VITAMINS AND COFACTORS

VITAMINS AND COFACTORS By Henry Wormser, Ph.D. PSC 3110 – Biochemistry I - Fall 2002 Reading material • Principles of Biochemistry with a Human Focus by Garrett and Grisham, First Edition, 2002, pages 453-468 • Handbook of NonPrescriptions Drugs, 11th edition, Chapter entitled “Nutritional Products” by Loyd V. Allen, Jr. Vitamins • a group of organic compounds needed in small quantities in the diet for normal activity of tissues • between 14 – 20 substances have been identified as vitamins • many vitamins act as cofactors, coenzymes or prosthetic groups for enzymes • most vitamins are derived from diet • no calories are derived from vitamins Vitamins • first vitamin discovered was thiamine or B1 • the term vitamin is derived from the fact that the substances are needed for life (vita) and because thiamine happened to be an amine the term was coined as such • however, not all vitamins are amines or nitrogen containing compounds Vitamins • vitamin requirements are usually expressed as RDA’s (recommended dietary allowances) • guidelines are provided by 2 organizations: • the Food and Nutrition Board of the National Academy of Sciences- National Research Council • the Food and Drug Administration (FDA) RDAs • applications of RDAs include: • evaluating the adequacy of the national food supply • establishing standards for menu planning • establishing nutritional policy for public institutions/organizations and hospitals • evaluating diets in food consumption studies • establishing labeling regulations • setting guidelines for food product formulation • developing materials for nutritional education RDAs • RDAs have limitations: • they are too complex for direct consumer use • they do not state ideal or optimal levels of intake • the allowances for some categories are based on limited data • the data on some nutrients in foods is limited • they do not evaluate nutritional status • they do not apply to seriously ill or malnourished patients Vitamin deficiencies • primary food deficiency • crop failure • food storage loss • food preparation loss • diminished food intake • • • • poverty anorexia food fadism chronic diseases Vitamin deficiencies • diminished absorption • absorption defect • parasites • malignancies • increased requirements • • • • rapid growth increased physical activity pregnancy hyperthyroidism • increased loss • drug therapy • diuresis • lactation Vitamin loss Loss is seen mainly in storage or food preparation • Vitamin A: sensitive to oxygen and light • Vitamin D: usually little loss • Vitamin E: sensitive to oxidation especially when heated or with alkali • Vitamin K: sensitive to acids, alkali, light and oxidizing agents • Vitamin C: very sensitive to oxidation, especially when heated in contact with metals • Vitamin B complex: water solubility results in loss in cooking water • Riboflavin is sensitive to light Vitamins • Vitamins are typically divided into 2 groups: – The fat soluble vitamins • A, D, E, and K – The water soluble vitamins • The B vitamins (B1, B2, B3, B6, B7, B12 and pantothenic acid) • Ascorbic acid (vitamin C) Bogus vitamins • • • • • • • Vitamin B4 Vitamin B10 Vitamin B11 Vitamin B15 Vitamin B13 Vitamin B17 Vitamin B19 adenine identical with folic acid “ “ “ “ pangamic acid orotic acid laetrile wormser’s secret formula Cofactors • provide “chemical teeth” for enzymes • sometimes referred to as coenzymes • enzymes: proteins with catalytic activity – simple enzymes: large protein (polypeptide) that catalyzes a reaction. The enzyme gets all the “tools” (chemical teeth) it needs from the amino acids. However, there are only 20 different amino acids – conjugated enzymes : apoenzyme + cofactor = holoenzyme EXAMPLE:Proteases: enzymes that cleave peptide bonds H N O R' H N H O R' H N N R H O H2O protease N OH R + H2N O Enzymes perform catalytic reactions such as hydrolysis; the side chains of amino acids participate in the reactions example of a simple enzyme Usually electron-rich side chains are involved in the catalysis CH 2OH H N CH 2 N Aliphatic chains are normally involved in hydrophobic interactions all these tools come from amino acids in the protein active site CH 2-COOH A serine protease enzyme such as chymotrypsin HYDROLYTIC CATALYSIS O N HN COOH O N COOH OR' NH R N H ASP HIS SER ASP HIS SER H2O R' R' N HN COOO O N COOH O NH 2 NH R HN O- R ASP HIS SER ASP HIS SER Example of a conjugated enzyme cofactor needed for reaction Zinc protease such as ACE H N Zn+2 OH O R' N N R H O PRODUCTS + ENZYME Cofactors • all water-soluble vitamins with the exception of vitamin C are converted/activated to cofactors • only vitamin K of the fat-soluble vitamins is converted to a cofactor • not all vitamins are cofactors; i.e., lipoic acid is not a vitamin • cofactors may also act as carriers of specific functional groups such as methyl groups and acyl groups The water soluble vitamins Pantothenic acid (vitamin B5) CH3 OH HO CH2 C CH3 CH C O H N CH2 CH2 COOH First recognized in 1933 as a growth factor for yeast (Roger J. Williams) Pantothenic acid • a yellow viscous oil (free acid) • stable to moist heat (not to dry heat) and to oxidizing and reducing agents • hydrolyzed in acid or alkaline medium • sources (numerous): liver, kidney, eggs, lean beef, milk, molasses, cabbage, cauliflower, broccoli, peanuts, sweet potatoes, kale (derive its name from everywhere) Pantothenic acid • serves in its activated form as the cofactor for coenzyme A (CoA) and the acyl carrier protein (ACP) • first phosphorylated