Vitamins & Coenzymes

1. TUMS Vitamins & Coenzymes Part one Dr. Azin Nowrouzi, PhD Tehran University of Medical Sciences

2. History • Dumas (1871) individuals fed on pure minerals, water, proteins, fats and carbohydrates did not maintain good health. • Lunin (1880) similar finding in animals, milk restored them to normal • Eijkman (1897) Beri-beri occurred in those fed on polished rice. Rice-polishings cured them. ..toxin in polished rice and antitoxin in rice polishings! • Grijns (1901) tried to isolate toxin but did not find any. There may be a protective substance in rice polishing. • Pokelharing, McCollum, Hopkins(1911) Corroborated the findings of Lunin. The protective substance present in milk were named “accessory factors” by Hopkins. • Funk (1911-1912) isolated crystalline substance from rice polishings that could cure polyneuritis in pigeons.

3. Casimir Funk, a Polish biochemist, isolated an anti-berberi substance from rice polishing. Named it vitamine An amine Vital for life Originally, it was thought these necessary compounds were all amines. Since they were vital to our health they became known as “vital amines”, ie. vitamines. When it was discovered that some were not amines, i.e., not ' --ines', the name was changed to:vitamins Origin of the word VITAMIN

4. What are Vitamins? Vitamins are micronutrients: • Nutritionally important organic compounds • Required in very small amounts. • Cannot be synthesized by the human body. • Do not enter into tissue structures unlike proteins. • Do not undergo degradation for providing energy unlike carbohydrates and lipids. Plants and animals synthesize vitamins. • Vitamins form through biochemical life processes of the plants and animals we eat. • Examples: • Most mammals can synthesize vitamin C; not humans and primates. • No mammal can synthesize B vitamins but rumen bacteria do. • 3.Some function as vitamins after undergoing a chemical change: • Provitamins (e.g., β-carotene to vitamin A).

5. Coenzymes, Cofactors, and Prosthetic groups • Vitamins bind the enzyme either loosely or tightly: • Coenzymes are lost upon dialysis because they bind the enzyme loosely. • When they bind enzymes tightly, they are considered prosthetic group. • The term cofactor includes such compounds but also includes other molecules such as metal ions that may be necessary for enzyme activity.

6. Classification • Fat-soluble vitamins are much more soluble in fats, hydrocarbons and similar solvents than in water • Vitamins A, D, K, E • Water-soluble vitamins are much more soluble in water than in “organic solvents”. • C, B complex

7. Classification, Requirements, Absorption Water-soluble • Absorption: at the small intestine. • Regulation of absorption: by either other vitamins or binding proteins in the small intestine. • Transported away from small intestine in blood. • Typically not stored; instead, kidney filters excess into urine • Thus, important to get these vitamins daily. • Toxicities almost unheard of.

8. Classification, Requirements, Absorption Oil-soluble • Absorption: Along with dietary fat in small intestine. • 40-90% absorption efficiency. • Absorption typically regulated by need: need absorption • Transported away from small intestine in chylomicra via blood and lymph (depending on size).

9. B Complex Vitamins

10. B Complex - General features • Present in all plant and animal cells. • Generally act as components of coenzymes in metabolism of carbohydrates, lipids and proteins, especially in energy-yielding reactions. • Dietary requirement is closely linked to metabolic rate. • Absorbed by passive diffusion (except B12) in small intestine and any excess is excreted in urine i.e. there is little or no tissue storage (except B12, some folic acid). • Must be continually supplied in diet (or by ruminal synthesis).

11. Vitamin B1 - Thiamine • Funk (1912) isolated the anti-berberi substance from rice polishing. • Jansen and Donath (1927) isolated thiamine in its pure form. • Williams (1936) elucidated the chemical structure of Thiamin: • A substituted pyrimidine and a thiazole coupled by a methylene bridge. • It is very water-soluble. • Readily decomposes in neutral solution. • Has the characteristic "meaty" odor and flavor. • The active form is thiamin pyrophosphate (TPP) • Thiamin is rapidly converted to thiamin pyrophosphate (TPP) in small intestine, brain and liver. • TPP is formed from thiamin by the action of thiamine diphosphotransferase (see the next slide). • Monophosphate (Thiamin -monophosphate (TMP) and -triphosphate (TTP) forms are also present. • Absorption is increased in times of deficiency, but reduced by thyroid hormone, diabetes, alcohol consumption. • Thiamine is carried by portal blood to the liver. • Free thiamine is present in plasma, but the coenzyme (TPP) is the primary cellular component.

12. Chemical structure 2-methyl-4-amino pyrimidine + 4-methyl-5-β-hydroxyethylthiazole Thiamine MgATP2- Thiamine diphosphotransferase MgAMP- TPP

13. Functions • Transketolase reactions of the Pentose phosphate pathway (hexose monophosphate shunt). • Oxidative decarboxylation of pyruvic acid. • Oxidative decarboxylation of –ketoglutaric acid. • TTP (thiamine triphosphate) is required for nerve function (unrelated to coenzyme activity).

