Vitamins and Coenzyme Functions-II Serkan SAYINER, DVM PhD. Assist. Prof. Near East University, Faculty of Veterinary Medicine, Department of Biochemistry [email protected] Water-Soluble Vitamins Vitamin C and B-complex Vitamins 3 Water-Soluble Vitamins ■ They are in different structures. ■ Small quantities serve as coenzymes in important reactions in metabolism (B-complex vitamins). ■ This group includes vitamin C and B-complex vitamins. ■ They are synthesized by plants and microorganisms. ■ Animals often can not synthesize quantities to meet tissue needs. ■ Mammalian store them at very low levels; insignificant. It is therefore necessary that they are continuously supplied by microorganisms in the food, liquid or gastrointinal tract. 4 Vitamin C (Anti-Scurvy Vitamin, Ascorbic Acid) ■ Overview; – It is a powerful reducing agent that participates in several important hydroxylation reactions. – Na+-coupled transporters help to facilitate entry of vitamin C into cells. – Glial cells in the brain regenerate vitamin C from DHA. – It is needed for collagen, carnitine, catecholamine, and bile acid biosynthesis. – Oxalate is a natural degradation product of vitamin C. – It uses Fe++ and Cu++ as cofactors, and it enhances intestinal Fe++ absorption. – It’s deficiency can result in "scurvy." – Although most mammals can synthesize vitamin C from glucose, it cannot be formed in primates, fish, flying mammals, songbirds, or the guinea pig. – It is a natural preservative added to pet food products. 5 Vitamin C (Anti-Scurvy Vitamin, Ascorbic Acid)

■ Scurvy, a vitamin C deficiency disease known since ancient times, was a particular problem for sailors in the 15th-19th centuries, who's diets were often less than adequate on the long voyages they endured. – These men would develop swollen legs blotched with capillary hemorrhages, decaying peeling gums with loose teeth, decreased capacity to heal wounds, depression, anemia, and fatigue. – Infantile scurvy (also known as Barlow's syndrome or disease), is associated with similar symptoms. ■ Although most vertebrates can synthesize vitamin C from glucose, it cannot be formed in primates, fish, flying mammals, songbirds, or the guinea pig. Because in these organisms there is no L- gulonolactone oxidase enzyme. Therefore, these animals require it in their diet. 6 7

Vitamin C is synthesized through the uronic acid pathway in carbohydrate metabolism. 8 Vitamin C

■ Ascorbic acid is found in plant and animal foods. ■ In fruits such as lemon, orange, grapefruit, grape, strawberry, blackberry, banana, melon, watermelon, rosehip, tomatoes, green peppers, cabbage, fresh potatoes and all green-leaved vegetables are rich sources. ■ On the other hand, animal foods are poor sources. ■ If vitamin C rich foods are stored against heat or stored for a long time, they lose their vitamins content significantly. 9 Vitamin C ■ Ascorbic acid is colorless, odorless, sour flavor. ■ Although it is soluble in water and alcohol, it is insoluble in benzene, ether and fats. ■ Its solutions are acidic. ■ The vitamin-based solutions, which are highly stable in crystalline form, are rapidly degraded by air and light. ■ Solvents which are highly resistant to strong acid environments are resistant to neutral and alkaline environments. Contanct with metals and air, and also increase in ambient temperature accelerates its oxidation. ■ The ascorbic acid, a monosaccharide derivative in the keto lactone structure shown by the C6H8O6 closed formula, resembles simple sugars with six carbons in terms of structure. 10 Vitamin C: Functional Groups

Ester Hydoxyl Grup C=C Double Bond 11 Vitamin C ■ Ascorbic acid has two isomers, L and D. ■ The L form, which is active in animals and humans, can easily be oxidized and converted into dehydro-L-ascorbic acid (DHA) by giving two hydrogens in vivo. Conversion is reversible. ■ The dehydro L form of vitamin and the dehydro-L-ascorbic acid have an activity of only 80% than L form. ■ The ability of these two forms to convert into one another gives ascorbic acid its redox mediator. 12 Vitamin C ■ Ascorbic acid is dehydroascorbic acid without entering the cell, it takes the form of ascorbic acid after entering the cell. ■ Orally taken vitamins are mostly absorbed by active transport from small intestines and passive transport at a small ratio. ■ When the vitamin taken from the body is transported to the blood, it is destroyed as much as CO2 and H2O as it is in some monosaccharides. ■ In ruminants, oral vitamin C is rapidly degraded by alkaline pH and microflora effect of rumen. Vitamins taken by mouth are therefore not taken into account when meeting the vitamin C requirements. 13 Vitamin C ■ Regardless of whether vitamin C is derived through the diet or from biosynthesis in liver (as in rodents), or the kidneys (as in reptiles), specific transport mechanisms are required to move it into dependent tissues. ■ Ascorbate enters cells via Na+-coupled vitamin C transporters (SVCT 1 or SVCT 2), and cellular efflux occurs by as yet undescribed mechanisms. ■ The oxidized form of vitamin C (dihydroascorbate-DHA), is thought to exit and enter cells via glucose transporters (GLUT 3 and GLUT 1), respectively. ■ In the CNS, glial cells regenerate vitamin C from DHA via reduced glutathione (GSH) oxidation, and then vitamin C is transported back into neurons. – Neurons exhibit a high level of oxidative metabolism, and thus require protection by this important water-soluble vitamin. 14