by ATP to 4’phosphopantothenate • next is the formation of 4’-phosphopantetheine by addition of cysteine and decarboxylation • adenylation by ATP forms dephospho-CoA • phosphorylation to the 3’-OH of the ribose generates CoA (coenzyme A) H N S O O CH 3 Acetyl CoA H N SH OH N O O P O O P O OOO H N O NH 2 N N N H2C N O H H OPO3 OH H H Coenzyme A Coenzyme A • performs a vital role by transporting acetyl groups from one substrate to another • the key to this action is the reactive thioester bond in the acetyl form of CoA • the thioester bond is stable enough that it can survive inside the cell, but unstable enough that acetyl-CoA can readily transfer the acetyl group to another molecule Example of an acetylation reaction CH 3 H3C N H3C OH CH 3 H3C N H3C O O CH 3 choline acetyl CoA CoA acetylcholine Acetylcholine is an important neurotransmitter in the autonomic nervous system (cholinergic) and in the brain Pantothenic acid • Deficiency: – rats • • • • graying of hair/fur in black rats dermatitis inflammation of nasal mucosa hemorrhage of adrenal cortex – humans • has not been encountered or extremely rare • difficult to induce with either synthetic diets and/or with antagonists (omegamethylpantothenic acid Pantothenic acid • vague symptoms in human deficiency: • numbness and tingling in feet “burning foot” • fatigue • GIT disturbances • available pharmaceutically as calcium pantothenate (d-isomer) and as racemic mixture • 5 - 7 mg/day appear to prevent signs of deficiency • appears to be non-toxic (up to 10-20 gm have been tolerated) Thiamine NH 2 S CH2 N N CH2-CH2-OH H3C N H3C THIAMINE Vitamin B1; antiberi-beri vitamin; antineuritic factor was the first water soluble vitamin discovered (Eijkman) Thiamine • has the odor and flavor of yeast • slowly destroyed by moist heat; more rapidly destroyed in a basic medium than in an acid one • source: whole cereals and grains; yeast; organ meat • pharmaceutical products use the hydrochloride or mononitrate salts Thiamine • active form is thiamine pyrophosphate (formed by the action of thiamine diphosphotransferase) • involved in the oxidative decarboxylation of pyruvic acid and a-ketoglutaric acid • involved in the transketolase reactions of the triose phosphate pathway • also required for nerve function (unrelated to coenzyme activity) Conversion of thiamine to TPP Typical reactions catalyzed by TPP Reactions in which thiamine pyrophosphate is a cofactor • Pyruvate decarboxylase • Alcohol fermentation – pyruvate to acetaldehyde • Pyruvate dehydrogenase • Synthesis of acetyl-CoA • Alpha-ketoglutarate dehydrogenase • Citric acid cycle • Transketolase reaction • Carbon-fixation reactions of photosynthesis • Acetolactase synthetase • Valine, leucine biosynthesis Thiamine pyrophosphate • the key portion of this cofactor is the thiazolium ring with its acidic hydrogen • the hydrogen is removed by the enzyme forming an ylid (anion next to cation) • the anion can then react with carbonyl groups in such molecules as pyruvate • the pyrophosphate functionality acts as a chemical handle which holds the cofactor in place within the enzyme thiazolium ring NH 2 C N N S O H3C N H3C H2C H2 C O P OO O P OOH thiamine pyrophosphate H NH 2 H3C C C N N S O H3C N H3C H2C H2 C O P OO O P OO- OH Hydroxyethyl thiamine pyrophosphate O Cl CH 3 Cl CH 3 Chemical mechanism for action of B1 in pyruvate dehydrogenase N H O- N O H S S pyruvate ylid acidic hydrogen Cl Cl CH 3 N S S OH HO O OCH 3 - CO2 N resonance Cl CH 3 CH 3 N N H + S S OH O H H O + H3C H ylid acetaldehyde Transketolase reaction CH2OH H CH2OH C HO H C C O H OH H H H C C C C OH transketolase OH OH TPP O HO H H H C C C C C O H OH OH OH + H H C C OH O CH2-OPO3H2 3-phosphoglyceraldehyde CH2-OPO3H2 D-xylulose-5-phosphate CH2-OPO3H2 D-ribose-5-phosphate CH2-OPO3H2 septulose-7-phosphate Transketolase reaction CH2OH H CH2OH C HO H C C O H OH H H C C C OH transketolase OH TPP H C OH HO H O C C C O H + OH CH2-OPO3H2 H C OH H C O CH2-OPO3H2 3-phosphoglyceraldehyde CH2-OPO3H2 D-xylulose-5-phosphate D-erythrose-4-phosphate CH2-OPO3H2 D-fructose-6-phosphate These reactions provide a link between the pentose phosphate pathway and glycolysis Activity of erythrocyte transketolase is commonly used as an index of thiamine deficiency Thiamine deficiency • earliest symptoms of thiamine deficiency include: – – – – – – constipation appetite suppression nausea mental depression peripheral neuropathy fatigue Thiamine deficiency (severe) • beri-beri (once associated with white polished rice diets and with highly milled wheat diets) • 2 clinical types • dry beri beri or neuritic beriberi – associated with polyneuropathy (depressed peripheral nerve function, sensory disturbance, loss of reflexes and motor control and muscle wasting • wet beri beri or cardiovacular beriberi – edema, congestive heart failure OH N H3 C N N S H3 C CH2-CH2-OH H3 C NH 2 N H3 C CH2-CH2-OH NEOPYRITHIAMINE OXYTHIAMINE These 2 compounds are potent antithiamine agents which may be used to induce symptoms of vitamin B1 deficiency in selected animals. Oxythiamine competitively inhibits thiamine pyrophosphate and becomes active after phosphorylation; neopyrithiamine prevents the conversion of thiamine to thiamine pyrophosphate Other clinical applications • Alcohol neuritis (peripheral neuropathy) • Sharp burning pain in the feet • Deep muscle tenderness with numbness • Coarse tremors, foot drop • Wernicke’s encephalopathy • Results from degeneration of basal ganglia due to chronic/heavy use of alcohol • Rigidity