatty acids): - Also called extramitochondrial system. The main product of this pathway is palmitate (16C).

A- Site: 1- Intracellular location: Cytoplasm. 2- Organ location: Many tissues including liver, adipose tissue, mammary gland, lung and kidney.

B- Requirements: - This pathway needs the following substrates, acetyl CoA, NADPH+H+

and group of enzymes called collectively fatty acid synthase complex.

1- Acetyl CoA: a- It is provided mainly by glucose through pyruvate. b- Acetyl CoA is formed in mitochondria and fatty acid synthesis occurs in cytoplasm. The acetyl CoA cannot diffuse to cytoplasm because mitochondrial membrane is impermeable to it. c- Acetyl CoA condenses with oxaloacetate, in the presence of citrate synthase to form citrate. Then citrate diffuses out of mitochondria to cytoplasm where it is splitted again, by citrate lyase, into acetyl CoA and oxaloacetate. 1) Acetyl CoA molecules are used for palmitate synthesis. 2) Oxaloacetate is converted to malate: (i) Malate may be exchanged with citrate to enhance fatty acid synthesis. (ii) Malate may be converted to pyruvate by malic enzyme. This reaction produces NADPH+H+, which is needed for fatty acid synthesis.

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Fatty acid synthesis

Glucose

Acetyl CoA NADPH + H+Malic enzyme

Pyruvate

Malate

MalateDehydrogenase

Oxaloacetate

Isocitrate DH

NADP+ NADPH + H+

-KetoglutarateIsocitrateCitrate

ATP-Citrate lyase

Cytosol

Mitochondria

Malate

Citrate

Citrate synthase

Acetyl CoA Oxaloacetate

Pyruvate

Pyruvate

Glycolysis

PDH

HO - CH - COOHCH - *COOH + NADP+ CH3 - C - COOH + *CO2 + NADPH + H+Malic enzyme

Malate Pyruvate

O

2- NADPH+H+: It is provided by: a- Hexose monophosphate pathway (HMP shunt). b- Action of malic enzyme on malate.

c- Action of cytoplasmic isocitrate dehydrogenase on isocitrate. It is similar to mitochondrial one but it used NADP+ as hydrogen carrier.

Isocitrate + NADP+ → α-Ketoglutrate + NADPH+H+

3- Fatty acid synthase complex:

a- This enzyme is a dimer i.e. formed of 2 subunits. b- Each unit, which is called monomer, contains 7 enzymes and a terminal protein called acyl carrier protein (ACP).

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CH3 - C ~ S - CoA + ATP + CO2 HOOC - CH2 - C ~ S - CoA + ADP + PiAcetyl CoA carboxylase

Acetyl CoA Malonyl CoA

OO

Biotin

8 Acetyl ~ CoA

7 ATP 7 CO2

Acetyl ~ CoACarboxylase

7 ADP + 7 Pi

Acetyl ~ CoA + 7 Malonyl ~ CoA

14 NADPH3H+

7 cyclesMultienzymecomplex

14 NADP+ + 7 CO2 + 8 CoA - SH

CH3(CH2)14COOHPalmitate

c- ACP is a protein contains the vitamin pantothenic acid in the form of phosphopantotheine. ACP is the part that carries the acyl group. d- Each monomer contains 2-SH groups, one provided by phosphopantotheine and attached to ACP. The other is provided by cycteine and attached to the enzyme 3-ketoacyl synthase. e- The 2 monomers are arranged head to tail, so the -SH group of ACP of one monomer is very close to the -SH group provided by 3-ketoacyl synthase of other the monomer.

C- Steps of cytoplasmic pathway:

1- Carboxylation of acetyl CoA to form malonyl CoA: a- Malonyl CoA is synthesized from acetyl CoA by acetyl CoA carboxylase in the presence of biotin and ATP. b- It is the rate limiting step in the pathway.

