Azin Nowrouzi, PhD

TUMS

6. PYRIDOXINE (vitamin B6)

Pyridoxal (PL) Pyridoxamine (PM)Pyridoxine orPyridoxol (PN)

Each of these forms can be phosphorylated at position 5 to form: PLP, PMP, and PNP.

Active formPyridoxal phosphate (PLP)

• PLP and PL account for 90% of the total B6 in the blood.• In the blood B6 is transported both in the plasma and the RBCs.• In the blood PLP is hydrolyzed to PL because only free PL gets inside the cells.• In muscle and other tissues, PL is converted back to PLP by a reversible reaction with the help of alkaline phosphatase and pyridoxal kinase.

• Active functional form is pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP). • For absorption, the “phosphorylated” form must be hydrolyzed to “dephosphorylated” form by the enzyme alkaline phosphatase in the intestine.• In the portal vein Vit B6 is present as PL, PM, PN.• In the liver they are converted back to phosphorylated forms. This conversion is catalyzed by the ATP requiring enzyme, pyridoxal kinase.

FunctionsFUNCTIONS: B6 is involved in: Amino acid metabolism

Transamination reactions required for the synthesis and catabolism of the amino acids.Decarboxylation reactions.

Breakdown of glycogen Glycogenolysis (cofactor for glycogen phosphorylase). 80-90% of body vit B6 is present in the muscles, most of it in PLP

(coenzyme) form bound to glycogen phosphorylase. Only 1 mol or less is present in the blood, Synthesis of epinephrine (adrenaline) and norepinephrine (noradrenaline)Synthesis of niacin (vitamin B3) from the amino acid tryptophan.

Covalent bonds of -amino acids made labile by

their binding to PLP-containing enzyme In the reactions of amino acid metabolism, the formyl (CHO) group of PLP condenses with -NH2 group of an amino acid and forms a Schiffs base. This linkage weakens or labilizes all the bounds around the -carbon of the amino acid.

The specific bond of an amino acid that is broken depends on the particular enzyme to which PLP is attached.

Mechanism of catalyzed reaction

Deficiency• Food sources:

– In animal foods major forms are PL and and PM along with their phosphorylated forms.

– In plants PN.– Bananas, beans, lentils, walnuts, salmon, chicken, beef, whole grain breads

and cereals, soybeans, liver, eggs, dairy products are excellent sources. • Requirements:

– The requirement for vitamin B6 in the diet is proportional to the level of protein consumption ranging from 1.4 - 2.0 mg/day for a normal adult.

– During pregnancy and lactation the requirement for vitamin B6 increases approximately 0.6 mg/day.

• TOXICITIES: – Megadoses of B6 (daily doses of >500mg) are used to treat pms symptoms.

They can cause neurotoxicity and photosensitivity in some individuals.• Deficiencies: are rare and usually are related to an overall deficiency of

all the B-complex vitamins.• Certain drugs form complexes with PL and PLP

– Penicillamine (used to treat rheumatoid arthritis and cystinurias). – Isoniazid (the hydrazide derivative of isonicotinic acid) is the primary drug for

chemotherapy of tuberculosis.

7. BIOTIN

• Biotin is relatively small, bicyclic (two-ring) compound formed from a tetrahydrothiophene (thiophene) ring ,

• and a second ring, which contains a ureido group. • The thiophene ring also has a valeric acid side chain. • Although eight different stereoisomers of biotin exist,

only one stereoisomer is found naturally and to have biological activity as a coenzyme. It is called d-(+)-biotin, D-biotin or simply biotin.

It is sometimes called vitamin H and also coenzyme R.

Holocarboxylase

In humans, the four holocarboxylases are : acetyl-CoA carboxylase, propionyl-CoA carboxylase, pyruvate carboxylase and beta-methylcrotonyl-CoA carboxylase. Biotin is chemically bonded in each of these enzymes via an amide linkage between the carboxyl group of the valeric acid side-chain in biotin and the epsilon-amino group of the lysine residue in the apocarboxylase.

The enzyme that catalyzes the formation of this covalent bond is called holocarboxylase synthetase.

Biotin CycleBiotin cycle: the chain of chemical reactions involved in the use and reuse of the vitamin biotin. One important role of biotinidase is:

1. To separate or free biotin from proteins to which it is bound in foods. Biotin in its free form can then be used by the body.

2. Biotinidase lets the body recycle or reuse the biotin over and over again so that we do not need to consume large amounts of this vitamin in our diets.

•Within cells, the carboxylases (pyruvate carboxylase, acetyl-CoA carboxylase, methycrotonyl-CoA carboxylase, propionyl-CoA carboxylase) are biotinylated via holocarboxylase synthetase. Biotin and apocarboxylases are the substrates. ATP and magnesium also participate in the reaction.