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? 15 Functions of Vitamin C ■ Functions are; – Powerful reducing agent (Antioxidant). – Enhance intestinal Fe++ absorption. – Reduce cataract formation. – Enhance leukocyte activity. – Participate in Cu++-dependent amidation reactions in polypeptide hormone biosynthesis (e.g., GH, CT, and MSH). – Participate in the amidation of C-terminal glycine residues in the brain by Cu++- dependent enzymes. – Act as a carrier of sulfate groups in glycosaminoglycan formation (the "ground substance" between cells in all organs). – Participate in hydroxylation reactions using O (with Fe++ or Cu++ as cofactors). • Hydroxyproline and hydroxylysine formation during collagen biosynthesis. • Carnitine biosynthesis from lysine and S-adenosylmethionine. • Dopamine hydroxylation during catecholamine biosynthesis. • Hydroxylation of steroid hormones, aromatic drugs, and carcinogens in liver microsomes. 16 Functions of Vitamin C ■ At the molecular level, ascorbate is a powerful reducing agent, like the fat-soluble vitamin E and as such possesses general importance as an antioxidant, thus affecting the body's "redox" potential. ■ It is used as a natural preservative in pet food products. ■ It is sometimes given to cats as a treatment to reverse the methemoglobinemia associated with acetaminophen toxicity. ■ The physiologic importance of vitamin C as an antioxidant has been documented in pond turtles. – They contain Vitamin C at high concentration in their brains.

– These animals exhibit a high tolerance for O2 depletion during diving. – Vitamin C may help to prevent oxidative damage to neurons during the reoxygenation period following a hypoxic dive. 17 Functions of Vitamin C ■ Other reactions involving Vitamin C include hydorxylation using molecular oxygen (O2), that also use either Fe++ or Cu++ as a cofactor.

■ Ascorbate is thought to play either of two roles in hydroxylation. 1. As a direct source of electrons for the reduction of O2, 2. As a protective agent for maintaining Fe++ or Cu++ in their reduced states.

■ Particularly important are hydroxylations involving hydroxyproline and hydroxylysine formation during collagen biosynthesis in connective tissue – The formation of collagen is important during growth and development, – Once physical maturity is achieved, there is relatively little collagen turnover; An exception is healing from tissue injury and scar formation. 18 19 Functions of Vitamin C ■ Vitamin C has a secondary function in connective tissue metabolism as a carrier for sulfate groups needed in glycosaminoglycan formation (i.e. chondroitin sulfate, dermatan sulfate). – These compounds help to form the gel matrix (or "ground substance") between cells in all organs. – There would seem to be an obvious connection between these needs for ascorbate in connec-tive tissue metabolism, and the basic symptoms of scurvy above ■ Although vitamin C supplementation is not considered to be essential in dogs, megadoses of ascorbate fed to the bitch during pregnancy, and provided to the offspring until young adulthood, have been associated with reducing the incidence of canine hip dysplasia in animals considered genetically at risk for this condition. – Newfoundland, Danua, Saint Bernard, Golden and Labrador Retriever, Mastiff, German Shepherd Dog etc. 20

Species: Canine Breed: Hybrid (Golden?) Sex: Male Weight: 45 kg Date of Birth: February 08, 2014

Diagnosis: Right leg hip dysplasia (right coxafemoral dysplasia)

Source: Neu Animal Hospital 21

Diagnosis: Right leg hip dysplasia (right coxafemoral dysplasia)

Source: Prof. Dr. Deniz SEYREK-İNTAŞ 22 Functions of Vitamin C ■ Vitamin C is also used in the biosynthesis of carnitine from lysine and S-adenosylmethionine. – Carnitine is involved in the transport of long-chain fatty acids across mitochondrial membranes.

■ Vitamin C is active in hepatic microsomal drug metabolism. – Both endogenous and exogenous steroids are hydroxylated and conjugated in the liver, as are certain nonsteroidal drugs (e.g., barbiturates) and suspected carcinogens. – The resulting hydroxylation makes these compounds more water-soluble, and thus more likely to be excreted from the body through bile or urine. – For example, the first step in hepatic bile acid formation from cholesterol is activated by vitamin C. 23 Functions of Vitamin C ■ Vitamin C is also concentrated in leukocytes. – Deficiencies in leukocyte ascorbate concentrations have been reported in some diabetics, leading to a decreased capacity for wound repair and response to infection.

■ Ascorbate is concentrated in the humor aqueous solution. There is a relationship between cataract formation tendency and low ascorbate bioavailability. – Vitamin E is known to play a role in this process together with vitamin C.

■ Vitamin C also plays an important role in biogenic amine (i.e., catecholamine) biosynthesis in the adrenal medulla, central (CNS), and sympathetic (SNS) nervous systems. – Vitamin C serves as a co-substrate in the hydroxylation of dopamine to norepinephrine, catalyzed by the enzyme dopamine β-hydroxylase. – Catecholamines are associated with the ability of animals to deal with stress, and they help to mobilize glycogen and triglyceride for energy purposes. 24 Functions of Vitamin C ■ Vitamin C has also been found to participate in Cu++-dependent amidation reactions in polypeptide hormone biosynthesis (e.g., growth hormone (GH), calcitonin (CT), and melanocyte stimulating hormone (MSH)). – It may be necessary for the evocation of increased numbers of cell surface acetylcholine receptors by muscle cells responding to nerve stimuli. ■ It plays a role in Fe++ metabolism, by enhancing conversion of dietary iron from the ferric (Fe+++) to ferrous (Fe++) state. – Since iron is more readily absorbed from the intestine in the Fe++ state, – It may also be involved in the mobilization of stored Fe++, especially from hemosiderin in the spleen. ■ The capacity of this vitamin to chelate Ca++ may mean it has a function as well in bone mineral metabolism. Animal experiments point to a role for this vitamin in tooth formation. 25 Vitamin C Catabolism ■ A major pathway of ascorbate catabolism is to oxalate.