of extremities • Complete or partial ophthalmoplegia • Sleep disturbances • Nausea and vomiting Other clinical applications • Korsakoff’s syndrome or psychosis • • • • • • Also a complication of chronic/heavy use of alcohol Usually follows DT’s (delirium tremens) Memory loss Delusions Disorientation Ocular palsies • Combined Wenicke-Korsakoff syndrome • Pregnancy neuritis • Certain gastrointestinal disorders Requirement for thiamine • Based on energy needs – 0.3 – 0.6 mg/1000 calories – Increased requirements: • Pregnancy and lactation • Eating large amounts of raw sea food (clams) – contain thiaminase • Stress situations (high level of exercise, fever, hyperthyroidism) • Drinking large quantities of tea (contains antagonist) Thiamine assay • biologic assay – in animals – time consuming and costly (curative or protective) • microbiologic using bacteria which require thiamine for growth • chemical/fluorescent assay – conversion of thiamine to thiochrome by alkaline ferricyanide H3C N N N N CH3 THIOCHROME S CH2-CH2-OH Lipoic acid • lipoic acid is a co-factor found in pyruvate dehydrogenase and a-ketoglutarate dehydrogenase, two multienzymes involved in a-keto acid oxidation • lipoic acid functions to couple acyl group transfer and electron transfer during oxidation and decarboxylation of a-ketoacids • no evidence exists of a dietary lipoic acid requirement in humans; therefore it is not considered a vitamin S H2C C H2 S CH COOH H2C SH HS CH C H2 COOH lipoic acid, oxidized form lipoic acid, reduced form S H2C C H2 S CH C O H N CH C O NH lipoamide complex (lipoyl-lysine conjugate) Lipoic acid exists in 2 forms: a closed-ring disulfide form and an open-chain reduced form; oxidation-reduction cycles interconvert these 2 species; lipoic acid exists covalently attached in an amide linkage with lysine residues on enzymes Riboflavin • vitamin B2, lactoflavin (ovo, hepato, verdo), vitamin G • a heterocyclic flavin linked to ribose analogous to the nucleosides in RNA • orange-yellow fluorescent compound • found in significant quantities in green leafy vegetables, milk and meats • heat stable, but easily destroyed by light • recommended intake is related to energy intake (kcal) – RDA 1 – 2 mg/day H2 C H H H H H3C C C C C N OH OH OH OH H N dimethylisoalloxazine ring system – confers some degree of planarity to the molecule and also color (yellow) O N H3C N O RIBOFLAVIN H Decomposition of riboflavin CH3 H3C N N N H3C N O LUMIFLAVIN (produced by photochemical cleavage of riboflavin under alk aline conditions) OHH H3C N COOH O OHH3C CH3 N O + UREA H3C H O N NH O O alloxan O H3C NHCH 3 NH 2 4-amino-1,2-dimethyl 5-methylaminobenzene Riboflavin • 2 cofactors are involved: – riboflavin phosphate (flavin mononucleotide, FMN) – flavin adenine dinucleotide (FAD) • involved in the metabolism of carbohydrates, fats and proteins (flavin dehydrogenases/flavoproteins) • hydrogen carriers in the respiratory chain NH 2 N O CH2 N N N H H2C C H C H C H C O O P OH O O P OH OH OH OH H H3C N N N H3C N O H O O H H H H OH OH FLAVINE ADENINE DINUCLEOTIDE Riboflavin reduced substance FAD dehydrogenases cytochrome electron system (electron transport chain) oxidized substance FADH2 Riboflavin H H3C N N N H3C N O FAD (oxidized form) hydrogen addition occurs in 2 steps H O H3C N N N H3C N H O H O FADH2 (reduced form) Riboflavin • Enzymes utilizing riboflavin cofactors: – – – – – – NADH dehydrogenase succinate dehydrogenase d and l-amino acid oxidases pyridoxine-5-phosphate oxidase glutathione reductase xanthine oxidase • In some enzymes, the cofactor is covalently bonded to an amino acid (dehydrogenases) Dehydrogenase reaction CO2CH2 CH2 CO2succinate FAD succinate dehydrogenase H FADH2 CO2C C CO2fumarate H Amino acid oxidases H2 O R H C NH 3+ FMN FMNH 2 NH 3 R C O CO2- CO2- most amino acids (except serine, threonine, basic, and dicarboxylic acids) can be deaminated by L-amino acid oxidases Xanthine oxidase OH N N N OH N HO N xanthine N OH N HO N N OH N H hypoxanthine N H uric acid N H xanthine oxidase Xanthine oxidase is a flavoprotein which also contains Fe and Mo Fatty acyl-CoA desaturase H H R SCoA FAD FADH2 H O fatty acyl-CoA desaturase O H R H SCoA Important step in the biosynthesis of unsaturated fats; this reaction is actually more complex than shown here and involves other cofactors, but FAD is a key cofactor for the enzyme Riboflavin deficiency • seldom seen in industrialized societies • deficiency when seen: • • • • • • • • cheilosis (vertical fissure in the lips) angular stomatitis (craks in the corner of the mouth) glossitis photophobia seborrheic dermatitis normochromic normocytic anemia usually encountered along with pellagra (niacin deficiency) newborns treated for hyperbilirubinemia by phototherapy (riboflavin is unstable to light) Biotin O H H H H (CH2)4-COOH N N S BIOTIN Biotin • an imidazole sulfur containing compound • sometimes referred to as vitamin B7 or vitamin H • widely distributed in foods (liver, kidney, milk, molasses) • a large portion of the daily need of biotin is met by synthesis by intestinal bacteria • deficiency is usually the result of a defect in utilization rather than simple dietary deficiency Biotin • like lipoic acid, biotin is converted to its coenzyme form (called biotinyllysine or biocytin) by formation of a covalent amide bond to the nitrogen of a lysine residue • like lipoic acid it performs a highly specialized function : adds a carboxyl group to substrates Biotin • • biochemical role: carbon dioxide fixation two step process: 