Biotinidase deficiency is a treatable, inherited metabolic disorder in which the body cannot process the vitamin biotin in a normal manner.

Functions

• Coenzyme involving CO2 transfer– acetyl CoA to malonyl CoA– Propionyl CoA to methylmalonyl CoA– Pyruvate to oxaloacetate

• Deficiency– Fatty liver and kidney syndrome, a fatal

metabolic disorder in chicks and turkey poults

• Raw egg white (avidin) can block absorption

Biotin (functions)• Coenzyme for several reactions involving CO2 fixation into various

compounds e.g.

Pyruvate to oxaloacetate (pyruvate carboxylase)

Propionyl CoA to methylmalonyl CoA (propionyl CoA carboxylase)

Acetyl CoA to malonyl CoA(acetyl CoA carboxylase) - initial step in de novo fatty acid synthesis.

Deficiency symptoms

• Rare because of widespread distribution in plant and animal food and significant lower gut synthesis.– Sources

• Yeast, rice, soybeans, peanuts, fish (herring and mackerel), mushrooms and bananas, safflower meal, liver and milk are rich sources.

• Can be induced by eating raw egg white – The fact is that nature created the egg in such a way

that its yolk is very rich in biotin. One of the highest concentration in nature. Eat the egg whole together with the egg white and you will be fine.

– Egg whites contain a glycoprotein called "avidin" which binds biotin - one of the B vitamins - very effectively. The cooking process deactivates the avidin in the egg, much the same way it deactivates every other protein in the egg white.

• Biotin deficiency is chief cause of fatty liver and kidney syndrome.

This baby developed severe biotin deficiency during intravenous feeding without biotin.

Aajonus Vonderplanitz, in his book “We Want to live” is a strong proponent of raw eggs.

How Biotin Works

1- Biotin carrier protein

2- Biotin carboxylase

3- Transcarboxylase

9. FOLIC ACID (folacin)• Folacin includes several derivatives of folic acid

(monopteroylglutamic acid).

• Folic acid is obtained primarily from yeasts and leafy vegetables as well as animal liver. Animals cannot synthesize PABA nor attach glutamate residues to pteroic acid, thus, requiring folate intake in the diet.

“Microorganisms Only can

synthesize Folacin”

Sulphonamides and antibiotics block PABA incorporation

by competitive inhibition

Structure

Folic acid exists in a polyglutamate form. Intestinal mucosal cells remove some of the glutamate residues through the action of the lysosomal enzyme, conjugase.

Humans and microorganisms:

Folic acid is reduced within cells (principally in the liver where it is stored) to tetrahydrofolate (THF or H4folate) through the action of folate reductase [or dihydrofolate reductase (DHFR) ] which is an NADPH-requiring enzyme.

Active functional form is:

Tetrahydrafolic acid (THF).

Active center (N5 and N10)

• Active center of tetrahydrofolate (THF). The N5 position is the site of attachment of methyl  and  formimino groups, the N10 the site for attachment of formyl  group and that both N5 and N10 bridge the methylene and methenyl groups.

Folate conversions

Carrier of one-carbon (e.g. methyl) groups The one-carbon units are added to, or removed from, metabolites such as:

• histidine • serine • methionine • purines.

Functions

• Role of N5,N10-methylene-THF in dTMP synthesis is the most metabolically significant function for this vitamin.

• Vitamin B12 and N5-methyl-THF in the conversion of homocysteine to methionine is important in helping cells to regenerate needed THF.

Participation of H4folate in dTMP synthesis

______Deoxyuridine______________          ________Deoxythymidine

____Monophosphate (dUMP)_______________Monophosphate (dTMP)_______         

Deficiency symptoms

• Identical to those for vitamin B12 deficiency: • Effect of folate deficiency on cellular processes

is upon DNA synthesis.– Impairment in dTMP synthesis and purine synthesis– Cell cycle arrest in S-phase of rapidly proliferating cells, especially

hematopoietic cells.• The result is megaloblastic leukemia as for vitamin B12 deficiency.

– The inability to synthesize DNA during erythrocyte maturation leads to abnormally large erythrocytes termed macrocytic anemia.

• Deficiency during pregnancy can cause neural tube defects such as spina bifidia.

Deficiency is rare due to the adequate presence of folate in food. Deficiency can occur when there is:1. Poor dietary habits as those of chronic alcoholics. 2. Impaired absorption or metabolism

• Certain drugs such as anticonvulsants and oral contraceptives and methotrxate can impair the absorption of folate.

