Vitamins 58.2

Vitamins 58.2. Vitamins soluble in water

HO S N CH3 5 1 2 1 2 Thiamine, Vitamin B 4 3 5 1 N 4 N + H3C 3-(4-Amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4- NH2 methylothiazolium chloride

A molecule of thiamine consists of a substituted thiazol ring attached to a substituted pyrimidine ring by a methylene bridge. The substituents in positions 2 and 4 in the pyrimidine ring and 4 and 5 in the thiazol ring are very important for the activity of thiamine.  The replacement of the methyl group by an ethyl group in the position 2 of pyrimidine does not have much influence on activity, whereas the C2 butyl derivative acts antagonistically. 2 HO S N CH3 5 1 2 1 2 4 3 5 N 4 N + H3C NH2

 The acetylation of the amino group in position 4 in the pyrimidine ring weakens action, while the replacement of the amino group by a hydroxyl group creates oxythiamine with antagonistic action

(antivitamin B1).  The hydroxyethyl group in the position 5 of the thiazol ring is very important for the activity of thiamine. Its elimination or replacement by another substituent results in the disappearance of activity.  The replacement of the hydrogen atom in the position 2 of the thiazol

ring by a sulfur atom (Thiothiamine, vitamin S-B1) does not influence activity.

In therapy thiamine analogues with an ‘open’ thiazol ring (acetiamine, benfotiamine, fursultiamine, prosultiamine) which act similarly to

thiamine, are also used. 3 Some of their properties are even better than those of thiamine, for example lower toxicity, higher stability, prolonged action and better absorption from the GT. In the body, they are a source of vitamin B1 and are used as analgetic agents.

HO S N CH S 3 CHO N CH O S 3 N N N N O H3C NH NH 2 CH3 2 PO3H2

Thiothiamine, Vitamin S-B1, SULBONE Benfotiamine, BIOTAMIN

O R CHO N CH O H C S 3 S CHO N CH 3 S 3 N N H C O N N 3 HO CH3 NH2 CH3 NH2 Acetiamine, THIANEURON Prosultiamine, R = -CH2-CH2-CH3; DITIAMINA

O Fursultiamine, Diavitan R = 4 The daily requirement of witamin B1 depends on a person’s age (low in children, 0.3–1.0 mg/24 h) and sex (slightly higher in men – 1.2–1.5 mg/24 h – than in women – approx. 1.1 mg/24 h). It also depends on nutrition (greater in the case of a diet rich in carbohydrates) and it is higher in pregnant women in (1.5 mg/24 h) and during lactation (1.6 mg/24 h).

A deficiency of vitamin B1 increases the concentration of pyruvic acid in the tissues and leads to the beriberi disease, which is characterized by the disturbance of the nervous system (peripheral sensomotor polyneuropathy and encephalopathy) with cardiac insufficiency, edemas and psychic disturbances.

In vitamin B1 deficiency medicinal products containing synthetic thiamine are used. They are administered mostly orally in daily doses of 3–9 mg and in serious cases intramuscularly, 10–20 mg once or twice daily.

5 Thiamine is needed for the metabolism of carbohydrates. It is also vital for the supply of energy necessary for the function of the nervous system, the cardiac muscle and skeletal muscles. In the body thiamine is phosphorylated to active thiamine pirophosphate (TPP) and thiamine triphosphate (TTP). TPP is a coenzyme for pyruvate decarboxylase, 2-ketoglutarate dehydrogenase and transketolase. TPP participates in the transfer of the aldehyde group in the pentose phosphate cycle.

After oral administration the transport of vitamin B1 depends on the dose. At concentrations < 2 mol active transport by a carrier occurs, while at concentrations >2 mol passive diffusion is observed. The absorption of thiamine is the greatest in the duodenum and the upper and middle sections of the small intestine. Thiamine is not absorbed from the stomach or the distal fragment of the small intestine. Thiamine is absorbed after its phosphorylation in the epithelium.

6 Vitamin B1, after oral administration, is not absorbed completely. Its maximal absorption is 8–15 mg/24 h. The absorption of thiamine is greater when it is administered in divided doses during meals. An excess of administered thiamine is eliminated in urine. The half-time of the -phase elimination of thiamine is 1.0 h. Thiamine is eliminated unchanged and as metabolites – thiaminecarboxylic acid, pyramine and others which have not been identified yet.

7 The stability of thiamine

The greatest stability is demonstrated by solutions of thiamine at pH 2, which can be stored for 6 months at 37 0C, without any effect on activity. Rapid degradation is observed at pH > 5, especially in the presence of atmospheric oxygen. The aqueous solutions at pH < 5 can be sterilized for 1 h at 100 0C. Chlorobutanol at a concentration of 0.5% is recommended for the stabilization of thiamine chloride in some pharmacopeal monographs.

8 + HO N CH H /H2O S 3 THIAMINE + N HO N H3C basic and NH2 4-Amino-5-hydroxy- neutral 5-(2-Hydroxyethyl)- environment 4-methylthiazol methyl-2-methyl- pyrimidin

H CHO N CH3 N N CH3 SH H C N N 3 N HO N CH3 NH2 NH2 HO H2S thiol form of thiamine Diazepine derivative O2

S The acidic hydrolysis of thiamine splits the bond N-C between the nitrogen atom of the thiazol ring and the carbon atom of the methylene group. In an alkaline environment a thiol form of thiamine is formed, which is very reactive and produces a benzodiazepine derivative after the elimination of hydrogen sulfide. In the presence of oxygen hydrogen sulfide is oxidized to elemental sulfur insoluble in water. 9 58.2.2. Riboflavin

CH2OH HO C H HO C H Riboflavin, VITAMINUM B2 HO C H 7,8-Dimethyl-10-(D-1-ribityl)-isoalloxazine CH2

H3C N N O NH H3C N O

The term riboflavin is made up of the names of its two components - ribitole and flavin. The word flavin is derived from flavus, the Latin word for yellow. The following relationship between the chemical structure and action of riboflavin is observed:

10  For biological activity the presence of two methyl groups in positions 7 and 8 is necessary; when they are absent or their position is changed action disappears. The replacement of the methyl groups by a chlorine atom produces compounds with antagonistic action.  A change of ribitol’s configuration from D to L causes action to disappear.  The imine group (-NH-) in position 3 is necessary for activity. When the hydrogen atom is replaced by a methyl group in the imino group action disappears.