14. Deficiency symptoms • Classic deficiency disease syndromes is beriberi in humans: • GI symptoms: • Inappetence, poor growth, nausea, vomiting, fever, diminished gastric motility. • Muscular weakness (through accumulation of lactic acid). • Neurological manifestations: • Progressive nervous dysfunction, Wernicke’s encephalopathy. • Cardiovascular manifestations: • Palpitation, cardiac hypertrophy and dilation, congestive cardiac failure. • Causes: • It can happen if raw fish containing microbial thiaminases are ingested. • Tea may contain antithiamine factors. • Chronic alcoholism often leads to thiamine deficiency. In alcoholics there is: Reduced intake Impaired absorption Impaired use Reduced storage Alcohol is high in calorie but low in vitamin B1. All this can lead to Wernike-Korsakoff syndrome. 4. All diuretics including, Furosemide has been shown to cause thiamine (B-1) deficiency. This can lead to "wet beriberi" which causes sodium retention, dilation of blood vessels and heart failure.

15. Thiamine Deficiency (B1) Beriberi Wet beriberi – dilated cardiomyopathy Due to peripheral dilation of arterioles Dry beriberi – peripheral neuropathy, atrophy

16. Alcohol & Wernicke-Korsakoff syndrome Ataxia (inability to coordinate muscular movements due to nervous disorders) and confusion Memory loss/confabulation (to fill in gaps in memory by fabrication) Alcohol dilated cardiomyopathy Opthalmoplegia – can’t follow light source Nystagmus -involuntary jerking of the eye

17. Sources • Widely distributed. • Brewers' yeast is very rich source. • Cereal grains are rich sources, especially in germ and seed coat. • Fresh green, leafy plants • Animal products (especially egg yolk, liver, kidney) are good sources. • Synthetic vitamin is usually available as thiamin hydrochloride.

18. Vitamin B2 or G = Riboflavin • Yellow, crystalline compound with yellow-green fluorescence in aqueous solution. • Only sparingly soluble in water. • Stable in acid or neutral, but not alkaline solutions. • Unstable in light. • Riboflavin is phosphorylated in the intestine to generate FMN (riboflavin 5’-phosphate) by the action of Flavokinase. Riboflavin + ATP FMN + ppi • FMN then reacts with ATP, yielding FAD: FMN + ATP FAD + ppi ppi = inorganic pyrophosphate. Flavokinase FAD synthetase

19. Chemical structure • Isoalloxazine ring system = dimethylbenzene + pteryn • Ribitol (Reduced ribose) attached to N10 Riboflavin

20. FMN FAD 5 5 1 1 riboflavin 5’-phosphate Flavokinase (riboflavin kinase) FAD synthetase FMN + ATP FAD + ppi

22. Chemical structure and atom numbering of the flavin mononucleotide

23. Riboflavin functions • Essential constituent of the • Flavoproteins • Flavin mononucleotide (FMN) • Flavin adenine dinucleotide (FAD) • These play key roles in hydrogen transfer reactions associated with • Glycolysis • TCA cycle • Oxidative phosphorylation.

24. Deficiency symptoms • Inappetence, poor growth, vomiting, skin eruptions and eye abnormalities in pigs. • Cheilosis/Angular stomatitis (fissure at the angle of the mouth) • Localized seborrheic dermatitis of the face • Vascular changes in the cornea • Purple smooth tongue due to loss of tongue papillae (Glossitis). 2. Poor growth and "curled toe paralysis" in chicks. • The toes frequently curl inward and they may be unable to stand. Cheilosis/Angular stomatitis Glossitis

25. Dietary Sources • Dairy products • organ meats (liver and heart) but not muscle meat. • Green leafy plants (especially alfalfa) • Yeast and animal products • Cereals are poor sources so poultry fed cereal-based rations should receive supplemental riboflavin.

26. Niacin = Vitamin B3 • Beta pyridine carboxylic acid • Two forms: Nicotinic acid and Nicotinamide. Nicotinamide is the amide derivative of nicotinic acid. • In most animal species (including humans) niacin can be synthesized from the essential amino acid, tryptophan. • Both forms contain a pyridine ring. Nicotinic acid Nicotinamide

27. Functions: Active coenzymes: nicotinamide-adenine dinucleotide (NAD+) nicotinamide-adenine dinucleotide phosphate (NADP+). Both are extremely important in hydrogen transfer reactions catalyzed by dehydrogenase enzymes. ATP synthesis, from oxidation of primary fuels (glucose, fatty acids and to a lesser extent, amino acids) (NAD+) Also important in reductive biosynthesis (NADP+) NAD+ NADP+

30. Deficiency symptoms 1. Pellagra in farm animals and humans (fiery inflammation of tongue, mouth and upper esophagus). 2. Poor growth, enteritis and dermatitis. 3. Occurs in people who subsist mainly on corn which is low in both niacin and tryptophan 4. The signs of pellagra include dermatitis, diarrhea, dementia (the three Ds) and loss of tongue papillae. Sources of B3 Most non-corn-based diets contain adequate amounts of nicotinamide or its precursor, tryptophan.