2- Synthesis of palmitate:

DR. MOHAMED BADR

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CO2

Acetyl - CoA Malonyl - CoAAcetyl - CoAcarboxylase

HS - Pan Cys - SHAcetyl

transacylaseCoA

Malonyltransacylase

CoA

Cn transfer fromHS - Cys Pan - SH

Fatty acid synthasemultienzyme complex

1

2

NADPHGENERATORS

1- HMP Shunt2- Isocitrate DH.3- Malic enzyme

OCys - S ~ C - CH31

2O

Pan - S ~ C - CH2 - COOHAcyl (acetyl) - malonyl enzyme

2 1to

-Ketoacylsynthase

CO2

Cys - SH1

2O

Pan - S ~ C - CH2 - C - CH3

O

-Ketoacyl enzyme(acetoacetyl enzyme)

-Ketoacylreductase

NADPH + H+

NADP+

Cys - SH1

2O

Pan - S ~ C - CH2 - CH - CH3

OH

-Hydroxyacyl enzyme

HydrataseH2O

Cys - SH1

2O

Pan - S ~ C - CH = CH - CH3

Unsaturated acyl enzymeNADPH + H+

NADP+ Enoyl reductase

Cys - SH1

2O

Pan - S ~ C - CH2 - CH2 - CH3 (Cn)Acyl enzyme

Palmitate

After cycling through

Steps Seven times1 4

ThioesteraseH2O

2

1

3

4

DR. MOHAMED BADR

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D- Comments on synthesis of plamitate:

1- A molecule of malonyl CoA combines with a pantotheine-SH group of the first monomer. This step is catalyzed by malonyl transacylase enzyme.

2- Also a molecule of acetyl CoA combines with a cysteine-SH group of the other monomer of fatty acid synthase, to form acetyl (acyl)-malonyl-enzyme. This step is catalyzed by acetyl (acyl) transacylase enzyme.

3- Then acetyl group attacks the malonyl group to liberate CO2 and to form 3 ketoacyl enzyme (acetoacetyl enzyme) that attached to pantotheine-SH group and to let the cysteine-SH group free.

4- The 3-ketoacyl group is reduced, dehydrated and then reduced again to form the corresponding acyl-enzyme.

5- The sequence of reactions is then repeated 6 more times to produce fatty acids of 6, 8, 10, 12, 14 and finally 16 carbons (palmitic acid).

6- Free palmitate is formed by the action of thioesterase enzyme of the multienzyme complex, which adds H2O and hydrolyzes the palmitoyl enzyme. 7- The overall reaction is as follows: E- Fate of palmitate:

1- Esterification: With glycerol to from acylglycerols or with cholesterol to form cholesteryl ester.

2- Chain elongation: Palmitate may be elongated to form a longer fatty acid.

3- Desaturation: i.e. synthesis of unsaturated fatty acid: Stearic acid (18 carbons) is derived from elongation of palmitate may undergo desaturation at C9-C10 to form oleic acid (unsaturated fatty acid).

4- Sphingosine formation: It is formed from palmityl CoA and the amino acid serine.

F- Regulation of fatty acid synthesis: - The rate limiting reaction in the cytoplasmic pathway is that catalyzed by the acetyl CoA carboxylase enzyme.

1- Long chain acyl CoA: Inhibits fatty acid synthesis through: a- It inhibits allosterically acetyl CoA carboxylase.

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Glucose

Pyruvate

TriacylglycerolLipolysis

Acyl CoA Glycerol Malonyl CoA

Acetyl CoACarboxylase

Acetyl CoACitrate

Mitochondria

Citrate

Citrate synthase

OxaloacetateAcetyl CoA

PHDH

Pyruvate

b- It inhibits transport of citrate from cytosol to mitochondria.

c- It inhibits pyruvate dehydrogenase (PDH) that synthesizes Pyruvate → Acetyl CoA → Fatty acid.