CH OH 2 Riboflavin is a precursor of riboflavine-5'-phosphate HO C H (flavin mononucleotide; FMN) and flavin adenine HO C H HO C H dinucleotide (FAD). The names nucleotide and CH2 dinucleotide are not correct because ribitol is not a pentose and is not fused to flavin with a glycoside H3C N N O bond but these terms are accepted because of their NH H3C N widespread use. O 11 . R 2H

H3C N N O NH H3C N . O H R FAD = Oxidized form H H3C N N O NH H3C N H R O H H C N N O 3 FADH2 . N H Reduced form H3C N . O H Free radical (FADH)

Enzymes containing riboflavin are called flavoproteins. They participate in oxidation-reduction reactions. Flavin coenzymes can exist in any of 3 different red-ox states: oxidised flavin (FAD), semiquinone (free adical) (FADH) or reduced (FADH2) flavin. FAD is converted to semiquinone by one-electron transfer. A second one-electron transfer converts semiquinone to the completely reduced dihydroflavin.

12 The presence of the three red-ox forms makes it possible for flavin coenzymes to participate in one- or two-electron transfers. Because of that flavoproteins calalyse many biochemical reactions together with various acceptors and donors of electrons such as:

 transmitters of 2 electrons (NAD+, NADP+, DT-diaphorase)  one- and two-electron transmitters e.g. quinones  one-electron transmitters (cytochrome proteins).

13 Examples of flavoproteins include:  dehydrogenases (acylo-CoA dehydrogenase, glutathione reductase, aldehyde dehydrogenase, mitochondrial glycerol-3- phosphate dehydrogenase, succinate dehydrogenase of the citric acid cycle, NADH dehydrogenase of the respiratory chain in mitochondria, dihydrolipoyl dehydrogenase)  monooxygenases (lactate oxygenase)  dioxygenases  oxidases (glucose oxidase, -amino acid oxidase, xanthine oxidase).

14 Vitamin B2 exists in many foods. Sources of this vitamin are liver, white meat, eggs, milk and fresh vegetables. The daily requirement of vitamin B2 is 1–3 mg. A deficiency of vitamin B2 inhibits growth in children. Symptoms of hypovitaminosis are inflammation of oral mucosa and the tongue, angular cheilitis (the cracking of mouth corners), seborrheic dermatitis.

Vitamin B2 is used in the treatment of cataract and inflammations of mucous membranes as well as in convalescence after devastating diseases:  orally in doses of 3–9 mg daily

 in severe deficits of vitamin B2, 5–10 mg daily (i.m.).

15 The stability of riboflavin When stored in the solid state and protected from light riboflavin is stable, even at an increased temperature. Under the influence of light and oxygen it is decomposed to lumiflavin. This reaction occurs easily in an alkaline environment under the influence of visible radiation. In neutral and acidic solutions, under the influence of light, lumichrome consisting of various lumiflavins is formed. R CH3 N N O H3C N N O H3C hv/OH- Figure 58.6. NH N H H3C N H3C N O O The decomposition of riboflavin Riboflavin Lumiflavin under the influence of light.

hv/H+

H H3C N N O + Lumiflavin NH H3C N O Lumichrom 16 58.2.3. Pantothenic acid

OH H C H 3 N Pantothenic acid, Vitamin B HO COOH 5 O (R)-N-(2,4-Dihydroxy-3,3-dimethyl-1-oxobutyl)--alanine. H3C

A molecule of pantothenic acid consists of -alanine, which is fused to 2,4-dihydroxy-3,3-dimethylobutanoic acid (pantoinoic acid) with an amide bond. Only a dextrorotatory R-isomer shows vitamin activity. Similar action is shown by a product of the reduction of pantothenic acid – dexpantenol, which is also used in therapy. In the body dexpantenol is easily oxidized to pantothenic acid. The introduction of a methyl group in position  (-methylpantothenic acid) or replacement of the carboxyl group by a sulfonic (-SO3H; pantoilotaurin) or benzoyl (phenylpantothenone) group creates compounds with antivitamin activity.

17 PANTOTHENOATE * Phosphorylation of pantothenoate ATP ADP Pantothenic acid is a component * Binding with cysteine 4-Phosphopantothenoate Cysteina ATP of coenzyme A (CoA) and acyl- ADP + P * Decarboxylation of cysteine rest 4-Phosphopantothenylcysteine carrying protein (ACP).

CO2 Serine Their biosynthesis consists of * Adenylation of 4-phospho- 4-Phosphopantotein ACP pantetheine ( Dephospho-CoA) ATP reactions shown in Figure 58.7. or binding with cystein by hydroxyl group ( ACP) PPi Dephospho-CoA The SH group participates in the ATP * 3'-Phosphorylation transfer of the acyl groups, while ADP Coenzyme A adenine nucleotide acts as the Pantothenic acid recognition site increasing the Pantoinic acid -Alanine Mercaptoetanoamine affinity and specificity of coenzymes O H3C OH O O - _ _ _ _ _ H _ _ _ _ H _ _ _ O P O CH C CH C N CH2 CH2 C N CH2 CH2 SH for enzymes that bind with it.