■ It has been estimated that under normal circumstances, approximately one-third of urinary oxalate may be derived from ascorbate catabolism.

■ Large doses of vitamin C (ascorbic acid) will enhance urine acidity, which promotes conversion of urate into uric acid, and oxalate into oxalic acid.

■ Vitamin C overload could also promote formation of calcium oxalate kidney stones in susceptible animals. 26 27 Vitamin C ■ In Deficiency; – Scurvy occurs. It is a deficiency disease which characterized general degeneration in connective tissue. It is Scurvy. • Due to functional disorders in mesenchymal cells in adults, disorders such as pain in the gums, spongiform appearance, loosening the teeth, edema in the teeth, pain in the joints, anorexia and anemia are seen. • Petechial hemorrhages as a result of the capsules breaking. • Ocular hemorrhage. • Saliva and lacrimal gland dryness. ■ In excess; – Disorders in the gastrointestinal tract. – Inadequate absorption of certain vitamins and minerals. – It can affect urine acidity and increase the conversion of urate to uric acid and oxalate to oxalic acid. – Calcium in the kidneys can trigger the formation of oxalate stones. 28

Scurvy

Source: Zoo Med 2008: Primate Medicince Source: Veterinary Gross Pathology Image Collection, Tufts Uni. 29

■ The B-complex vitamins are B-complex central to the metabolism of all mammalian cells. Vitamins ■ They act as coenzymes in specific reactions. • Vitamin B1 (Thiamin) • Vitamin B (Riboflavin) ■ Glycolysis, the hexose 2 monophosphate shunt (HMS), the • Vitamin B (Nicotinamide, Niacin) 3 tricarboxylic acid (TCA) cycle,

• Vitamin B5 (Pantothenic acid) and lipid metabolism are some examples. • Vitamin B6 (Pryridoxine, Pyridoxal, Pryridoxamine) ■ They are generally associated • Vitamin B7 (Biotin) with their respective enzymes

• Vitamin B9 (Folic acid) through covalent bond formation near the active site. • Vitamin B12 (Cobalamin) 30

Vitamin B1 ■ It is also called as Thiamine or Anti-beriberi Vitamin. ■ Overview – Thiamin diphosphate is used to facilitate oxidative decarboxylation and transketolase reactions. – Thiamin deficiency, which limits aerobic metabolism, can be fatal. – Thiamin is activated to its coenzyme form in brain and liver tissue. – Several foods exhibit thiamin antagonist activity. – Erythrocytes can be used to assess thiamin deficiency; Erythrocytic transketolase activity. 31

Vitamin B1 ■ The first discovered water-soluble vitamins (1897). It is a vitamin that many vertebrates and some microorganisms need to be found in their nutrients. ■ Thiamine is composed of one mole of thiazole coupled with a methylene bridge to one mole of pyrimidine. Christiaan Eijkman 11.08.1858 - 5.11.1930 1929 Nobel Prize for Physiology or Medicine 32

Vitamin B1 ■ The thiamine has a characteristic of sulfur odor with a bitter taste. Very soluble in water, partially soluble in alcohol, insoluble in organic solvents. ■ Thiamine in fresh foods is less stable than dry foods as moist environments increase degradation. ■ The thiamin is heat labile, if the fishes are cooked at 83ºC for at least 5 minutes, the enzyme becomes denatured. ■ It is produced by some microbes, found in most plant and animal tissues. ■ The coenzyme form (thiamine diphosphate/pyrophosphate-TPP) is required for various reactions in metabolism. ■ Approximately 80% of thiamin in the body is found in the diphosphate form. ■ It is not stored in the body. 33

Vitamin B1 Food Sources ■ The main thiamin sources are; – Grain bark, – Brewer's yeast, – Peanut, – Cotton seeds, – Soybean, – Rough clover, – Shell rice, – Peas and walnuts. 34

Absorption and Metabolism of Vitamin B1 ■ The absorption of thiamine is carried out in the gastrointestinal tract. The excretion is through urine as various metabolites. ■ Although the mechanism of thiamin absorption is not fully known, it is suggested that it can be absorbed by both active and passive transport. – It has been reported to be absorbed by active transport with sodium at low concentrations and with passive transport at high concentrations by intestinal mucosa. ■ In ruminants, free thiamine is absorbed by active transport from the rumen mucosa and bound to the plasma proteins (thiamin binding protein) and delivered to the liver by portal circulation. In the liver, it turns into thiamine pyrophosphate (TPP). It occurs in the form of free vitamin form in plasma, but the coenzyme form of TPP is lost in the kidneys by losing its phosphorus. 35

Functions of Vitamin B1 ■ There are two general types of reactions in mammals that utilize the activated coenzyme form of this vitamin: 1. Oxidative decarboxylation • Conversion of pyruvate to acetyl-CoA in glycolysis; Coenzyme of Pyruvate dehydrogenase • Conversion of α-ketoglutarate to succinyl-CoA in the TCA cycle; Coenzyme of α- ketoglutarate dehydrogenase • Decarboxylation of α-ketocarboxylic acid derivatives of the branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), particularly in brain and muscle tissue. 36

Functions of Vitamin B1 2. Transketolase Reactions • It is the coenzyme of Transketolase which is an important enzyme in the hexose monophosphate shunt (HMS), and also in the "dark reactions" of plant photosynthesis, where CO2 is converted to carbohydrate. • Since erythrocytes depend heavily on HMS. • A deficiency of this vitamin can be judged by measuring erythrocytic transketolase activity. • Given the above discussion, thiamin-deficient animals would be expected to exhibit accumulation of substrates involved in the above reactions (e.g., pentose sugars, pyruvate, α-KG=, and the α-ketocarboxylate derivatives of the BCAAs). 37 Transketolase Reactions 38