1. Binding of CO2 to biotin – N-carboxybiotin 2. Transfer of CO2 to a substrate – Activation of biotin requires enzyme, CO2, ATP and Mg++ Biotin Biotin-dependent enzymes: • Pyruvate carboxylase (synthesis of oxaloacetate for gluconeogenesis and replenishment of the citric acid cycle) • Acetyl CoA carboxylase (fatty acid biosynthesis) • Propionyl-CoA carboxylase  b-methylcrotonyl-CoA carboxylase • holocarboxylase synthase (multiple carboxylase) Reactions involving biotin enzymes O H3C C CO2-O2C O CH2 C oxaloacetate O SCoA -O2C CH2 C SCoA CO2- pyruvate O H3 C C acetyl CoA O H3C CH2 C SCoA -O2C malonyl CoA O CH C CH3 methylmalonyl CoA O O CO2- propionyl CoA HCO3- + NH4+ + ATP H2N C O P OH OH carbamyl phosphate Biotin • deficiency: • quite uncommon • can be induced by feeding raw egg white (avidin) • avidin is a protein which binds tighly with biotin (MW 70,000) • symptoms are: anorexia, nausea, muscle pain, fine scaly desquamation of the skin • requirements: 150 – 200 mcg/day • therapeutic use: in babies with infantile seborrhea (cradle cap) and Leiner’s disease Pyridoxine (vitamin B6) CH2OH HO CH2OH H3C N PYRIDOXINE A pyridine derivative Other forms of B-6 CHO HO CH2OH HO CH2NH 2 CH2OH H3C N H3C N PYRIXOXAL PYRIDOXAMINE Collectively, pyridoxine, pyridoxal and pyridoxamine are known as vitamin B6 Pyridoxine • vitamin B6, rat “acrodynia factor”, antidermatitis factor • widespread occurrence • pyridoxine: mostly in vegetable products • pyridoxal and pyridoxamine: mostly in animal products • pyridoxine is stable in acid solution, but unstable in neutral or alkaline solutions (destroyed by light) CH2O H HO CH2O H H3 C N pyridoxine CH2-NH2 CHO HO CH2O H HO CH2O H H3C N pyridoxal H3C N pyridoxamine CHO HO CH2 O O P OH OH HO CH2-NH2 CH2 O O P OH OH H3 C N H3 C N pyridoxal phosphate CO O H HO pyridoxamine phosphate CH2O H H3 C N pyridoxic acid Pyridoxal phosphate • pyridoxine is converted to pyridoxal phophate by phosphorylation and oxidation to the aldehyde • pyridoxal phosphate is then attached to the holoenzyme via a covalent bond to a lysine residue (a Schiff’s base) • the Schiff’s base bond is readily broken and reformed • this reversibility is very important in the biochemical action of this cofactor Biochemical functions H O N O CH 2OH CH 2OH HO H HO CH 2OPO3 HN H CH 2OPO3 H3C N H H3C N H HO H3C N H Able to catalyze the breakdown of amino acids Pyridoxal phosphate Biochemical functions: Decarboxylation of amino acids Transaminase reactions Racemization reactions Aldol cleavage reactions Transulfuration reactions Conversion of tryptophan to niacin Conversion of linoleic acid into arachidonic acid (prostaglandin precursor) 8. Formation of sphingolipids 1. 2. 3. 4. 5. 6. 7. Lys CO 2H HN H R NH 2 HO HO H3C N H Lys NH 2 R O OHN H Decarboxylation of amino acids - CO2 R R H3C N H N H resonance stabilization N H HO H HO H3C N H H3C N H H+ Lys R H N H HO H H HO H N H H3C N H R NH 2 Lys NH 2 H3C N H Important transaminases • ALT ( alanine aminotransferase) • formerly known as SGPT (serum glutamate pyruvate transaminase) • alanine + alpha-ketoglutarate = pyruvate + glutamate • increased serum level in liver injury Important transaminases • AST (aspartate aminotransferase) • formerly known as SGOT (serum glutamate oxaloacetate transaminase) • aspartate + alpha-ketoglutarate = oxaloacetate + glutamate • elevated when heart and/or liver are damaged Important decarboxylases - CO2 SERINE TYRO SINE ETHANO LAMINE DO PA - CO2 DO PAMINE ACETYLCHO LINE EPINEPHRINE - CO2 TRYPTO PHAN HISTIDINE - CO2 - CO2 5-HT HISTAMINE SERO TO NIN GLUTAMIC ACID CYSTEINE GAMMA AMINO BUTYRIC ACID (GABA) - CO2 CYSTEINE SULFINIC ACID TAURINE Mechanism for transamination reaction R H C N H C COOR C N C COONH2 CH2 N H aldimine N H ketimine R C O N H pyridoxamine phosphate COOalpha-keto acid Pyridoxine • deficiency: – difficult to produce in humans – may be accomplished artificially with a pyridoxine antagonist (deoxypyridoxine) – symptoms include: nausea and vomiting, seborrheic dermatitis, depression and confusion, mucous membrane lesions, peripheral neuritis, anemia Pyridoxine antagonists NH-NH 2 N N O N Hydralazine (antihypertensive) isoniazid (antitubercular) cycloserine (antitubercular) N H O C NH NH 2 H2N O CH3 H HS C C COOH CH3 NH 2 penicillamine (antirheumatic; Wilson's disease) Pyridoxine can antagonize the antiparkinsonian use of L-DOPA Brain L-DOPA L-DOPA L-dopamine B6 stimulates this reaction outside of the brain CO2 L-dopamine use carbidopa: an inhibitor of DOPA decarboxylase in combination with DOPA: Sinemet 10/100 or Sinemet 25/250 Pyridoxine deficiency • can be monitored by measuring the level of xanthurenic acid in the urine • this is related to a decrease in kynureninase activity (pyridoxal phosphate is the coenzyme) • kynurenine, a breakdown product of tryptophan is normally converted to kynurenic acid – but in B6 deficiency it is shunted to form xanthurenic acid XANTHURENIC ACID OH N COOH OH Pyridoxine • requirements: • children: 0.5 – 1.2 mg • adults: 2.0 mg • pregnancy: 2.5 mg – Requirement for B6 is proportional to the level of protein consumption • therapeutic uses: • • • • deficiency to counterract the effects of antagonists certain rare forms of anemia in women taking oral contraceptives (estrogen shifts tryptophan metabolism Discovered in 1913 from yeast; also known as vitamin B3 1915 – 1920: Irving Golberg demonstrated that lack of niacin causes pellagra COOH N NICOTINIC ACID one of the simplest vitamin; like B6 also a pyridine derivative CONH2 N NICOTINAMIDE