O - H3C O O O P NH2 Figure 58.7. O N N Adenine O N N O P O CH2 The biosynthesis of coenzyme A and O - O O Ribozyl-3'-phosphate ACP from pantothenic acid. -O P O OH 18 O - Coenzyme A is a transporter of the acyl group in the citric acid cycle, in the oxidation and synthesis of fatty acids and in the reactions of acetylation of endogenic substances and drugs, e.g. in the synthesis of acetylcholine and cholesterol. Coenzyme A influences the function of the GT, the regeneration of the epithelium and the growth of hair and nails. The daily requirement of pantothenic acid is 8–10 mg. It is supplied in a normal diet. Spontaneous pantothenic acid avitaminosis is not observed because it exists commonly in nature and is synthesised by certain intestinal bacteria. Pantothenic acid is found in yeast, liver, yolk of eggs, legumes, grains of cereals, vegetables, milk and fruits.

19 Calcium pantothenate (CALCIUM PANTOTHENICUM) and Dexpantenol (BEPANTHEN) are used in streptomycin poisoning, in postoperative intestinal atonia, paralytic ileus, rheumatoidal diseases and locally in the damage of the cornea, in skin, hair and nail diseases, in pharyngitis, rhinitis, chronic and acute sinusitis, and as supplement to foods and vitamin products. Pantothenic acid for pharmaceutical products is obtained synthetically. The amide bond in pantothenic acid is sensitive to acidic and alkaline hydrolysis.

20 58.2.4. Pyridoxine

Pyridoxine, R = -CH2OH; VITAMINUM B6 N CH 1 3 6 2 3-Hydroxy-4,5-di(hydroxymethyl)-2-methyl-pyridin 5 HO 4 3 OH Pyridoxal; R = -CHO R Pyridoxamine; R = -CH2NH2

In therapy pyridoxine is used as vitamine B6 but vitamin properties are demonstrated by three pyridine derivatives: pyridoxine, pyridoxal and pyridoxamine. In the body, they undergo reversible transformation under the influence of enzymes:

pyridoxine pyridoxal pyridoxamine

21 Pyridoxal shows the greatest activity. The oxidation of the aldehyde group to the carboxyl group eliminates activity. The replacement of substituents at C4 and C5 by methyl or alkoxymethyl groups produces compounds with antagonistic action

The active form of vitamin B6 is pyridoxal 5-phosphate (PALP), which in the body exists in two tautomeric forms.

- H O N CH3 O - N CH3 + - - O P O O P O OH O - O CHO O CHO

PALP is the coenzyme of many enzymes (transaminases, decarboxylases, racemases and others), participating in nonoxidative tansformation of amino acids.

22 Enzymes dependent on PALP participate in the following reactions:  transamination, leading to the degradation and synthesis of amino acids  - and -decarboxylation, as a result of which biogenic amines are formed: dopamine, noradrenaline, histamine, serotonine, tyramine, GABA  - and -elimination, leading to the synthesis of ketoacids, e.g. pyruvate is formed from serine by -elimination of ammonium  racemization  aldol reaction. The intermediate product in these reactions is Schiff’s base.

23 The daily requirement of vitamin B6 is ~1.25 mg and is supplied in a normal diet. Sources of vitamin B6 are yeast, rice bran, crop germ, meat, celery, lettuce and peppers. A deficit of witamin B6 can be caused by wrong nutrition or the use of certain drugs, for example isoniazid, hydralazine or penicillamine. These drugs deactivate pyridoxal phosphate because together with PALP they form Schiff’s base.

The following symptoms are observed in vitamin B6 deficiency:  decreased synthesis of serotonine and noradrenaline, which can lead to neuropathy and depression  increased elimination of xanthurenic acid in urine (PALP is the coenzyme of kinureninase).

The symptoms of B6 avitaminosis are nausea, vomiting, damage of the skin and mucous membranes, psychical disturbances, convulsions, polyneural inflammation and anemia. 24 In therapy synthetic pyridoxine is used. Pyridoxine is applied:  in the treatment of convulsions in children caused by the hereditary mutation of the apoenzyme of cerebral glutaminic acid decarboxylase  in congenital anemia, caused by the genetic defect of the apoenzyme of the synthesis of -aminolevulinic acid, whose coenzyme is PALP  in the treatment of acrodynia (pain and inflammation of the skin on the ends of the nose, hands and feet)  as an auxiliary drug in the treatment of changes of the skin and mucosa, leucopenia and agranulocytosis  during convalescence after devastating diseases. Pyridoxine is well absorbed from the GT. In the blood 80% of PALP is bound with proteins. The main metabolite of pyridoxine is inactive 4-pyridoxine acid. 25 58.2.5. Biotin

2 HN1 3NH Biotin, Vitamin B7, Vitamin H H * * H cis-Tetrahydro-2-oxothiene-[3,4]imidazoline-4-valeric acid 6 5 4 * OH S O Biotin is composed of an tetrahydroimidazolone ring fused with a tetrahydrothiophene ring. A valeric acid subsituent is attached to one of the carbon atoms of the tetrahydrothiophene ring. In one molecule of biotin three asymmetric carbon atoms exist, so 8 optic isomers are possible. Additionally cis/trans isomerism is possibile. Only cis D-biotin shows vitamin activity. The length of the chain in the thiophene ring is also very important. The shortening or elongating of this chain or introducing a double bond into it produces compounds with antagonistic action. 26 Biotin exists in many natural foods. Its best sources are yolk of eggs, yeast, liver and kidneys. Biotin is also synthesised by saprophytic intestinal bacteria. Biotin is a cofactor responsible for the transfer of the carboxylate group in several carboxylase enzymes:  alfa and beta acetyl-CoA carboxylase  -methylcrotonyl-CoA carboxylase  propionyl-CoA carboxylase  pyruvate carboxylase.

27 O Biotin-lysine complex = HN NH H H H biocitine N S O O ~1,5 nm

Biotin is the prosthetic group of the enzyme with which it is bound by the -amino group of the lysine rest. A chain composed of 10 atoms separates the biotin ring from the  carbon atom of lysine to a distance of approx. ~1.5 nm. This chain is responsible for the ability of biotin to transfer the carboxylate group.