31. Adenine is no longer considered a vitamin ! purine (pyrimidine + imidazole) FAD NAD NADP CoA S-AdoMet  PAPS (Phosphoadenosine phosphosulfate) ATP Vitamin B4- Adenine

32. Energy charge (EC) within cells The energy charge can have a value ranging from 0 (all AMP) to 1(all ATP). Most cells maintain EC at a constant value with very little variation: The energy charge of most cells range from 0.8 to 0.95. As EC drops catabolic, energy producing pathways, such as Glycolysis increase in rate, while anabolic, energy consuming pathways decrease in rate. The opposite occurs as EC increases, resulting in a tight control around an optimal value, as seen in the figure. It is evident that control of these pathways has evolved to maintain the energy charge within rather narrow limits. In other words, the energy charge like the pH of a cell is buffered.

33. Vitamin B5 / pantothenic acid • Chemical nature • Dipeptide derivative of the amino acid β-alanine and a butyric acid derivative.

34. Coenzyme A and Acetyl coenzyme A • Essential constituent of coenzyme A, Pantothenic acid combines with ATP and cysteine in the liver to generate CoA-SH. • CoA-SH transfers activated acyl groups, R-(C=O)-, such as acetyl group by binding them as a thioester. Acyl transfer is important in the TCA cycle and de novo fatty acid synthesis.

35. Vitamin B5 deficiency • Deficiency symptoms • 1. Poor growth, diarrhea, loss of hair, characteristic "goose-stepping" in pigs. • 2. Poor growth and feather development, dermatitis in chickens. • Sources • Widely distributed in plants (especially legumes and cereal) and animal products. • Deficiency has been observed in pigs fed a low protein (14%) corn-soybean ration fortified with minerals and vitamins except pantothenic acid.

36. Lipoic Acid & DihydroLipoic Acid (DHLA) lipoic acid = Internal disulfur of 6,8-dithiooctanoic acid. Lipoic Acid (LA) is part of a redox pair. oxidized form reduced form

37. Structure PDH = Pyruvate dehydrogenase complex

38. Lipoic acid • Alpha Lipoic acid is a natural substance found in certain foods and also produced in the human body. • Alpha Lipoic acid is a disulfide compound found naturally in mitochondria as the coenzyme for pyruvate dehydrogenase and-ketoglutarate dehydrogenase.

39. The coenzyme function for Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase

40. Clinical Uses for R Alpha Lipoic Acid • Perhaps the best use of R-alpha lipoic acid is as a life extension nutrient. It acts as: • an anti-oxidant • anti-glycation agent • blood sugar normalizer • mitochondria activator • glutathione enhancer. • Dosage of R Alpha Lipoic Acid • As a nutritional supplement, doses of 50 to 100 mg per day are generally recommended. • As a Therapeutic agent, higher doses may be used. • In Germany, dosages of 600 mg per day are prescribed for preventing the damaging effects of hyperglycemia in diabetes. • Larger doses, 1200 mg given intravenously, have been used to treat aminita mushroom poisoning. • R Alpha Lipoic Acid Side Effects and Precautions • Clinical research has shown no evidence of carcinogenic effects with administration of alpha lipoic acid. Serious side effects have not been observed, even at high doses. Minor side effects include skin reactions and gastrointestinal effects, such as nausea and vomiting.

41. Pyruvate dehydrogenase complex (PDH) PDH The reaction is: Pyruvate + NAD+ +CoASH Acetyl CoA + NADH + H+ + CO2 5 non-protein molecules (coenzymes) required for this enzyme catalyzed reaction are: NAD+ and CoASH (coenzyme A); (these are present in the equilibrated reaction formula, as can be seen above) TPP (thiamine pyrophosphate), Lipoic acid and FAD (flavin adenine dinucleotide) participate in the reaction but do not show up in the equilibrated reaction formula. E1 = Pyruvate dehydrogenase E2 = Dihydrolipoamide acyltransferase E3 = Dihydrolipoamide dehydrogenase

42. Mechanism of the reaction catalyzed by PDH complex

43. E1 uses TPP to release CO2 and produce HydroxyethylTPP (HETPP)

44. E2 uses lipoic acid to transfer the hydroxyethyl group from TPP to CoASH in order to produce AcetylCoA

46. 6. PYRIDOXINE (vitamin B6) Each of these forms can be phosphorylated at position 5 to form: PLP, PMP, and PNP.

47. Active (coenzyme) form Pyridoxal phosphate (PLP) • 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. • 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.

48. Functions • FUNCTIONS: 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.

49. 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.

50. Mechanism of catalyzed reaction