2- Insulin: Stimulates fatty acid synthesis through: a- Insulin stimulates transport of glucose into cells e.g. adipose tissue. This increases the amount of pyruvate → acetyl CoA → fatty acid synthesis.

b- Insulin activates both pyruvate dehydrogenase complex and acetyl CoA carboxylase.

c- Insulin inhibits lipolysis through inhibition of cAMP → No long chain fatty acids that inhibit lipogenesis.

II- Microsomal pathway for fatty acid synthesis:

III- Mitochondrial pathway for fatty acid synthesis:

A- This pathway occurs in mitochondria under anaerobic conditions i.e. in absence of oxygen, by elongation of acyl CoA.

IV- Synthesis of unsaturated fatty acids:

A- Nonessential unsaturated fatty acids: - These are fatty acids which contain one double bond e.g. palmitoleic acid (16:1) and oleic acid (18:1).

DR. MOHAMED BADR

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- Synthesis of oleic acid (oleyl CoA): - It is synthesized – in the microsome – from stearyl CoA (active

stearic acid).

B- Essential fatty acid: - These are unsaturated fatty acids which contain more then one double bond. - They are essential because they cannot be formed in the body and should be taken in the diet e.g. linoleic acid (18:2), linolenic (18:3) and arachidonic acid (20:4).

- Sources of essential fatty acids: - Vegetable oils as corn oil and olive oil.

- Functions of essential fatty acids: a- They are important for normal growth.

b- They enter in the structure of phospholipids mainly in the second position. Phospholipids have many functions as: 1- They enter in the structure of cell membranes. 2- They act as lipotropic factors i.e. prevent accumulation of fat in liver. 3- Dipalmityl lecithin acts as surfactant in lungs. 4- Cephalin is important for coagulation.

Stearyl - CoA + enzymeAcyl transferase

CoA-SH

Stearyl - Enz

HydroxylaseO2 + NADPH + H+

NADP+ + H2OHydroxy stearyl - Enz

HydrataseH2O

Oleyl - Enz

Acyl transferase

Oleyl - CoA + Enzyme

Microsomal synthesis of oleyl CoA

CoASH

DR. MOHAMED BADR

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c- Essential fatty acids combine with cholesterol forming esters, which are rapidly metabolized by the liver. This prevents precipitation of free cholesterol along the endothelium of blood vessels → prevents atherosclerosis.

d- Linoleic , linolenic and arachidonic acids give rise to a group of compounds called eicosanoids. They include the prostanoids, leukotrienes and lipoxins. Prostanoids include prostaglandins, prostacyclin and thromboxane.

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CH3 - C - CH2 - COOHO

CH3 - CH - CH2 - COOHOH

CH3 - C - CH3

O

Ketone bodies

- These are 3 compounds formed by the liver and include: 1- Acetoacetate.

2- β-Hydroxybutyrate.

3- Acetone.

- Functions (Importance) of ketone bodies:

A- Ketone bodies are used as a source of energy. They are converted into acetyl-CoA which is oxidized in tricarboxylic acid (TCA) cycle.

B- In prolonged fasting and starvation, ketone bodies can be used as a source of energy by most of tissues including skeletal muscles, cardiac muscles and kidneys.

C- Liver does not contain enzymes for ketone bodies oxidation (Ketolysis). Thus liver cannot oxidize them.

- Synthesis of ketone bodies (ketogenesis): A- Site: 1- Organ location: Liver 2- Intracellular location: Mitochondria.

B- Precursor: - Acetyl CoA (derived from fatty acids oxidation and ketogenic amino acids).

C- Steps: - Acetoacetate is the first ketone body produced. Then both β-hydroxy butyrate and acetone are derived from it.

1-Formation of acetoacetyl CoA: a- From condensation of 2 acetyl CoA molecules. b- It results in the course of β-oxidation of fatty acids (last 4 carbons).

DR. MOHAMED BADR

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2- Formation of HMG-CoA (β-hydroxyl methyl glutryl CoA):- By condensation of third molecule of acetyl CoA in the presence of HMG CoA synthase.