28 Biotin-dependent enzymes are carboxylases: pyruvate, acetyl-CoA, propionyl-CoA, -methylcrotonyl-CoA. In most biotin-dependent reactions the source of the carboxylate group is a hydrocarbonate ion. Large amounts of hydrocarbonate ions exist in biological fluids, but because their electrophilicity is weak hydrocarbonate must be activated first. ATP and Mg(II) ions participate in the activation of hydrocarbonate ions. The carbonate-phosphate anhydride created in this reaction transfers the carboxylate group to the N1 atom of the biotin ring. Next the carboxylate group is transferred to the substrate. (Fig. 58.8).

29 - HCO3 ATP Mg2+ ADP 1 O Acetyl-CoA O O O - H N N H O O O _ _ P C - H3C C SCoA O - R Anhydride of S phosphoric and Biocitine rest carbonic acid 2 3 O -O N NH O O _ _ P H2C C SCoA S R COO- Carboxybiocitine rest Malonyl-CoA Figure 58.8. The role of biotin in the carboxylation of acetyl-CoA.  – The activation of hydrocarbonate by ATP,  – the carboxylation of biotin,  – the transcarboxylation (the transport of the carboxylate group from biotin to 30 the substrate). Biotin as the coenzyme of carboxylases participates in the biotransformation of:  pyruvate to oxalacetate, which is very important for gluconeogenesis  acetyl-CoA to malonyl-CoA, which is an important substrate in the synthesis of fatty acids  propionyl-CoA to methylmalonyl-CoA, which participates in the citric acid cycle  -methylocrotonyl-CoA to -methylglutaryl-CoA, a precursor of malonic acid, which is a substrate in the biosynthesis of steroids.

31 Biotin is absorbed from the small intestine as a result of active transport and passive diffusion. Aprox. 80% of biotin is bound with plasma proteins. The concentration of free and bound biotin in the blood is 200–1200 ng/l. The level of biotin in erythrocytes is only ~10% of its concentration in plasma. The elimination of biotin occurs in the kidneys and intestines. Its half-time depends on dosage and for an oral dose of 100 g/kg of body mass it is ~26 h. This period is shorter (10–14 h) for the same dose in people with a deficit of biotin. The daily requirement of biotin is 10–30 g in children (depending on age) and 30–100 g in adolescent and adults. 32 Biotin deficiency can be caused by:  excessive consumption of raw egg-whites over a long period (months to years); egg-whites contain high amounts of avidin, a protein that binds biotin strongly and irreversibly. The biotin- avidin complex is not broken down or liberated during digestion, but removed from the body in feces. When cooked, the egg- white avidin becomes denaturated and entirely non-toxic;  the deficit of holocarboxylate synthase catalysing the attachment of biotin to the lysine rest of carboxylate apoenzyme;  oral therapy using antibiotics and sulphonamides.

33 People with type 2 diabetes often have low levels of biotin. Biotin may be involved in the synthesis and release of insulin. Initial symptoms of biotin deficiency include: dry skin, seborrheic dermatitis, fungal infections, rashes including erythematous periorofacial macular rash, thin and brittle hair, hair loss or total alopecia. If left untreated, neurological symptoms can develop, including mild depression, which may progress to profound weakness and, eventually, to somnolence, changes in mental status, generalized muscular pains (myalgias), hyperesthesias and paresthesias. Biotin is most often used together with other vitamins of the B group in psoriasis, seborrhea and dermatitis. In therapy biotin obtained synthetically is used.

34 58.2.6 Cobalamins (Vitamins B12)

O H2N The chemical structure of vitamins B NH2 12 CH O 3 O is based on a corrin ring, which is CH H2N 3 R NH similar to the porphyrin found in heme, N N 2 H3C + O chlorophyll, and cytochrome. The H3C Co CH H2N N N 3 central metal ion is cobalt. Four of the O CH3 H3C six coordination sites are provided by CH3 NH2 O the corrin ring, and the fifth by a HN O CH3 dimethylbenzimidazole group. The N CH 3 sixth coordination site, the center of O N CH3 -O P O HO reactivity, is variable, being a cyano O group, a hydroxyl group, a methyl O HO group or a 5’-deoxyadenosyl group (the C5’ atom of the deoxyribose forms the R = -CN; Cyanocobalamin; covalent bond with Co). R = OH; Hydroxycobalamin 35 Vitamin B12 is naturally found in foods, including meat (especially liver and shellfish), eggs and milk products.

Vitamins B12 are necessary for the biosynthesis of nucleinic acids. Vitamins B12 are absorbed as a result of active transport. An internal factor (glycoprotein secreted by parietal cells) is indispensable for the absorption of vitamins B12. The cobalamin-internal factor complex is transported to the ileum, where it binds with the specific receptors in the epithelium of the intestine. After the dissociation of this complex to cobalamin and the internal factor, cobalamin is transported to mucous cells and from them to the portal circulation. The amount of vitamin B12 that is absorbed depends on the concentration of the internal factor, the pancreas function and the density of receptors in the ileum. The maximal concentration is observed 4–8 h after administration.

36 The maximal concentration is observed 4–8 h after administration.

The absorption of vitamin B12 is decreased by aminoglycoside antibiotics, aminosalicylic acid, biguanids, chloramphenicol, colestyramine, potassium salts, methyldopa and antiepileptic drugs.

Vitamin B12 is not absorbed in Addison’s disease.

After absorption, vitamin B12 is transported with the blood in a form bound with -globuline – transcobalamin, and reaches the liver and other tissues.

In the body, vitamin B12 (inactive) is transformed to two active coenzymes – 5'-deoxyadenosylcobalamin and methylcobalamin. These coenzymes are formed in various compartments – methylcobalamin in cytosol and adenosylcobalamin in the mitochondria.

37 CH3 HS S

+ + NH NH3 3 -OOC -OOC Homocysteine Methionine

Metionine synthase

Methylocobalamin

5-Me-THF THF

Methylcobalamin participates in the coupled conversion of:  homocysteine to methionine and  methylotetrahydrofolate (Me-THF) to tetrahydrofolate (THF).