Functions of Vitamin B1 ■ TPP plays an important role as the coenzymes of the two enzymes involved in carbohydrate metabolism, which catalyze the decarboxylation of ketoacids and biosynthesis and degradation reactions of ketone bodies. ■ In these reactions, the thiazole ring of TPP is carried in a covalently bonded active aldehyde group. Mg2+ is required as a cofactor. ■ Thiamine is also necessary for the transmission of neural impuls and for the basic functions of nerve cells. This is independent of their basic function in the krebs cycle and pentose phosphate pathway. – When nerve cells are stimulated, TPP and TTP degrade TMP or free thiamine, and higher levels of TMP and free thiamine promote Na and K transport in the membranes. 39

Functions of Vitamin B1 ■ Provision of Na passive transport in excitable membranes such as ganglionic cell membranes is important for impulse transport. ■ It has also been reported that thiamine is also effective on insulin biosynthesis and that significant decreases occur in pancreatic insulin secretion in rats with thiamine deficiency. ■ Some foods contain thiamine antagonist, which is thiaminase. These include raw tuna, salmon, shellfish, rice bran, coffee, tea and bracken ferns. This enzyme inactivates thiamine into two metabolites ■ If cats, for example, are fed excessive amounts of raw fish in home-prepared diets, they may develop signs of thiamin deficiency. – Additionally, some symbiotic microbes found in the digestive tract also produce thiaminase. 40

Vitamin B1 Deficiency ■ Thiamin deficiency is associated with, – Beriberi in primates, – Polioencephalomalacia in ruminant animals and horses, – Chestak's paralysis in the fox, mink, and cat. – Polyneuritis in avians, – Opisthotonos in pigeons, – Generalized symptoms of deficiency include peripheral neuropathy (most marked in the extremities), weakness, tenderness and atrophy of muscles, fatigue, and decreased attention span. – Affects on the CNS are largely a result of its importance to aerobic metabolism, and when thiamin is deficient there is conversion to anaerobic glycolysis, local production of lactic acid, and neuronal dysfunction. – Affected patients may exhibit vestibular "seizures," fixed and dilated pupils, loss of physiologic nystagmus, plus stupor or coma. The condition may become fatal if left untreated. – Within the human population, thiamin deficiency is most often associated with alcoholism. 41

Beriberi Disease

Source: Dahliasagucio 42 43

Vitamin B2 ■ It is also called Riboflavin. ■ Overview – It is discovered by Paul Györy in 1920. – Riboflavin is used to produce FMN and FAD, and stored forms of this vitamin tend to decompose in the presence of light. – FAD and FMN are coenzymes containing iron or molybdenum. – FAD and FMN production is enhanced by thyroid hormones. – Riboflavin, like bile acids, exhibits an enterohepatic circulation (EHC). – Erythrocytes can be used to assess riboflavin deficiency; Erythrocytic glutathione reductase activity. 44

Vitamin B2 ■ Riboflavin is an orange-yellow compound with a closed formula C17H20N4O6, odorless, bitter flavor, melt-resistant to a temperature of about 280 ºC. ■ Riboflavin is an isoalloxazine derivative. It contains a side chain called dimethyl isoalloxazine which combines with ribitol. ■ Riboflavin, also known as the growth factor, is also called lactoflavin because it is first isolated from the milk. ■ All plants and animals require riboflavin in their metabolism. 45

Vitamin B2 ■ Riboflavin is found in three forms; free vitamin and two coenzyme forms which are FMN and FAD. ■ Riboflavin functions as part of two coenzymes, Flavin adenine dinucleotide (FAD), and riboflavin 5'-monophosphate (Flavin mononucleotide, FMN). ■ FMN is formed by ATP-dependent phosphorylation of riboflavin (flavokinase enzyme), whereas FAD is synthesized by a further reaction with ATP in which the AMP moiety of ATP is transferred to FMN (FAD pyrophosphorylase). ■ FAD and FMN are usually tightly bound to their respective Apoenzyme protein, and contain one or more trace elements as essential cofactors (usually iron or molybdenum). Metalloflavoproteins are capable of reversible reduction, thus yielding FADH2 and FMNH2. 46

Vitamin B2 Food Sources ■ Riboflavin is synthesized in green plants, yeast, fungi and some bacteria. ■ Rapidly growing, green leaved plants and animal feeds, especially rough clover is a good source. ■ While cereals and cereal products contain lower levels, milk, eggs, liver, heart, kidney and meat are rich riboflavin sources for humans. 47

Absorption and Metabolism of Vitamin B2 ■ In mucosal cells, riboflavin is phosphorylated to FMN by flavokinase enzyme (ATP-dependent phosphorylation). FMN is transported to the liver by binding to plasma albumin with portal circulation. FMN is converted to FAD in the liver. These events occur primarily in the liver. ■ Riboflavin can not be stored in significant amounts in animal tissues as it is in other water-soluble vitamins. ■ It varies the concentrations in the tissues and liver, kidney and heart contains riboflavin in higher amount than other tissues. ■ One-third of total body flavins accumulate in the liver. When riboflavin is consumed in large quantities with food, a large portion of the free riboflavin is excreted in the urine, and a small amount in stool, bile and perspiration. ■ Degradation products are excreted as hydroxymethyl derivatives. 48

Functions of Vitamin B2 ■ FMN and FAD are prosthetic groups (coenzyme) of redox enzymes known as flavoproteins. – Ferrodoxine reductase, succinate dehydrogenase, NADH-KoQ reductase, Succinyl-KoQ reductase, glycerol-3-phosphate dehydrogenase, xanthine oxidase, acyl-CoA dehydrogenase, Glutathione reductase ■ These enzymes are essential for oxidative degradation of pyruvate, fatty acids and amino acids and also for electron transport chain. ■ Since riboflavin can not be synthesized in animal tissues, the requirement is met with either food or microbial synthesis. ■ Diseases, drug use, alcohol, heavy metals and exercise are factors affecting the need for riboflavin. 49

Functions of Vitamin B2 ■ While riboflavin absorption decreases in hyperthyroidism, it increases in hypothyroidism.