Oxidation of nicotine yields nicotinic acid H N [OXIDATION] HNO 3 N nicotine N nicotinic acid COOH This reaction does not occur in vivo – strictly a laboratory reaction Nicotinic acid • niacin, vitamin B3, niacinamide, antipellagra vitamin • both form are active: the free acid and the amide • sources: organ meat (largest source), fish, yeast, dried fruit, nuts, cereal grains, some vegetables • pellagra-inducing diets: corn meal, corn starch, sweet potatoes, rice, syrup, pork fat (once a common diet in southern states among sharecroppers) Coenzyme forms NH 2 O N NH 2 O N O H H OH OH H OOH H OH OR H2C O P O O P O CH 2 O H N N N H H H NAD - OXIDATION REACTIONS R=H R = PO3 NADPH - REDUCTION REACTIONS Two cofactor forms of niacin: NAD and NADP; these cofactors are not tightly held by the enzyme and may be reused for reaction after reaction Biochemical function O C NH 2 N N H H O C NH 2 + + H NAD + or NADP+ NADH + H+ or NADPH + H+ In the older literature NAD+ is referred to as DPN or coenzyme I NADP+ is referred to as TPN or coenzyme II Oxidized and reduced forms Sparing action of tryptophan Tryptophan can substitute for niacin: 60 mg of tryptophan is equivalent to 1 mg of niacin; 60 gm of protein contains 600 mg of tryptophan which then represent 10 mg of niacin TRYPTOPHAN FORMYLKYNURENINE KYNURENINE 3-HYDROXYANTHRANILIC ACID 3-HYDROXYKYNURENINE B6-dependent reaction NICOTINIC ACID COOH CH2 CH O NH 2 CH CH2 COOH NH 2 N H tryptophan O NH 2 CH CH2 NH 2 blocke d by de ficie ncy OH of riboflavin 3-hydroxyk ynurenine blocke d by de ficie ncy of pyridoxine blocke d by de ficie ncy of thiamine H N C H N-formylk ynurenine O COOH CH2 NH 2 k ynurenine NH 2 CH COOH O alanine COOH COOH NH 2 OH 3-hydroxyanthranilic acid CO2 N Pellagra • Early stages: • • • • • Anorexia Indigestion Muscle weakness Reddened skin Rough skin • Advanced stages • 3 D’s of pellagra: dermatitis, diarrhea, dementia Clinical uses of nicotinic acid • pellagra symptoms from: • • • • • gastric ulcer or carcinoma diarrhea isoniazid therapy carcinoid syndrome Hartnup disease (impairment of tryptophan absorption) • peripheral vasodilator (nicotinic acid or nicotinyl alcohol) • hypolipidemic agent (only nicotinic acid in large doses – lowers both triglycerides and cholesterol (Niaspan, Nicobid) Carcinoid syndrome • a slow growing neoplasm of enterochromaffin cells (ileum, stomach, bronchus) • tryptophan metabolism is altered resulting in excess serotonin synthesis • symptoms include: • • • • • facial flushing edema of head and neck abdomina cramps and diarrhea asthmatic symptoms cardiac insufficiency • urinary 5-HIAA (5-hydroxyindole acetic acid) is high (5HIAA is a metabolite of serotonin; serotonin is derived from tryptophan) Cautions concerning the use of nicotinic acid in large doses • as an acid, it can erode gastrointestinal mucosa leading to ulceration • it also causes a depletion of glycogen stores and fat reserves in skeletal and cardiac muscle • additionally, there is an elevation in blood glucose and uric acid production • for these reasons, nicotinic therapy is not recommended for diabetics or persons who suffer from gout Ascorbic acid • vitamin C; anti-scorbutic vitamin (scurvy) • structure is reminiscent of glucose • produced in plants from glucose via the uronic pathway • the enzyme gulonolactone oxidase converts gulonolactone to ascorbic acid • exists in the enolic and ketonic forms • sources: citrus fruits, tomatoes, green peppers, strawberries, cantaloupe, cabbage, turnips, peas, lettuce and aspargus ASCORBIC ACID AND DEHYDROASCOBIC ACID O HO O HO CH CH2OH OH O CH CH2OH OH O O O Ascorbic acid • Biochemical functions: – Production and maintenance of collagen • Proline --------hydroxyproline • Lysine -------- hydroxylysine – Mitochondrial electron-transport chain (cytochrome C) – Metabolism of tyrosine • Tyrosine ----- p-hydroxyphenylpyruvic acid---- 2,5dihydroxyphenylacetic acid (homogentisic acid) Proline hydoxylase: (collagen formation) O O N vitamin C; O2 proline hydroxylase N HO Dopamine-beta hydroxylase ( neurotransmitter formation) OH HO NH 2 HO NH 2 O2; Vitamin C HO dopamine beta hydroxylase dopamine HO norepinephrine Anti-oxidant properties of vitamin C: helps prevent damage to cellular proteins and DNA O HO OH O O HO O O OH HO OH O O Normal metabolic processes in the cell lead to the generation of reactive oxidizing agents such as superoxide Superoxide can react with and damage protein and DNA, leading to cellular changes that can lead to premature aging and cancer Vitamin C reacts with superoxide, thus preventing this damage Ascorbic acid – conversion of folic acid to THFA – hydroxylation reactions of cholesterol to cholic acid – hydroxylation of tryptophan to 5hydroxytryptophan – regulation of cholesterol biosynthesis in the adrenal gland – aids in the absorption and utilization of iron – antioxidant properties may inhibit formation of nitrosamines during digestion of protein Ascorbic acid • defiency: scurvy – hemorrhage from mucous membranes, mouth and GIT, skin and muscles – gingivitis: swelling, tenderness, redness and ulceration of gums – loosening or loss of teeth – swelling of joints – rarefaction of bones and dentine Ascorbic acid • requirements: • children: 30 mg • adults: 40 –80 mg • pregnancy: 100 mg • therapeutic uses • scurvy • idiopathic methemoglobinemia • questionable use: common cold Vitamin B12 Vitamin B12 • • • • cyanocobalamin (Redisol) hydroxocobalamin (Alpha redisol) function deficiency • hematological sequelae • neurological sequelae