38 In the lack or deficit of methylcobalamin  homocysteine accumulates in the body; homocysteine is a risk factor in sclerosis  a deficiency of methionine is observed; methionine participates as adenosylmethionine in the conversion of

 phosphatydyletanolamine to lecitin in the cell membrane  etanolamine to choline, which is a precursor of ACh; a deficiency of methionine may cause neurologic disturbances

 a deficiency of THF is observed, which results in a decreased synthesis of purine and pyrimidine bases and a defect of DNA synthesis leading to anemia.

39 Deoxyadenosylcobalamin participates in the intramolecular rearrangement of L-methylmalonyl-CoA to succinyl-CoA.

_ - CH3 CH2 COO H C COO- 5'-Deoksyadenosylocobalamine H C H C C O S CoA Methylmalonyl-CoA mutase O S CoA L-Malonyl-CoA Succinyl-CoA

In a deficiency of vitamin B12 the rate of this transformation is decreased and the level of propionyl-CoA and methylmalonyl- CoA, which can be used in the synthesis of fatty acids, is increased.

Vitamin B12 is slightly soluble in water and accumulated in the liver. 40 Vitamin B12 deficiency can be caused by:  its insufficient amount in food (vegetarian diet)  deficiency of the internal factor (pernicious anemia, Addison’s anemia, Biermer’s anemia)  intestinal diseases  congenital (inborn) deficiency of transcobalamin II  a disturbance of the intrahepatic circulation.

Vitamin B12 is administered:  orally, only to eliminate its deficiency in food and to people with an increased requirement of this vitamin  intramuscularly or subcutaneously to people with a high deficiency and megaloblastic anemia and with neurological symptoms. 41 58.2.7. Nicotinamide

N Nicotinamide, Vitamin PP NH2 Pyridine-3-carboxamide O

Nicotinamide is known as vitamin PP, or antipellagric witamin. Pellagra is characterized by dermatic changes (reddening, rupture, exfoliation), gastrointestinal changes (anorexia, nausea, vomiting, diarrhea, porphyria) and neural changes (sleeplessness, headaches, dysmnesia, depression). Nicotinamide is synthesised by microorganisms, among others by intestinal flora. A precursor of nicotinamide in the body is tryptophan. 42 The recommended daily allowance of vitamin PP is 2-12 mg for children, 14 mg for women, 16 mg for men, and 18 mg for pregnant or breast-feeding women. The food sources of vitamin PP are animal products (liver, heart, kidneys, chicken, fish – tuna and salmon, milk, eggs), fruits and vegetables (leaf vegetables, broccoli, tomatoes, carrots, dates, sweet potatoes, asparagus, avocados), seeds (nuts, whole grain products, legumes, saltbush seeds) and fungi (mushrooms, brewer’s yeast). Nicotinamide in these products exists as NAD and NADP. In therapy synthetic nicotinamide is used. The properties of vitamin PP are also found in nicotinic acid. Nicotinamide plays an important role in biochemical reactions. It is a component of two coenzymes - nicotinamide-adenine dinucleotide (NAD+) and nicotinamide- adenine dinucleotide phosphate (NADP+). Reduced forms of these coenzymes are NADH and NADPH. The synthesis and decomposition of NAD+ and NADP+ is shown in Figure 58.9.

43 deamidase NICOTINAMIDE Nicotinate Figure 58.9. 1 5-Phosphoribosyl- 2 1-pirophosphate The biosynthesis and decomposition of 6 PPi NAD+ and NADP+: Nicotinate mononucleotide (NMN) NAD+ glycohydrolase ATP – deamination of nicotinamide to 3 nicotinate, PPi + Desamido-NAD – conversion of nicotinate to Glutamine ATP 4 nicotinate mononucleotide (NMN), Glutamate AMP + PPi 5 – adenylation of NMN, NAD+ NADP+ ATP ADP – transamination (transmission of Oxidized form Reduced form amine group of glutamine to nicotinate pro-R pro-S O O H H rest), N H2 N H2 + + N N – phosphorylation of NAD in O CH2 - O P O O position 2' of adenosyl rest, NH2 O N N - O P O HO OH – hydrolysis of N-glycoside bond by N N + O CH2 NAD glycohydrolase. O

HO OH * 44 Nicotinamide nucleotides act as transmitters of 2 electrons and play an important role in red-ox reactions, catalysed by dehydrogenases. A reactive center of the coenzyme is the position C4 of the pyridine ring. This position is chiral. Enzymes requiring nicotinamide as an coenzyme are stereospecific. For example, the position C2 of ethanol is pro-chiral. Dehydrogenase transfers stereospecifically the pro-R hydrogen atom of ethanol to the position pro-R of NADH.

H O HR S H O S H NH2 Alcoholic NH HR C OH + 2 + dehydrogenase C O + N N CH3 CH3 R R Ethanol

45 Lactate can exist as D and L, but lactate dehydrogenase of mammals is stereospecific for L-lactate and transfers the hydrogen atom to the position pro-R of NADH.

O H HS O COO - Lactate COO- R NH NH2 H + 2 dehydrogenase HO C + C O + N N CH3 CH3 R R L-Lactate

NAD+- and NADP+-dependent dehydrogenases catalyse 6 types of reactions:  simple transmission of the hydrogen atom by alcoholic, lactate and malate dehydrogenases

CH C + 2H+ + 2e-

OH O 46  oxidation of -hydroxy acids (isocitrate, 6-phosphogluconate dehydrogenases)

- - CH CH COO C CH COO C CH2 + CO2 OH R O R O R  oxidation of aldehydes (aldehyde dehydrogenase) - H H2O O C C + 3 H+ + 2 e- O O  reduction of isolated double bonds