■ Riboflavin excretion in the urine increases in diabetic patients.

■ Alcohol is a potent FAD antagonist.

■ Metals such as iron, copper and zinc form chelates with riboflavin and FAD and inhibits its absorption. 50

Vitamin B2 Deficiency ■ Riboflavin deficiency causes several non-specific signs and symptoms in animals, including mucus membrane inflammation, alopecia, dermatitis, anemia, photophobia, corneal vascularization, and cataracts. – Rats: vascularization on the stomach, congenital disorders – Chickens: Paralysis Source: Medlibes – Pigs: Hair loss, dermatitis, sensitivity to light, growth disorders occur. – Dogs: Growth regression, alopesia, drowsiness, dermatitis, hypoplastic anemia, bradycardia – Chickens: Growth stops, stroke and death ■ Deficiency of this vitamin does not usually lead to life-threatening conditions. Source: NCSU ■ Erythrocytic glutathione reductase activity has been used to assess riboflavin deficiency. 51 52

Vitamin B3 ■ Also called Nicotinic acid/Niasin, Nicotinamide, Vitamin PP and Antipellagra Factor. ■ Genel Bakış – First identified by Hugo Weidel in 1873, it was revealed by Conrad Elvehjem in 1937 that was the preventive factor of pellegra. – It is a component of NAD+ and NADP+. – Dietary nicotinamide, niacin, and Trp can all give rise to NAD+. – The Trp content of some food sources may be quantitatively more important in generating NMN, and thus NAD+, than niacin itself. – Coenzymes generated from niacin are best associated with dehydrogenase reactions. – Niacin deficiency can result in pellagra. 53

Vitamin B3

■ Vitamin B3 is also called vitmain PP. This name is given by taking the initials "pellegra preventive" because of the prevention of the pellagra disease that occurs in the absence of vitamin. ■ Nicotinic acid (niacin) can be converted to nicotinamide in the organism, and nicotinamide can also be converted to nicotinic acid. ■ Chemically it is one of the simplest structured vitamins. 54

Vitamin B3 ■ There are anti-vitamins and antagonists with a basic pyridine structure. Two of the most important are 3-acetyl pyridine and pyridine sulfonic acid. ■ This water-soluble vitamin is a component of the most central electron carrier substances in living cells, the nicotinamide adenine dinucleotides (i.e., NAD+/NADH, and NADP+/NADPH). – Therefore, there are functions in several important metabolic pathways (e.g., the Embden Meyerhoff pathway (EMP), the hexose monophosphate shunt (HMS), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and fatty acid biosynthesis and oxidation). – These electron carriers play a widespread role in many dehydrogenase enzyme reactions occurring in both the cytosol and within mitochondria. – NAD+- linked dehydrogenases catalyze redox reactions in oxidative pathways, whereas NADP+-linked dehydrogenases (or reductases) are found in pathways concerned with reductive biosynthesis. 55

Vitamin B3 Food Sources ■ Niacin is most commonly found in meat and liver.

■ In addition, yeast, green vegetables, tea, coffee, walnuts, nuts, wheat, rye and legumes are rich sources. ■ Milk, dairy products, eggs and fruits are insufficient for niacin. 56

Absorption and Metabolism of Vitamin B3 ■ Niacin and nicotinamide are effectively absorbed in physiological and pharmacological quantities. ■ Those reaching the intestinal lumen in the form of coenzymes (NAD, NADP) are absorbed after being hydrolyzed. ■ Niacin is transported through the structure of the red blood cells and rapidly passes into the kidney, liver and adipose tissue. ■ In blood, brain, liver and kidney cells, both niacin and nicotinamide are converted into coenzyme forms. ■ Storage in the body is of insignificant levels. ■ Coenzymes have a wide distribution in the body, but concentrated is in the liver. ■ The main route of excretion of niacin and its metabolites is the urinary tract. 57

Absorption and Metabolism of Vitamin B3 ■ Dietary nicotinamide, niacin (nicotinate), and tryptophan (Trp) can all give rise to nicotinate mononucleotide (NMN), a precursor to both NAD+ and NADP+, by enzymes present in most cells. ■ Dietary nicotinamide, which is naturally present in most plant and animal foods, must first undergo deamidation to nicotinate (which is added to many animal pet foods and complete feeds). ■ This compound is then converted to desamido-NAD+ by reaction first with 5-phosphoribosyl-1-pyrophosphate (PRPP), forming NMN, and then by adenylylation with ATP. The amido group of glutamine (Gln) then contributes to form the coenzyme NAD+, which may be further phosphorylated to form NADP+. ■ In many animals, NMN can also be synthesized from the essential amino acid Tryptophan (Trp), which is taken from food sources. It can not be synthesized in a large part of cats and fish. – Although only about 1/60th of dietary Trp is normally utilized in this manner, this amount may increase 3-fold with pregnancy. 58

Absorption and Metabolism of Vitamin B3 ■ It should be noted that the Trp content of some food sources may be quantitatively more important in generating NMN, and thus NAD+, than niacin itself. – For example, cow's milk normally has about 11 "niacin equivalents" in its Trp content, yet only one niacin equivalent in its niacin content (an 11:1 ratio). – For beef this ratio is about 21:2, eggs 19:1, wheat flour 5:2, yet for corn it is about 2:5.