3- Formation of acetoacetate by HMG CoA lyase. - Acetoacetate is either: a- Spontaneously decarboxylated into acetone. b- Reduced by hydroxybutyrate dehydroganase into β-hydroxybutyrate.

Fatty acyl CoA2CH3C - CoA

O

2 Acetyl CoA

CH3C - CH2 - C - CoAAcetoacetyl CoA

CH3C - CoAAcetyl CoA

CoA H

HOHHMG CoA

synthase

HO - C - CH2 - C - CH2 - C CoA

HMG CoA

HMG CoA lyase CH3C - CoA

CH3C - CH2 - C OH

Acetoacetate3-Hydtoxybutyratedehydrogenase

Spontaneous

NADH + H+

NAD+

CH3 - C - CH3 CH3 - C - CH2 - C - OH

3-HydroxybutyrateAcetone

Synthesis of ketone bodies. HMG =hydroxymethylglutaryl CoA.

O O

O

O OOH

CH3

O

O O

CO2

O O

OH

H

DR. MOHAMED BADR

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D- Conditions that increase ketogenesis: 1- Fasting and starvation. 2- Carbohydrate poor diet. 3- High fat diet. 4- Diabetes mellitus.

E- Regulation of ketogenesis:

1- Conditions causing excessive lipolysis in adipose tissue leads to very high level of plasma free fatty acids (FFA).

2- The liver in both fed and fasting states has the ability to extract 30% of the free fatty acids passing through it. Thus at high concentration of plasma FFA, the amount extracted by the liver is increased.

3- In Liver, FFA is activated to acyl CoA which will take one of tow pathways: a- Estrifiication to form acylglycerols or phospholipids. b- β-oxidation to give acetyl CoA.

4- Acetyl CoA is oxidized in citric acid cycle, but if the amount of acetyl CoA molecules is more then the capacity of TCA, they are used to form ketone bodies.

5- After meal, insulin is secreted. This inhibits lipolysis and in turn inhibits ketogenesis.

6- During fasting anti-insulin hormones are secreted e.g. glucagon. This stimulates lipolysis and in turns ketogenesis.

Regulation of Ketogenesis Adipose tissue Blood Liver

TGlLipolysis

FFA

Insulin Glucagon

FFA → Acyl CoA TGEstrification

-Oxidation

Acetyl CoA Citric acid cyclyKetogenesis

Ketone bodies

DR. MOHAMED BADR

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Amino acidCatabollsm

Fatty acidOxidation

Glycolysis

2 Acetyl CoA

CoAAcetoacety CoA

Acetyl CoA

CoA

-Hydroxy - -methylglutaryl CoA (HMG CoA)

Acetyl CoACO2

AcetoacetateAcetone

-Hydroxybutyrate

NADH + H+

NAD+

Peripheral Tissues(e.g. muscle)

Acetoacetyl CoA 2 Acetyl CoA

Thlophorase

Succlnate

Succlnyl CoAAcetoscetate

-Hydroxybutyrate

TGA Cycle

NAD+NADH

Acetoacetate

-Hydroxybutyrate

Liver

Blood

Oxidation of ketone bodies (ketolysis)

A- Site: Extrahepatic tissue because liver does not contain enzymes for ketolysis. Mitochondria are the site for ketolysis. B- Steps: 1- Acetone is volatile and removed in the expired air.

2- β-hydroxybutyrate is converted into acetoacetate by hydroxybutyrate dehydrogenase enzyme.

3- Acetoacetate is then converted into acetoacetyl CoA by: a- Thiophorase enzyme in the presence of succinyl CoA. b- Acetoacetate thiokinase (synthetase) enzyme.

4- Acetoacetyl CoA is splitted into 2 molecules of acetyl CoA which are oxidized in citric acid cycle.

- Blood ketone bodies and ketosis:

A- Blood ketone bodies concentration is less than 3 mg/dl.

B- Ketonemia: Is the increase of blood ketone bodies above normal concentration.