CH CH C C + 2 H+ + 2 e-

 oxidation of -CH-NH- bonds by folate reductase

CH NH C N + 2 H+ + 2 e-

47 A deficiency of vitamin PP is observed:  in people whose main food is  maize (corn), because it contains vitamin PP in the form of niacitin, which is not assimilated by people  sorgo, which contains a large amount of leucine which inhibits cholinate phosphoribosyltransferase  an enzyme responsible for the conversion of tryptophan to NAD+  in people receiving isoniazid or pyrazinamide; these drugs act antagonistically to vitamin PP (antagonistic action is also demonstrated by pyridine-3-sulfonic acid and its amide, 3- acetylpyridine, quinolinic acid)  in the carcinoid syndrome, in which tryptophan is transformed to serotonine  in Hartnup disease, where the absorption of tryptophan is

disturbed. 48 58.2.8. Folic acid

OH 9 10 O N CH N COOH 4 5 2 N 3 6 H COOH 2 N 1 8 7 H H2N N N Folic acid, Acidum folicum, Pteroilglutaminic acid

N-[4-[[(2-amino-4-hydroxypteridin-6-yl)methyl]-amino]benzoyl]-L-glutaminic acid

A molecule of folic acid consists of pteroic acid (comprised of 2- amino-4-hydroxy-6-methylpteridine and 4-aminobenzoic acid) bound with L(+)-glutaminic acid by an amide bond. The introduction of the amine group or another amino acid into position 4 instead of glutaminic acid produces compounds with antagonistic activity.

49 Natural folic acid, synthesized by plants and microorganisms, contains most often from 2 to 7 glutaminic acid rests, connected by peptide bonds in position . In mammals monoglutaminate dominates. Pteroylglutaminates are active and in the body are transformed into pteroylmonoglutamate. Sources of folic acid are liver, spinach, wheat germ, turnip greens, dried beans and peas, fortified cereal products, sunflower seeds and other fruits and vegetables. Mammals cannot synthesise PABA and connect pteroic acid with glutaminic acid, so they must receive folic acid in food. Folic acid is reduced to dihydrofolic acid by folate reductase and then dihydrofolic acid is reduced to tetrahydrofolic acid (THF) by dihydrofolate reductase in the presence of ascorbic acid.

50 FOLIC ACID The active forms of THF are: DIHYDROFOLIC ACID (DHF)  N5-formyl-THF DHF reductase inhibitors 10 e.g. methotrexate  N -formyl-THF H or R H or R  N5, N10-methenyl-THF OH O 9 5 10 N CH2 N COOH  N , N -methylene-THF N 5 10 N COOH H H2N N N  N5-methyl-THF TETRAHYDROFOLIC ACID (THF)

N5-formyl-THF is known as folinic acid, while N10-formyl-THF as ‘active formic acid’.

51 H N 5 N Formylmethionine 10 N O H H Methylene tetrahydrofolate is N5-Formyl-THF formed from THF by the addition H N of methylene groups existing in one HCHO + THF 5 Purines-C2 N 10 H N of three carbon donors: O H N10-Formyl-THF formaldehyde, serine or glycine.

H2O H Methyl THF can be obtained from N Histidine + THF 5 Purines-C8 N methylene THF by reduction of the 10 N N5,N10-Methenyl-THF methylene group. NADPH + H+ NADP+ Formyl THF is synthesised by H N Serine + THF 5 Thymidine oxidation of methylene THF. N 10 N N5,N10-Methylene-THF

NADH + H+

NAD+ H N 5 Methionine N 10 N CH3 H 52 N5-Methyl-THF 5-Methyl-THF participates together with methylcobalamin in the conversion of homocysteine to methionine. N5,N10-Methenyl-THF and N5-formyl-THF are donors of monocarbon fragments in the biosynthesis of purines (the C8 and C2 atoms respectively). N5-Formyl-THF and N10-formyl-THF are donors of the –CHO group in the biosynthesis of formylmethionine, which makes possible the beginning of the initiation stage in the biosynthesis of proteins. N5,N10-methenyl-THF is the donor of the methyl group in the biosynthesis of dTMP (deoxythymidine monophosphate) from dUMP (deoxyuridine monophosphate)

53 The recommended daily allowance of folic acid is 25100 g for children (depending on age), 150200 g for young and adult people. A greater amount is required for pregnant (400 g per day) and breast-feeding (280 g per day) women. A deficiency of folate causes megaloblastic anemia. Folic acid for pharmaceutical products is obtained synthetically. The absorption of folic acid is a result of active transport and partially of passive diffusion (2030%). In the portal circulation THF is found. In the liver the methylation of folate is observed. The level of folate in plasma (mainly 5-methyl-THF but also THF and 10-formyl-THF) is 717 ng/ml. In erythrocytes polyglutamates of folate are present. The concentration of folate in erythrocytes is 40 times greater than in plasma. 5-Methyl-THF crosses the blood-brain barrier and its concentration in the cerebrospinal fluid is 23 times greater than in plasma. 54 58.2.9 Ascorbic acid

Ascorbic acid (Acidum ascorbicum, VITAMINUM C) can be considered as: HO OH H  a furan derivative: 5(R)-5-[(S)-1,2- * O 1 * 5 2 O dihydroxyethyl]-3,4-dihydroxy-(5H)- 4 3 furan-2-one H HO OH

HO OH HO OH 5 H 5 H * O * O  the enolic form of -lactone of 3- * 4 1 O * 4 1 O 3 2 3 2 oxo-L-gulonic acid H H HO OH O OH

55 To discuss the relationship between the chemical structure and activity of ascorbic acid the numeration of carbon atoms is based on that used in gulonic acid. The endiol group is responsible for the acidic and red-ox properties of ascorbic acid. More acidic properties are shown by the hydroxyl group at the C3 atom (pKa=4.17) than at the C2 atom (pKa=11.57), because the C=O group stabilizes the adjacent =C-OH group. HO OH H R R O O O The endiol group of O O O H H H O ascorbic acid is sensitive to -O OH .O OH HO . oxidation. Ascorbate Radical of monodehydroascorbic acid