– Additionally, pyridoxal phosphate (the active form of vitamin B6) is needed to convert Trp to NMN. Therefore, in some dietary situations, a vitamin B6 deficiency may lead to a deficiency of NMN. ■ Excess dietary leucine (e.g., from sorghum) has also been reported to contribute to niacin deficiency by inhibiting the key enzyme that converts Trp to NMN. ■ Symptoms of niacin deficiency can also be seen by administering certain drugs that divert Trp toward direct serotonin (5- hydroxytryptamine) formation. – Antidepressants (such as fluoxetine, paroxetine, sertraline) 59

Functions of Vitamin B3 ■ Nicotinamide is found in tissues as nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). ■ NAD+ and NADP+ act as coenzymes of oxidoreductase enzymes. ■ NAD+ and NADP+ coenzymes are of great importance for oxidation- reduction systems, which are particularly involved in the carbohydrate, protein and lipid metabolism. Some important reactions they have been taking; – Carbohydrate metabolism: Glycolysis, Krebs cycle – Lipid metabolism: Glycerol synthesis and degradation, Fatty acid synthesis and oxidation, Steroid synthesis – Protein metabolism: Synthesis and degradation of amino acids, oxidation of carbon chains via the Krebs cycle – Synthesis of rhodopsin and photosynthesis. 60

Functions of Vitamin B3 ■ One of the important functions of Niacin is to participate in the structure of the "glucose tolerance factor" which is an organic chromium complex and increases the insulin response of the organism.

■ An essential amino acid, tryptophan insufficiency, indirectly leads to niacin deficiency. In such cases, sudden loss of appetite, severe diarrhea, dehydration and death are seen.

■ It is suggested to give 5-6 grams of niacin per animal per day for cows that has high milk yield and ketosis. Since the synthesis in this animal can not be met the requirement. It was also found that these practices also increased the amount of milk fat. 61

Vitamin B3 Deficiency ■ Niacin deficiency results in weakness (lassitude), indigestion and inappetence, and later in the classic signs of "pellagra"; the "3 Ds" – dermatitis, diarrhea, and dementia. ■ Black Tongue disease in dogs are seen. ■ Gastrointestinal problems, diarrhea, dehydration, ulceration in the tongue and mouth, weight loss and loss of appetite are seen.

Source: Canidae Source: Hellenicdermatlas 62 63

Vitamin B5 ■ It is also named as Pantothenic Acid and Antidermatitis Factor. ■ Overview – It is discovered by Roger J. Williams in 1933. – Pantothenic acid gives rise to two coenzymes, 4- phosphopantetheine and coenzyme A (i.e., CoA.SH). – 4-Phosphopantetheine is a prosthetic group for acyl carrier protein, which participates in fatty acid biosynthesis. – The reactive thiol (-SH) group of CoA.SH serves as a carrier (and activator) of acyl groups, most notably in degradative energy- yielding pathways. 64

Vitamin B5 ■ Pantothenic acid is an amide formed from pantoic acid (2,4- dihydroxy-3,3-dimethyl butyric acid) and β-Alanine.

■ Pantothenic acid is incorporated into the biologically active form of the coenzyme A (CoA) and 4-Phosphopantetheine (the acyl carrier protein-ACP). – Both compounds participate in very important reactions in the metabolism of carbohydrates, lipids and proteins. 65 66

Vitamin B5 ■ Pantothenic acid is derived from the word "pantos", which means everywhere in Greek. ■ Since the quantities contained in foodstuffs do not meet the requirements of monogastric animals and birds during the growing and laying periods, they have to be added to the feedstuffs. ■ However, levels obtained with foodstuffs are sufficient for humans and ruminants.

■ Food Sources: Meat, heart, kidney, liver, eggs, milk, rice, soybean, yeast. 67

Absorption and Metabolism of Vitamin B5 ■ Pantothenic acid may be present in linked or free form in foods. – The bound forms of the coenzymes are coenzyme A and 4- Phosphopantetheine (acyl carrier protein (ACP)). The linked form must be converted to free form before absorption. – Coenzymes are hydrolyzed with intestinal pyrophosphatase and phosphatase to pantotheine, pantotheine and pantothenate are absorbed by diffusion, and pass to portal circulation. The pantothenate brought to the liver by the portal circulation is incorporated into the coenzyme A structure. ■ Approximately 80% of the vitamin in animal tissues is found in the form of coenzyme A. ■ When taken too much, it is released into the urine as free vitamin. ■ Despite its presence in the liver and kidney, it is not stored in the tissues. ■ It is found in the form of coenzyme A inside red blood cells, while in serum free form. 68

Functions of Vitamin B5 ■ Thioesters of CoA.SH play a central role in pathways of energy metabolism, and 4-phosphopantetheine is an important prosthetic group in acyl carrierprotein (ACP), which participates in reactions concerned with fatty acid biosynthesis. Some examples of reactions involving these pantothenic acid derivatives are as follows: – The first step in the TCA cycle: Ttransfer of an acetyl group from acetyl-CoA to oxaloacetate, thus forming citrate – The fifth step in the TCA cycle: Conversion of succinyl-CoA to succinate. – Activation of long-chain fatty acids in the cytoplasm and propionate in mitochondria – Mitochondrial β-oxidation of fatty acids – Cytoplasmic synthesis of cholesterol from 3-hydroxy-3-methylglutaryl-CoA (HMG- CoA). – Mitochondrial synthesis of ketone bodies – Acetylcholine biosynthesis – Porphyrinbiosynthesis – Fatty acid or acetate transfer to polypeptides, including some enzymes, receptors, and hormones. 69