Vitamin B12 • synthesized by bacteria only • red in color, levorotatory and stable to heat • commercially available either as cyano or hydroxocobalamin • stored in the liver as the coenzyme • absorbed only in the presence of the intrinsic factor (a glycoprotein released by parietal cells) • transported to tissues via transcobalamin II • present in foods such as liver, fish, eggs, milk • absent in vegetables and fruits Vitamin B12 • by far the most complex vitamin in structure • made up of a planar corrin ring (4 pyrroles) • the only vitamin that possesses a metal ion (cobalt) as part of its structure • the major cofactor form of B12 is adenosylcobalamin or 5’deoxyadenosylcobalamin • small amounts of methylcobalamin also occur (intermediate in methyl transfer reactions) Vitamin B12 • the corrin ring is similar to the porphyrin ring system found in hemoglobin except that in corrin 2 of the pyrroles are linked directly (without methylene bridges) • the cobalt is coordinated to the 4 pyrrole nitrogens • one of the axial cobalt ligands is a nitrogen of the dimethylbenzimidazole group • the other axial ligand may be CN, OH, CH3 or the 5’-carbon of a 5’-deoxyadenosyl group H 2 NCOCH 2 CH 2 H 2 NCOCH 2 CH 3 CH 3 CH 2 CONH 2 corin nucleus H 2 NCOCH O H 3C H 3C H N 2 N CN Co CH 2 CH 2 CONH N 2 cobalt coordinated N CH 3 CH 3 CH 2 CH 2 CONH H 2C CH 3 CH 3 2 H N H 3C O O P O O OH N CH 3 benzylimidazole N CH 3 HO VITAMIN B 12 Vitamin B12 • biochemical functions (mediated by coenzymes) • mutase reaction (rearrangement reaction – methylmalonyl CoA to succinyl CoA (lipid metabolism) • methylation reactions – uracil to thymine – homocysteine to methionine – aminoethanol to choline • activation of amino acids for protein synthesis • ribonucleotides to deoxyribonucleotides for DNA synthesis in certain bacteria Causes of B12 deficiency • Pernicious anemia (autoimmune gastritis against parietal cells - loss of intrinsic factor) • rarely due dietary deficiency • N2O/oral contaceptive drugs • intestinal parasite • gastrectomy • chronic gastritis • Schilling test Diagnosis of B12 deficiency • Schilling test • distinguishes deficiency caused by pernicious anemia with that caused by malabsorption • compares absorption in radiolabeled B12 with intrinsic factor and radiolabeled B12 without intrinsic factor • in pernicious anemia the B12 with intrinsic factor will be absorbed while the B12 by itself will not • in malabsorption neither will be absorbed Manifestation of B12 deficiency • macrocytic megaloblastic anemia • megaloblasts are abnormal erythroid precursors in bone marrow (most cells die in the bone marrow) • reticulocyte index is low • hyperchromic macrocytes appear in blood • anemia reflects impaired DNA synthesis • other cells may be involved (leukopenia, thrombocytopenia • spinal cord degeneration (irreversible) • • • • swelling, demyelination, cell death neurological disease results from deficient methylmalonyl-CoA mutase this cannot be treated with folic acid!! Treatment of B12 deficiency • use IM cyanocobalamin or hydroxocobalamin • administer daily for 2 - 3 weeks, then every 2 4 weeks for life • monitor reticulocytosis early to assure treatment is working (reticulocyte count should go up) • monitor potassium levels to ensure hypokalemia does not occur due to excessive RBC synthesis Folic acid • • • • MOA deficiency use drug interactions with folic acid Also known as folacin, vitamin M and pteroylglutamic acid Widely distributed in leaves (foliage) of plants O C OH N N H2N N N FOLIC ACID N H N H COOH CH COOH Chemically composed of pteroic acid (pteridine and PABA) and glutamic acid FOLIC ACID • absorbed by both active and passive transport • on the average we absorb 50 -200ug per day (about 10 -25% of dietary intake) • storage is in the form of 5-methyl THF (5 -20 mg) • found in green vegetable, dietary yeasts, liver, kidney • bacteria synthesize their own folic acid (dihydropteroate synthetase) Folic acid • Biochemical functions – one carbon fragment transfer (formyl, methyl, hydroxymethyl) • • • • • • conversion of homocysteine to methionine conversion of serine to glycine synthesis of thymidylic acid synthesis of purines (de novo) histdine metabolism synthesis of glycine PURINE CARBONS DERIVED via FOLATE NH 2 N N N H N H2N N O N N DNA N DNA ADENINE (A) GUANINE (G) BIOCHEMICAL ACTIVATION OF FOLIC ACID FOLIC ACID 7,8-DIHYDROFOLIC ACID (DHFA) N5, N10-METHYLENE TETRAHYDROFOLIC ACID TETRAHYDROFOLIC ACID (THFA) N5-FORMYL TETRAHYDROFOLIC ACID (LEUCOVORIN, FOLINIC ACID, CITROVORUM FACTOR) OTHER FORMS OF THFA: N5-METHYL THFA 5-FORMIMIDO THFA N N10-FORMYL THFA N5, N10-METHENYL THFA Deficiency of folic acid • Inadequate intake • defective absorption (most common) • • • • • • sprue gastric resection and intestinal disorders acute and chronic alcoholism drugs (anticonvulsants and oral contraceptives) pregnancy pellagra Deficiency of folic acid • abnormal metabolism of folates • folic acid antagonists (dihydrofolate reductase inhibibitors - methotrexate, pyrimethamine, trimethoprim) • enzyme deficiency • vitamin B12 deficiency • oral contraceptives • increased requirement • pregnancy, infancy METHOTREXATE H NH2 N N H2 N N N CH2 N O CH3 METHOTREXATE N CH COOH (CH2 )2 -COOH Inhibits enzyme dihydrofolate reductase (DHFR) which is necessary for maintaining pool of reduced folates required for DNA synthesis METHOTREXATE – also known as amethopterin or MTX – a potent inhibitor of dihydrofolate reductase which catalyzes the conversion of folic acid to tetrahydrofolic