+ e, + H+ - e, - H+

R O O H O O Dehydroascorbic acid 56 As a result of the loss of one hydrogen atom by ascorbic acid a monodehydroascorbic acid radical, stabilized by mesomerism, is formed. The loss of another hydrogen atom results in the creation of dehydroascorbic acid. Monodehydroascorbic radicals can also undergo spontaneous dismutation to ascorbic acid and dehydroascorbic acid. The C4 and C5 carbon atoms are asymetric, so 4 isomers are possibile  L- and D-ascorbic acids and L- and D-isoascorbic acids. There is a relationship between antiscorbutic action and the configuration of the chiral carbon atoms. Apart from the endiol group the R configuration at the C4 atom is necessary. This configuration is demonstrated by natural L(+)-ascorbic acid and D(+)-isoascorbic acid (araboascorbic acid), which shows 20 times weaker action than L(+)-ascorbic acid. Potency is strongly increased by the S configuration at the C5 atom. D-Ascorbic acid and L-isoascorbic acid with the S configuration at the C4 atom do not show antiscorbutic57 action. When more carbon atoms or a carbinol group are introduced into the chain activity decreases but does not disappear. The replacement of the hydroxyl group at the C6 atom by a chlorine atom decreases antiscorbutic action but to a lesser degree than other changes D-isoascorbic and L-glucoascorbic acids act antagonistically. The daily requirement of vitamin C is the greatest of all vitamins and is, on average, 1 mg/kg of body mass. A greater requirement occurs in breast-feeding women (100 mg daily). Sources of vitamin C are fresh fruits and vegetables, especially currants, strawberries, citruses, cabbage, parsley, spinach, cuckooflower, tomatoes and green peppers. Foods such as eggs, meat, bean, corn, grain products do not contain any vitamin C.

58 Primates and guinea pigs do not synthesize vitamin C because in their bodies L-gulonolactone oxidase, which catalyses the metabolism of L-gulonolactone to 2-oxo-L-gulonolactone, does not exist. Other mammals can synthesize vitamin C.

D-Glucose Figure 58.12. D-Glucuronic acid The role of L-gulonolactone oxidase in the biosynthesis of OH OH ascorbic acid H OH H OH L-Gulonolactone O oxidase O O O H H HO OH HO O L-Gulono--lactone 2-Oxo-L-gulono--lactone

ASCORBIC ACID 59 7080% of vitamin C received with food is absorbed mainly in the duodenum and in the proximal segment of the small intestine. The active transport of vitamin C is disturbed in intestinal dysfunction, vomiting, anorexia, alcoholism and in smokers. 25 % of vitamin C is bound with plasma proteins. The concentration of vitamin C in thrombocytes and lymphocytes is higher than in erythrocytes and plasma. ASA and other salicylates, when regularly administered, block the absorption of vitamin C by thrombocytes and decrease its concentration in plasma and thrombocytes. Vitamin C is transported through the portal vein to the liver and other tissues. The greatest amount of vitamin C is absorbed by organs with high metabolic activity such as the adrenal glands, hypophysis, pancreas, thymus, retina, spleen, stomach and lungs.

60 ASCORBIC ACID In the body, vitamin C is oxidized to

Dehydroascorbic acid dehydroascorbate. It is a reversible reaction and vitamin COOH H O COOH C is regenerated partially under the C O HO H H OH COOH influence of glutathione. + H OH C O HO H -CO2 COOH HO H Additionally, dehydroascorbic acid H C OH CH2OH CH OH 2 HO C H L-Treonic acid Oxalic acid is metabolized to 2,3-diketo-1- L-Xylose CH2OH gulonic acid (reversible reaction), 2,3-Diketo-1-gulonic acid which is next metabolized to L-treonic acid, oxalic acid, L- - CO + H O 2 2 xylonic acid and L-xylose COOH COOH (Fig. 58.13). HO H H OH + H OH H OH HO H HO H

CH2OH CH2OH L-Xylonic acid L-Lixonic acid

61 The red-ox system ascorbic acid  dehydroascorbic acid plays the role of a specific hydrogen donor and a transmitter of electrons in the cells. It forms a common red-ox system with cytochromes a and c, pyrimidine and flavin nucleotides and with glutathione. These properties are responsible for the participation of vitamin C in microsomal reactions of hydroxylation catalysed by oxidases, the control of the mitochondrial and microsomal respiratory cycle, the control of oxidative potential in cells, the biosynthesis of folic acid, maintaining the active forms of iron and copper [Cu(II) and Fe(II)] and in antioxidative reactions (a scavanger of free radicals). Additionally, vitamin C facilitates the absorption of Ca2+ ions, stimulates the synthesis of prostaglandins and is a modulator of immunity.

62 The following hydroxylases are ascorbic acid-dependent oxygenases: dopamine -hydroxylase, prolyl and lysyl hydroxylase, steroid 7-hydroxylase, 4-hydroxyphenylpyruvate dioxygenase (metaloprotein containing Cu(II) ions) and homogentisate dioxygenase (metaloprotein containing Fe(II) ions). Dopamine -hydroxylase participates in the biotransformation of DA to NA. The steroid 7-hydroxylase catalyses the biotransformation of cholesterol to 7-hydroxycholesterol in the biosynthesis of bile acids.

NADPH+ NADP Ascorbate O 7 + 2 7 7-Hydroxylase OH Cholesterol 7-Hydroxycholesterol 63 Prolyl and lysyl hydroxylases are peptide hydroxylases, because hydroxylation is possibile only after the binding of proline/lysine with polypeptide. These hydroxylases require the presence of molecular oxygen, Fe(II) ions, -ketoglutarate as a co-substrate and ascorbate. -Ketoglutarate Succinate

Fe(II) O2 Ascorbate

Pro Pro OH

During this reaction one oxygen atom is attached to the proline molecule and one to the succinate molecule. Hydroxyproline and hydroxylysine play an important role in the biosynthesis of collagen. 64 4-Hydroxyphenylpyruvate and THYROZINE -Ketoglutarate homogentisate dioxygenases Thyrozine aminotransferase 1 B6 participate in the catabolism of Glutamate 4-Hydroxyphenylpyruvate tyrosine. 4-Hydroxyphenylopyruvate [O] Disturbances of tyrosine dioxygenase 2 Ascorbate catabolism cause such catabolic CO2 disorders as: Homogentisate [O]   type II tyrosinemia, Homogentisate 1,2-dioxygenase 3 Ascorbate   neonatal tyrosinemia and Maleylacetoacetate   alcaptonuria (congenital metabolic disorder) (Fig. 58.14). Figure 58.14. The participation of ascorbic acid in the catabolism of tyrosine.