Vitamin B5 Deficiency ■ Symptoms of pantothenic acid insufficiency in experimental studies conducted in humans and animals; – Decrease in growth and decrease in feed consumption, skin lesions, various disorders in the nervous system, digestive system disorders, decrease in antibody synthesis, decrease in resistance to infections, and inadequate adrenal function. – Growth problems in chickens, coat discolored and dermatitis occur, reproductive failure occurs. – In Pigs; hair loss, diarrhea, gastrointestinal disorders, growth retardation, respiratory and digestive failure are seen. – Dogs have anemia and fatty liver. – No apparent deficit symptoms of pantothenic acid have been observed in humans. 70

Vitamin B6 ■ Overview – It is discovered by Paul György in 1934.

– Vitamin B6 refers to a group of chemically very similar compounds that can be converted into each other in biological systems; vitamers. These include pyridoxol (pyridoxine), pyridoxal and pyridoxamine.

– Vitamin B6 is used in muscle glycogenolysis, – It is used in erythrocytes and bound to hemoglobin. – Pyridoxal phosphate is used in transamination reactions. – Although rare in animals, a vitamin B6 deficiency can result in increased amounts of amino acid metabolites appearing in urine, and it can reduce conversion of Trp to NAD+. 71

Vitamin B6

■ Three forms of vitamin B6, which have three different active forms, pyridoxol (pyridoxine), pyridoxal, and pyridoxamine, differ only in terms of the functional groups at position carbon 4. ■ In pyridoxol is an alcohol, pyridoxal is an aldehyde and pyridoxamine is an amine.

Pyridoxal Pyridoxine Pyridoxamine

– Pyridoxine, the form most prominent in plants, and for the phosphorylated coenzyme derivatives. – Pyridoxal and pyridoxamine phosphate, common forms found in animal tissues 72

Vitamin B6 ■ Coenzyme forms of Vitamin B6 are pyridoxal phosphate (PLP) and pyridoxamine phosphate. ■ Pyridoxine, pyridoxamine and pyridoxal are major transport forms available to cells, and account for most of the vitamin in plasma. ■ Once inside cells they become phosphorylated. In erythrocytes, this vitamin becomes concentrated about four- to five-fold, where it is bound mainly to hemoglobin (it may enhance O2 binding). 73

Vitamin B6 Food Source

■ Vitamin B6 acts as a coenzyme for many enzymes that are involved in carbohydrate and lipid metabolism and protein metabolism.

■ In general, meat, liver, green leafy vegetables, cereals, rice husks, hard-shelled foods are rich sources of vitamin B6. 74

Absorption and Metabolism of Vitamin B6 ■ It is absorbed by the passive diffusion from the jejenum (largely) and ileum.

■ Vitamin B6 is phosphorylated when taken into the cell.

■ Absorbed B6 compounds are rapidly transported to the liver and are often converted to pyridoxal phosphates (PLP). This is the most active form of vitamin in the metabolism; its coenzyme form. ■ The pyridoxal and pyridoxal phosphate in the circulation is mainly transported in the plasma by binding to albumin and erythrocyte hemoglobin. ■ This vitamin is found in erythrocytes bound to hemoglobin. It supports oxygen binding. ■ It is excreted via urine in the form of pyridoxic acid. 75

Functions of Vitamin B6

■ Vitamin B6 coenzymes participate in over 100 different enzyme- catalyzed reactions, most of which occur in all living cells, and some of which are only present in liver and kidney cells (main sites of gluconeogenesis). ■ They are involved with muscle glycogenolysis, and with amino acid metabolism, where they function in numerous transamination, decarboxylation, dehydratase, and side-chain cleavage reactions. ■ Pyridoxal phosphate (PLP) is an important coenzyme which aids the action of muscle glycogen phosphorylase, the enzyme mediating glycogen breakdown. ■ Muscle phosphorylase may account for as much as 70- 80% of total body vitamin B6 in mammals. 76

Functions of Vitamin B6

■ Transamination reactions involving vitamin B6 include those concerned with the synthesis of nonessential amino acids, and also those concerned with the first step in amino acid catabolism. Transaminases are rate-limiting for the catabolism of specific amino acids in the liver. ■ Decarboxylation reactions involve the synthesis of several important substances, including neuroactive amines (e.g., serotonin, histamine, and γ-aminobutyric acid (GABA)), Δ-aminolevulinic acid (dALA) (the first step in heme biosynthesis). – These enzymatic reactions are particularly important when they occur in vital organs such as the brain, heart, and liver.

– due to vitamin B6 insufficiency in the brain, glutamate decarboxylase and Δ- aminobutyric acid transaminase enzymes are inactived. Neurological disorders such as convulsion occurs as a result of enzyme inactivation. 77

Functions of Vitamin B6

■ B6 vitamin is involved, – In the synthesis of sphingomyelin, lecithin, carnitine and taurine. • Taurine is important as a bile acid conjugate, and also in brain and eye function. – In the reactions of deaminases (especially serine, threonine and cysteine). – In the reactions of sulfuric amino acids. – In the synthesis of niacin from tryptophan, – In the synthesis of Δ-aminolevulinic acid from glycine and succinyl CoA, which constitute the first step of porphyrin synthesis, – In the biosynthesis of arachidonic acid from linoleic acid in the metabolism of essential fatty acids, – In the conversion of Phenylalanine or tyrosine to epinephrine and norepinephrine. – Also in performing the activities of the rasemases that enable the microorganisms to use D-amino acids and are not found in mammalian tissues. 78

Vitamin B6 Deficiency ■ Deficiencies due to lack of pyridoxine are rare in animals. ■ Due to its importance in amino acid catabolism, a deficiency of this vitamin can result in urinary excretion of increased levels of some amino acid metabolites. + ■ A lack of B6 can also reduce NAD formation from tryptophan. ■ General deficiency symptoms include hyperirritability and convulsive seizures in infants, and inflammation of the oral cavity and oily dermatitis in adults. ■ In rats deficiency leads to dermatitis in some parts of the body and decrease in growth rate. ■ Degenerations and hypersensitivity in the nervous system. ■ In chickens, the growth rate and laying yield decrease. 79 80 81

Vitamin B7 ■ It is also known as Biotin, Vitamin H and Coenzyme R or Beauty Vitamin. ■ Overview – Biotin participates in carboxylation reactions.