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CH3 - C - CH2 COOH + NAHCO3 CH3 - CH3 - C - CH2 - COONa + H2CO3

Acetoacetate Sodiumbicarbonate

Sodium aceto -acetare

Carbonicacid

CO2

H2O

O O

1- It occurs when the rate of formation of ketone bodies (ketogenesis) is grater then the rate of their oxidation (ketolysis). 2- The concentration of blood ketone bodies may reach up to 90 mg/dl. 3- If the condition is severe, ketonemia may lead to acidosis (ketosis).

C- Urine ketone bodies: Less than 40 mg/day.

D- Ketonurea: Is the increase of urine ketone bodies concentration above normal concentration. 1- It usually occurs with ketonemia. 2- The concentration of urine ketone bodies may reach up to 5000 mg/day.

E- Causes of ketonemia and ketonurea: 1- Starvation. 2- Severe diabetes mellitus.

F- Ketosis (= Ketoacidosis): It is a condition of metabolic acidosis result from ketonemia.

1- Mechanism: a- Increase ketone bodies in blood is neutralized by blood buffers mainly bicarbonate (HCO3

-).b- Bicarbonate will be depleted and this leads to decreased blood pH (acidosis).

2- Acidosis may causes transfer of K+ ions from intracellular fluid to blood leading to hyperkalemia. 3- Ketotic coma: In severe cases of ketosis as in uncontrolled diabetes mellitus coma may by developed and the condition may be fatal.

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Hydrocarbon "tail"

12

3

45

678

910

1112

13

14 15

1617

18

19

20

21 22

23

24

25

26

27

A B

C D

OH

Site of attachmentof fatty acid incholesteryl ester.

Structure of cholesterol showing siteof attachment of fatty acid incholesteryl esters.

CH3 - C ~ S - CoA + CH3 - C ~ S - CoA CH3 - C - CH2 - C ~ S - CoAThiolaseO

Acetyl CoA Acetyl CoACoASH

Acetoacetyl CoA

O O O

Cholesterol metabolism

I- Structure: - Cholesterol is an animal sterol. - It is an alcohol having -OHgroup at C3.

II- Sources of cholesterol: A- Endogenous: Cholesterol is formed in the body almost in all cells from acetyl-CoA (about 700 mg/day).

B- Exogenous: Cholesterol occurs only in food of animal origin such as egg yolk, meat, liver and brain. Diet supplies about 400 mg/day.

III- Synthesis of cholesterol: A- Location: 1- Intracellular location: Cytosol. 2- Organ location: a- Liver is the major site of cholesterol synthesis. b- Other tissues e.g. intestine, adrenal cortex, gonads and skin.

B-Precursor: Active acetate (Acetyl-CoA).

C- Steps: 1- Formation of acetoacetyl CoA by condensation of two molecules of acetyl CoA:

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C - CH2 - C ~ S - CoA

OCH3

O Acetoacetyl - CoA

CH3 - C ~ S - CoAO

Acetyl - CoA

CoA-SHHMG - CoA synthase

H2O

HOOC - CH2 - C - CH2 - C ~ S - CoA

OCH3

OH-Hydroxy - -Methylglutaryl - CoA

2 NADPH + 2H+

2 NADP+ + CoA-SH

HMG - CoA Reductase

HOOC - CH2 - C - CH2 - CH2 - OH

CH3

OHMevalonate

HO

10

13 17

Mevolanate

Sequaline

Lanosterol

Dwsmosterol

Cholesterol

2- Conversion of acetoacetyl CoA to mevalonate:

3- Conversion of mevalonate to cholesterol:

D- Regulation of cholesterol synthesis:

- HMG CoA reductase is the key enzyme for cholesterol synthesis. It isregulated through: 1- Feed back inhibition: Cholesterol (the end product of the pathway) acts as feed back inhibitor of HMG CoA reductase enzyme. Thus it decreases more cholesterol synthesis.

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2- Feed back regulation: Cholesterol (either synthesized by the cell or reaching it from diet) inhibits HMG CoA reductase gene. This decreases transcription and synthesis of HMG CoA reductase.