3. An increased demand for the vitamin. • Pregnancy • Folate will nearly double by the third trimester of pregnancy.

VITAMIN B12 (cobalamin)• Vitamin B12, is also called cobalamin,

cyanocobalamin and hydroxycobalamin.

• It is built from : 1. A nucleotide and 2. A complex tetrapyrrol ring structure

(corrin ring)3. A cobalt ion in the center.4. A R- group

• When R is cyanide (CN), vitamin B12 takes the form of cyanocobalamin.

• In hydroxycobalamin, R equals the hydroxyl group (-OH).

• In the coenzyme forms of vitamin B12,– R equals an adenosyl group in

adenosylcobalamin.– R equals a methyl (-CH3) group in

methylcobalamin.

• Vitamin B12 is synthesized exclusively by microorganisms (bacteria, fungi and algae) and not by animals and is found in the liver of animals bound to protein as methycobalamin or 5'-deoxyadenosylcobalamin.

• Known as the "red" vitamin because it exists as a dark red crystalline compound, Vitamin B12 is unique in that it is the only vitamin to contain cobalt (Co3+) metal ion, which, gives it the red color.

• The vitamin must be hydrolyzed from protein in order to be active.

• Intrinsic factor, a protein secreted by parietal cells of the stomach, carries it to the ileum where it is absorbed.

• It is transported to the liver and other tissues in the blood bound to transcobalamin II.

• It is stored in the liver attached to transcobalamin I.– It is released into the cell as

Hydroxocobalamin (see the next slide)• In the cytosol it is converted to

methylcobalamin.• Or it can enter mitochondria and be

converted to 5’-deoxyadenosyl cobalamin.

Dorothy Crowfoot Hodgkin(1910-1994)

Dr. Stadtman in her lab

In the cytosol

In mitochondria

Functions• Only two reactions in the body require vitamin B12 as a

cofactor: 1. During the catabolism of fatty acids with an odd number of

carbon atoms and the amino acids valine, isoleucine and threonine the resultant propionyl-CoA is converted to succinyl-CoA for oxidation in the TCA cycle. – methylmalonyl-CoA mutase, requires vitamin B12 as a cofactor in

the conversion of methylmalonyl-CoA to succinyl-CoA.– 5'-deoxyadenosine derivative of cobalamin is required for this

reaction

2. The second reaction catalyzed by methionine synthase converts homocysteine to methionine – This reaction results in the transfer of the methyl group from N5-

methyltetrahydrofolate to hydroxycobalamin generating tetrahydrofolate and methylcobalamin during the process of the conversion.

CH3-THF

Methionine SAM

SAH

methyltransferases

CH3-

Methylacceptor

Homocysteine

MSTHF

B12CH2-THF

CBS

cystathionine

cysteine

B6

B6

Folate cycleFolate cycle

TransulfurationTransulfurationpathwaypathway

Methionine cycleMethionine cycle

Methylacceptor

Methionine and Folate cycles are interrelated

Deficiency symptoms• Pernicious anemia in humans (inability to absorb B12

because of lack of gastric intrinsic factor).• Neurological disorders due to progressive demyelination

of nerve cells. – This results from increase in methylmalonyl-CoA.– Methylmalonyl-CoA is a competitive inhibitor of malonyl-CoA in

fatty acid biosynthesis.– Can substitute malonyl-CoA in any fatty acid biosynthesis and

create branched-chain fatty acid altering the architecture of normal membrane structure of nerve cells.

• Sources– Synthesized only by microorganisms, so traces only are present

in plants; liver is a rich source.– B12 is found in organ and muscle meats, fish, shellfish, dairy

products, eggs and in fortified foods like breakfast cereals.

• It is derived from glucose via uronic acid pathway. Enzyme L-gluconolactone oxidase is responsible for conversion of gluconolactone to ascorbic acid.

• This enzyme is absent in primates, including humans, some bats…. • The active form is ascorbic acid itself.

Vitamin C (Chemical nature)

VITAMIN C• Vitamin C is L-ascorbic acid, which is a colorless,

crystalline acid with strong reducing properties.• Functions• Vitamin C has antioxidant properties similar to those of

vitamin E, – Protects cells from free radicals.– Protects iron from oxidative damage, thus enhancing iron (Fe2+)

absorption in the gut.

• The main function is as a reducing agent.– It has the potential to reduce cytochrome a and c of the

respiratory chain and molecular oxygen and nitrates.

• It is required for various hydroxylation reactions e.g. proline to hydroxypoline for collagen synthesis (see next slide).