65 By reducing Fe(III) ions to Fe(II) ions ascorbic acid increases the absorption of iron which is necessary for the production of hemoglobin and erythrocytes. Because of that ascorbic acid is helpful in the treatment of anemia caused by iron deficiency. The red-ox potential of the reaction ascorbic acid  dehydroascorbic acid is E'0 = +0.1 V, which makes possible the reduction of reactive 1 oxygen forms: singlet oxygen ( O2), superoxide anion radical, hydroxyl radical and other radicals. It is active in the first, second and third defense line. The antioxidative action of vitamin C is used increasingly in the prophylaxis and therapy of many disorders in which overproduction of free radicals is observed. These disorders include: neurodegenerative disorders (Parkinson’s disease, Alzheimer’s disease, multiple sclerosis), inflammatory diseases of the gastric tract (Crohn’s disease, ulcerative inflammation of the colon), circulatory system diseases (angina pectoris), neoplastic disease, rheumatism and many others. 66 Vitamin E is the first defense line against free radicals. As a result of its reaction with superoxide radical hydrosuperoxide and a vitamin E radical are formed. The vitamin E radical is reduced by ascorbic acid. This reaction is probably mediated by coenzym Q. The so formed monodehydroascorbic acid radical is scavanged in the reaction of dismutation.

67 OH OH H O O ROO O RX HO - O OH L-Ascorbate

OH OH H O O L-ascorbic acid OH OH HO OH O RXH ROOH H O O O Reaction of dismutation

O_ OH OH Radical of OH monodehydro- H O ascorbic acid O Dehydroascorbic acid

O O Figure 58.15. The role of ascorbic acid and -tocopherol in the defense of cell 68 membranes against free radicals It is thought that ascorbic acid inhibits the initiation and promotion phases of neoplastic cells. (Fig. 58.16).

Normal cell Figure 58.16. Promotion Ascorbic acid The inhibition of tumor formation by ascorbic acid. Neoplastic cell

Initiation Ascorbic acid

Multiplication of neoplastic cells

Tumor

69 Possible anticancerogenic mechanisms of action demonstrated by ascorbic acid:

 antioxidative action  inhibition of the formation of nitrosoamines  stimulation of the immunologic system  modulation of the cancerogenic effect  protection from chromosome duplication caused by cancerogens  inhibition of the synthesis of DNA, RNA and proteins in neoplastic cells.

70 The beneficial action of ascorbic acid has been observed in the treatment of neoplasms of the mouth, pharynx, esophagus, stomach, breast, lung, colon and uterine cervix. In the physiological state, a high concentration of ascorbic acid is observed in the stomach wall, on the luminal side. In people with chronic gastritis, infection caused by Helicobacter pylori or neoplasm of the stomach, low concentrations of vitamin C in plasma and in the stomach are often observed. Infection caused by H. pylori stimulates the formation of chronic atrophic gastritis and leads to insufficient production of gastric acid and a significant decrease in the level of vitamin C in the stomach. The deficiency of gastric acid facilitates an accumulation of bacteria which reduce nitrates to nitrites.

71 Nitrites in reactions with amines Not damaged gastric mucosa and N-substituted amides form Helicobacter pylori nitrosamines. Atrophic gastritis Nitrosamines are very cancerogenic for the pharynx and especially for pH >5 the stomach. Number of microorganisms >107/l Vitamin C prevents the formation of Nitrate Nitrite nitrosamines and because of that Ascorbic acid can be helpful in the protection against H. pylori infections and can Nitrosoamine reduce the risk of neoplasms of the pharynx and the stomach (Fig. Gastric tumor 58.17).

In neoplasic diseases some specialists recommend supplementing

vitamin C by administering daily doses as high as 1 to 5 g. 72 The deficiency of ascorbic acid is accompanied by susceptibility to infections, mucous bleeding, subcutaneous hemorrhages, edema, arthralgia and difficult healing of wounds and fractures. A long-term deficiency of vitamin C leads to anemia and scurvy. When vitamin C is used in high doses, the following effects are observed:  decreased absorption of copper because of the inhibition of the activity of ceruloplasmin  deactivation of superoxide dismutase  release of iron from its reserves in tissues  acidification of urine, which can cause the formation of urate, citrate and oxalate calculus in the urinary tract  disturbances of the gastric tract and diuresis  hemolysis of erythrocytes may occurs in the cases of deficiency of glucoso- 6-phosphate dehydrogenase. 73 In pharmaceutical products ascorbic acid obtained synthetically is used. Ascorbic acid in the solid phase and in pharmaceutical products is stable, whereas in solutions it is rapidly degraded. In the presence of atmospheric oxygen autooxidation to dehydroascorbic acid is observed. This acid is hydrolysed to 2,3-diketogulonic acid which is oxidized to oxalic acid. The products of autooxidation of ascorbic acid are the same as its main metabolites. Anaerobic degradation is the effect of dehydratation, hydrolysis and decarboxylation. The final product of these reactions is furfural. The degradation of ascorbic acid is catalysed by metal ions. The rate of this reaction in aqueous solutions dependens on the concentration of hydrogen ions, substrate charge and solvent polarity.

74 O H O H

H O H H O H O O O O Dehydratation O O H H - 2H2O O O HO OH HO H O + H O Ascorbic acid Hydrolysis 2

OH Decarboxylation COOH CHO O - CO , - H O O 2 2 O Furfural

Figure 58.18. The anaerobic degradation of ascorbic acid