3- Hormonal regulation: a- Glucagon: Inhibits HMG CoA reductase. b- Insulin: Stimulates HMG CoA reductase.

4- Inhibition by drugs: - Lovastation and mevastation are drugs, which inhibit HMG CoA reductase by reversible competitive inhibition. They are used to decrease plasma cholesterol levels in patients with hypercholesterolemia.

IV- Functions of cholesterol: A- Cholesterol enters in the structure of every body cell (cell membrane). B- Cholesterol is the precursor of: Vitamin D3, steroid hormones and bile acids: 1- In skin → 7-dehydrocholesterol → Vitamin D3. 2- Gonads: a- Ovaries: Estrogens and progesterone. b- Testis: Testosterone and androgens. 3- Adrenal cortex: Glucocorticoids and mineralocorticoids. 4- Liver: bile acids.

Note: - Primary bile acids: cholic and chenodeoxy cholic acid. - Secondary bile acids: Deoxycholic acid and lithocholic acid. V- Excretion of cholesterol: - About one gram of cholesterol is excreted daily: A- ½ gram is secreted as such with bile which transports it to the intestine for elimination.

B- ½ gram is converted to bile acids which are excreted in the feces.

C- Some cholesterol is synthesized by intestinal cells and modified by bacteria before excretion. Bacterial enzymes reduce cholesterol into coprostanol, which excreted into feces.

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VI- Transport of cholesterol: A- Cholesterol is hydrophobic. It is transported in plasma in the more soluble lipoprotein forms: LDL, VLDL and HDL.

B- Free cholesterol is removed from tissues by HDL and transported to the liver to be excreted.

C- Cholesterol ester is the storage form of cholesterol: It is formed either in tissues or plasma.

1- In tissues, cholesterol is esterified by ACAT enzyme (acyl CoA cholesterol acyl transferase):

Cholesterol + Acyl CoA → Cholesterol ester + CoASH

2- In plasma, cholesterol is esterified by LCAT enzyme (lecithin cholesterol acyl transferase). LCAT is associated with HDL.

Cholesterol + lecithin → Cholesterol ester + lysolecithin

D- In plasma, the cholesterol ester is transferred form HDL to VLDL and LDL by cholesterol ester transfer protein. This protein allows the transfer triacylglycerol in the opposite direction. Thus, much of the cholesterol ester formed by LCAT in HDL finds its way to the liver via VLDL remnants (IDL) or LDL.

VII- Plasma cholesterol: A- Cholesterol present in plasma is either free or esterified (cholesteryl ester). 1- Total plasma cholesterol: 140-220 mg/dl. 2- Free plasma cholesterol: 25-126 mg/dl.

B- Cholesterol is transported in plasma in the forme of lipoproteins. Most of cholesterol is transported with LDL and to lesser extent with HDL.

C- Hypercholesterolemia: 1- Definition: It is increased plasma cholesterol concentration above 200 mg/dl.

2- Causes: a- Diet rich in carbohydrate, cholesterol and satururated fatty acids.

b- Hypothyroidism as thyroxine stimulates conversion of cholesterol to bile acids.

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c- Diabetes mellitus as insulin stimulates lipoprotein lipase. Thus deficiency of insulin as in diabetes mellitus decreases clearance of plasmalipoprotein.

d- Kidney affection (nephritic syndrome) unknown mechanism.

e- Obesity.

f- Obstructive jaundice due to decreased excretion of cholesterol and bile acids.

g- Familial hypercholesterolemia.

1D- Hypocholesterolemia: 1- Definition: It is decreased plasma cholesterol concentration below 140 mg/dl 2- Causes: a- Prolonged fasting due to decreased activation of HMG-CoA reductase.

b- Diet rich in unsaturated fatty acids and poor in saturated fatty acids, carbohydrate and cholesterol.

c- Liver diseases, where most plasma cholesterol is synthesized.

d- Hyperthyroidism.

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