acid (THFA) – THFA acts as an acceptor of a one-carbon unit from either formate or formaldehyde – 5-formyl THFA is also known as folinic acid or the citrovorum factor (leucovorin) – THFA one-carbon carriers are important in the synthesis of purines, thymine, choline, and other important cellular constituents – MTX is used in treating acute lymphocytic leukemia in children, choriocarcinoma, osteogenic sarcoma, carcinomas of the head, neck, bladder and testis – in lower doses: treatment of psoriasis and rheumatoid arthritis NH2 N H2 N N CH2 CH3 PYRIMETHAMINE (DARAPRIM) Cl H2 N N N NH2 OCH3 CH2 OCH3 OCH3 TRIMETHOPRIM – – – – diaminopyrimidines inhibitors of dihydrofolate reductase have activity in both bacterial and protozoal organisms more effective if used in combination with another drug pyrimethamine is more selective for protozoal enzyme than trimethoprim – used in treatment of malaria and PCP The fat soluble vitamins By Henry Wormser Professor of Medicinal Chemistry Fat soluble vitamins • Vitamins A, D, K and E are the fatsoluble vitamins • excessive use of vitamins A and K can lead to toxicities • fat soluble vitamin tend to be stored in fatty tissues of the body and in the liver Vitamin A • Exits in 3 forms: • all trans-retinol • long chain fatty acyl ester of retinol (main storage form) • retinal (the active form in the retina) • retinoic acid is also considered to be physiologically active • provitamin A or carotene can be converted to retinol in vivo Vitamin A • recommended intakes are expressed in retinol equivalents (RE) 1 RE = 1 mcg of retinol = 6 mcg of b-carorene = 12 mcg other carotenes • older usage expressed activity in USP units or International units (IU). These were based on biological activity in the vitamin a-deficient rat (1 IU = 0.3 mcg of retinol) Vitamin A contains 5 conjugated double bonds which are key to some biological actions Isolated in impure form by McCollum in 1915 CH3 H3C CH3 CH3 CH2OH CH3 RDA: 0.7 mg VITAMIN A (RETINOL) Vitamin A • Diseases of deficiency: – Nigh blindness and xerophthalmia (dry eye) – Skin disorders – Lack of growth • Hypervitaminosis: – A serious potential problem (CNS disorders; birth defects) H3C H3C CH 3 CH 3 CH 3 H3C CH 3 CH 3 CH 3 CH 3 b-carotene liver O2 H3C CH 3 CH 3 CH 3 H3C CH 3 CH 3 CH 3 H O OH CH 3 CH 3 retinol (from diet) retinal (active form in vision) H3C CH 3 CH 3 CH 3 H3C CH 3 CH 3 CH 3 COOH O CH 3 vitamin A acetate (R = CH3) vitamin A palmitate (R = C16H33 R O CH 3 retinoic acid ("hormonally-active form") Vision and the role of vitamin A • photoreception is the function of 2 specialized cell types: rods and cones • both types of cells contain a photosensitive compound called opsin – in rod cells opsin is called scotopsin and the receptor is called rhodopsin or visual purple – rhodopsin is a serpentine receptor imbedded in the membrane of the rod cell; it is a complex between scotopsin and 11-cis retinal Vision and the role of vitamin A • intracellularly, rhodopsin is coupled to a Gprotein called transducin • when rhodopsin is exposed to light, it is bleached releasing the 11-cis-retinal from opsin • absorption of photons by 11-cis-retinal triggers the conversion to all-trans-retinal (one important conformational intermediate is metarhodopsin II); also there is a change in conformation of the photoreceptor Vision and the role of vitamin A • these transformations activate a phosphodiesterase (which hydrolyzes c-GMP to GMP) • c-GMP is necessary to maintain the Na+ channels in the rods in the open conformation • with a decrease in c-GMP, there occurs a closure of the Na+ channels, which leads to hyperpolarization of the rod cells with concomittant propagation of nerve impulses to the brain CH3 H3C CH3 11-cis H O CH3 H3C N Schiff's base H RHODOPSIN (11-cis retinal + opsin) H lysine chain of opsin N N 1. light 2. isomerization of retinal 3. change in shape of rhodopsin O CH3 H3C CH3 N H H3C 11-trans retinal CH3 H N N H signal transduction nerve impulse Additional role of retinol • retinol also functions in the synthesis of certain glycoproteins and mucopolysaccharides necessary for mucous production and normal growth regulation • this is accomplished by phosphorylation of retinol to retinyl phosphate which then functions similarly to dolichol phosphate Retinoic acid (Retin-A) is important for cellular differentiation; It controls cellular growth – particularly cell growth Used in the treatment of acne; also used as an anti-wrinkle agent (Retin A, Retin A micro, Avita, Renova) CH3 H3C CH3 CH3 COOH CH3 RETINOIC ACID (RETIN A) Also used orally to treat acute promyelocytic leukemia (APL) Product used is Vesanoid (10 mg capsules) Isotretinoin or accutane is a modification of retinoic acid; it contains a 13-cis double bond and is orally effective Used in the treatment of severe acne CH3 H3C CH3 CH3 COOH CH3 ISOTRETINOIN (ACCUTANE) An aromatic analog of retinoic acid; orally effective and used in the management and treatment of psoriasis CH3 H3C CH3 CH3 COOH CH3O CH3 ACITRETIN (SORIATANE) Etretinate (Tegison) CH3 H3C O CH3O CH3 CH3 CH3 OC2H5 Esterified form of acitretin; also used orally in the treatment of recalcitrant psoriasis; 10 and 25 mg capsules Alitretinoin (Panretin) H3C CH 3 CH 3 CO 2H CH 3 CH 3 9-cis-retinoic acid (Alitretinoin) Currently used as a 0.1% gel for the topical treatment of cutaneous lesions in patients with AIDS-related Kaposi sarcoma BEXAROTENE (Targretin) H3C CH2 CH3 CH3 H3 C CH3 COOH Bexarotene (Targretin) • indicated