Hydroxylation of proline and lysine residues in collagen

• Vitamin C is required for the maintenance of normal connective tissue as well as for wound healing because synthesis of connective tissue is the first event in wound tissue remodeling.

Other activities• Several other metabolic reactions require

vitamin C as a cofactor: – The catabolism of tyrosine and the synthesis

of epinephrine from tyrosine and the synthesis of the bile acids.

– It is also believed that vitamin C is involved in the process of steroidogenesis.

• The adrenal cortex contains high levels of vitamin C which are depleted upon adrenocorticotropic hormone (ACTH) stimulation of the gland.

Roles in the bodySources• Citrus fruits and green leafy vegetables • Vitamin C is readily absorbed and so the primary cause of vitamin C

deficiency is poor diet and/or an increased requirement.

Deficiency symptoms1. Scurvy

– Bleeding gums– Small red spots on skin– Rough skin– Wounds fail to heal– Weak bones and teeth– Anemia and infections

2. Stress (e.g., infections, smoking)– Mechanism unknown, but vitamin C requirements increase during stress

3. Common cold?4. Disease prevention?

– Cancer, heart disease

Periodontal disease

Vitamin Chemical name B1

B2

B3

BB44

B5

B6

B7

B8

B9

BB1010

BB1111

B12

Thiamine

Riboflavin

Nicotinamide (niacin)

Adenine (no longer considered a vitamin)Adenine (no longer considered a vitamin)

Pantothenic acid

Pyridoxine

Biotin

InositolInositol

Folacin (folic acid)

pp-aminobenzoic acid (PABA) / H1-aminobenzoic acid (PABA) / H1 L-carnitine / b-hydroxy-g-trimethylammonium butyrateL-carnitine / b-hydroxy-g-trimethylammonium butyrate

(or choline)(or choline)

Cyanocobalamin

Vitamin B-complex

8. Inositol (Vitamin Bh) • Inositol is part of the vitamin B-complex.• Since it is not essential in the human diet, it cannot be

considered a vitamin. • Inositol is naturally present in foods high in fiber (wheat,

legumes, bran, etc). • It is required for:

– For proper formation of cell membranes. – For formation of lecithin– It is not a vitamin per se, but it works synergistically with

many vitamins and nutrients.• functions closely with a B complex vitamins, Choline, folacin,

Vitamins B-6 and B-12. – Inositol is necessary for proper function of nerves, brain, and

muscles in the body. – This nutrient is also often used to counter depression;

indeed, serotonin needs inositol for proper functioning. – Exists in all human cells, where it plays an important role in

cell proliferation and differentiation. • Low levels of this nutrient may result in depression and

some research has shown that increased levels of Inositol appear to be a promising treatment for depression.

10. P-amino benzoic acid (Bx)• A substance required for the synthesis of folic acid by

many organisms. • PABA is an essential nutrient for some bacteria and is

sometimes called Vitamin BX.• Para-aminobenzoic acid is included in the B-vitamin

complex, although it is not an essential nutrient for humans and it varies in its activity from other B vitamins.

• Although humans lack the ability to synthesize folate from PABA, it is sometimes marketed as an essential nutrient under the premise that it can stimulate intestinal bacteria.

• It also absorbs ultraviolet light and is used as aminobenzoic acid, as a topical sunscreen. Abbreviated PAB or PABA.

• The potassium salt is used as drug against fibrotic skin disorders.

Structure

Folic acid

PABA (vitamin Bx)

11. L-CARNITINE (B11)• L-carnitine

– Is made in the body from the amino acids lysine and methionine,

– Is needed to release energy from fat. – It transports fatty acids into mitochondria, the

powerhouses of cells. – In infancy, and in situations of high energy

needs, such as pregnancy and breast-feeding, the need for L-carnitine can exceed production by the body.

• Therefore, L-carnitine is considered a "conditionally essential" nutrient.

L-carnitine

• L-carnitine’s actions appear to be particularly important in the heart.

• As an example, patients with diabetes and high blood pressure were given 4 grams of L-carnitine per day in a preliminary study.

• After 45 weeks, irregular heartbeat and abnormal heart functioning decreased significantly compared with nonsupplemented patients.

Sources of L-carnitine• Dairy and red meat contain the greatest

amounts of carnitine. Therefore, people who have a limited intake of meat and dairy products tend to have lower L-carnitine intakes.

• Carnitine deficiencies are rare, even in strict vegetarians, because the body produces carnitine relatively easily.

• Rare genetic diseases can cause a carnitine deficiency.

• Also, deficiencies are occasionally associated with other diseases, such as diabetes and cirrhosis.

• Most people do not need carnitine supplements. For therapeutic use, typical amounts are 1–3 grams per day.