for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma • usually the patients receiving this drug have failed to respond to other treatment protocols • pregnancy (Category X drug) Adapalene (Differin) HO 2C Used as a 0.1% gel in the treatment of acne vulgaris Adapalene OCH 3 Tazarotene (Tazorac) EtO 2C S N C C H3C CH3 Topical treatment of patient with facial acne vulgaris of mild to moderate severity; gel 0.05%, 0.1% Vitamin A toxicity • vitamin A is higly toxic when taken in large amounts either acutely or chronically • may occur with 200 mg (666,000 IU) in adults or half this amount in children • signs include headache, nausea and vomiting, increased cerebrospinal fluid pressure, blurred vision and bulging of the fontanelle in infants Chemical name Abbreviation Generic name Vitamin D2 D2 ergocalciferol Vitamin D3 D3 Cholecalciferol 25hydroxyvitamin D3 1,25-dihydroxy vitamin D3 25(OH)D3 calciferol 1,25-(OH)3 Calcitriol 24,25-dihydroxy 24,25(OH)2D3 vitamin D3 Secalcifediol Vitamin D • There are 2 major precursor forms: • 7-dehydrocholesterol • ergosterol • UV irradiation affords cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) • Discovery: • 1890 – sunlight prevents rickets • 1924 – Steanbock and Hess found that irradiating certain foods produced vitamin D2 • 1970 – hormonally active form of vitamin D discovered Vitamin D • RDA – 20 mg (required in minute amounts) • disease of deficiency: rickets • Malformation of bones – due to improper bone mineralization • Hypervitaminosis • Toxic dose only 10X higher than the RDA • Causes hypercalcemia – can lead to cardiac arrest • vitamin D is not a vitamin (or a cofactor) – it is a steroid hormone OH HO 7-DEHYDROCHOLESTEROL CH3 PRE-D 3 D3 (CHOLECALCIFEROL) CH2 HO Biological functions • Calcium homeostasis – it is critical for the body to maintain the proper calcium level in the blood stream – Intestinal calcium absorption: acts as a signal to tell intestinal cells to take up more calcium from the gut – Bone calcium mobilization • Signals osteoclast (bone cells) to release calcium into the blood stream in response to low calcium levels Biological functions • Cellular differentiation – much less well understood – signal to bone marrow cells to change into other cells Problem: 1a,25(OH)2-D3 causes hypercalcemia high levels 1a,25(OH)2 vitamin D3 leukemia cell normal white blood cell derived from bone marrow grows at the proper rate Various analogs of vitamin D Potential use: -anti-cancer agent -immunosuppressive OH HO synthetic analog of vitamin D CH3 CH3 H3C CH2 HO OH DOXERCALCIFEROL (HECTOROL) Doxercalciferol (Hectorol) • a synthetic vitamin D analog that undergoes in vivo metabolic activation to 1-a,25dihydroxyvitamin D2 • Activation does not require involvement of the kidneys • Used in hyperparathyroidism in patients undergoing chronic renal dialysis • Initial dose 10 mcg orally 3 times per week CH3 H OH H PARACALCITOL (ZEMPLAR) HO OH PARICALCITOL (Zemplar) CH3 A synthetic vitamin D analog indicated for the prevention and treatment of secondary hyperparathyroidism associated with chronic renal failure HO H OH H PARACALCITOL (ZEMPLAR) OH H3 C OH CH3 H CH2 HO OH CALCIPOTRIENE (DOVONEX) Calcipotriol (Dovonex) a vitamin D derivative approved for the treatment of psoriasis. Mechanism of action is unknown. Receptor affinity is similar to that of calcitriol, but is less than 1% as active in regulating calcium metabolism HO OH H3 C CH2 OH Calcipotriene • An analog of vitamin D3 with a modified side-chain containing a 24-OH group and a cyclopropyl group • binds strongly to the D3 receptor on keratinocytes in skin and it suppresses their proliferation (used in psoriasis) • has only about 0.5% of the activity of D3 on calcium and phosphorus metabolism Dihydrotachysterol (DHT) A reduction product of vitamin D-2 Used in the management of hypoparathyroidism has only 1/450th the antirachidic activity of vitamin D-2 H3C CH3 HO Vitamin K • the koagulation vitamin • exists in 2 forms: – plant origin: phylloquinone or vit K1 – bacterial origin: menaquinones or vit K2 • also certain synthetic quinones have vitamin K activity – menadione (vitamin K3) – menadiol sodium phosphate (vitamin K4) O CH3 CH3 O CH3 3 CH3 PHYTONADIONE (VITAMIN K 1; PHYLLOQUINONE) O CH3 CH3 O CH3 n = 1 -12 CH3 MENAQUINONE (VITAMIN K 2 SERIES) O CH3 O MENADIONE (VITAMIN K 3) CO 2 CH 2 CH 2 CO 2 O 2C CH CH 2 CO 2 O2 OH CH 3 O CH 3 O R OH WARFARIN & OTHER ANTICOAGULANTS O R NAD NADH ANTIVITAMIN K ACTION OF ORAL ANTICOAGULANTS Vitamin E • alpha (E1), beta (E2) and gamma(E3) tocopherol • sources: plant oils (corn, peanut, wheat germ), green leafy vegetables, meat, eggs • value resides in the antioxidant properties of vitamin E (may prevent the formation of peroxides) ALPHA TOCOPHEROL CH3 CH3 H3C O CH3 HO CH3 ALPHA TOCOPHEROL CH3 CH3 CH3 Found in a variey of different sources (primarily vegetable fats) Vitamin E • Estimated requirements: 5 mg/day + 0.6 mg/day of unstaurated fat • Biological function – antioxidant for fatty acids – Acts like vitamin C; prevents lipid peroxidation and/or damage to cells by lipid hydroperoxides Uses for vitamin E • hemolytic anemia in premature infants, unresponsive to B12, Fe and folic acid • macrocytic megaloblastic anemia seen in children with severe protein-calorie malnutrition Other coenzymes O CH3O CH3 CH3 CH3O O Coenzyme Q (Ubiquinone) CH2 CH C CH2 H 10 Serves as entry into the electrontransport chain H H2 N N H H OH OH OH Tetrahydrobiopterin N CH CH CH3 N N Involved in the conversion of phenylalanine to tyrosine 09/12/02