– Biotin, pantothenate (B5), and cobalamin (B12) are needed by herbivores to move propionate into hepatic gluconeogenesis. – Biotin-supplemented diets are sometimes fed to young, growing animals. – Raw egg white reduces intestinal biotin absorption. 82

Vitamin B7 ■ Biotin is a heterocyclic compund which has two rings of 5 atoms containing sulfur (S). ■ This water-soluble B- complex vitamin is a widely distributed imidazole derivative found in plants in the free form, but in animals it is linked to protein. 83

Vitamin B7 Food Sources ■ Biotin is naturally found in free form in vegetables, fruits, milk and rice bran, and is found in animal tissues, in plant seeds and in yeast due to large amounts of protein. ■ Ruminants and rabbits meet the need for microbial synthesis. ■ Vegetable sources such as fresh vegetables and fruits; animal sources such as liver, kidney, pancreas, eggs, yeast and milk are rich sources of biotin. ■ Corn, cereals such as wheat, meat and fish are insufficient in terms of biotin. 84

Absorption and Metabolism of Vitamin B7 ■ During digestion, biotin is initially released from protein as a lysine- adduct (biocytin), and either further digested to free biotin, or absorbed as such and hydrolyzed within intestinal mucosal cells. ■ The enzyme involved in this process (sometimes referred to as biotinidase), may also be the biotin carrier protein (BCP) associated with this vitamin both intracellularly and within serum. ■ Intestinal absorption of this vitamin in the jejunum is thought to occur by facilitated diffusion at low concentrations, and by simple diffusion at high concentrations. ■ Although biotin deficiencies are rare, they can be induced through use of broad-spectrum oral antibiotics over a long period of time (which reduces microbial biosynthesis), or through excessive consumption of raw eggs. 85

Absorption and Metabolism of Vitamin B7 ■ Raw egg whites contain the active form of avidin, a protein that binds tightly with biotin, thus making it unavailable for absorption from the digestive tract. Therefore, raw eggs should not be fed to young animals who need biotin for proper growth and development. – Since heat denatures avidin, cooked egg consumption is not associated with biotin deficiency. ■ The biotin in blood circulation is transported to liver, kidney and muscles, and binds to the carboxylases in the cytosol and mitochondria. ■ It is found all cells but mostly concentrated in liver and kidney. ■ The distribution between cells varies according to the localization of biotin-dependent enzymes. 86

Functions of Vitamin B7

■ Biotin makes CO2 fixation in carboxylase reactions, or transfers a molecule of carboxyl group to another molecule.

■ Biotin, an essential coenzyme in carbohydrate, fat and protein metabolism, is of great importance for gluconeogenesis in situations where adequate carbohydrates are not available. In particular, biotin, pantothenic acid and cobalamin are involved in the transport of propionate to the hepatic gluconeogenesis pathway in herbivores.

■ It is the coenzyme of carboxylase, decarboxylase, transketolase enzymes. 87

Functions of Vitamin B7 ■ The function of biotin is to participate in carboxylation reactions. 1. First, a carbonic phosphoric anhydride is formed through combination of a high energy - phosphate from ATP, CO2 derived from HCO3 , magnesium (Mg++), and potassium(K+). 2. Next, biotin carboxylase (sometimes referred to as holoenzyme synthetase), and manganese (Mn++) help to facilitate movement of the activated carboxyl group of carbonic phosphoric anhydride to biotin, thus forming carboxybiotin. 3. This compound now becomes available to various carboxylases (or transcarboxylases) for one carbon transfer. 88

Functions of Vitamin B7

■ There are four important biotin-driven "CO2-fixing" reactions that occur in animal cells. 1. Conversion of pyruvate to oxaloacetate (OAA, via pyruvate carboxylase) • Enzymes are especially important for hepatic and renal gluconeogenesis and muscle tissue during exercise. 2. Conversion of propionyl-CoA to methylmalonyl-CoA (via propionyl-CoA carboxylase). • Propionate is an important source of hepatic gluconeogenesis in herbivores. • It is converted to succinyl-CoA via methylmalonyl-CoA and incorporated into the TCA cycle. Cobalamin is required in this reaction. 3. Formation of acetoacetate from leucine (via β-methylcrotonyl- CoA carboxylase). • Synthesis of Ketone bodies. 4. Formation of malonyl-CoA from acetyl-CoA in fatty acid biosynthesis (via acetyl-CoA carboxylase). 89

Vitamin B7 Deficiency ■ Biotin deficiency is not usually caused by simple dietary deficiency, but by defects in utilization. Symptoms can include dermatitis, alopecia, and muscle weakness. In young animals, extra demands on biotin, a vitamin needed for proper growth and development, are sometimes met by feeding biotin-supplemented diets. – Biotin is very important for the functions of thyroid and adrenal glands, reproductive system and the nervous system. – In avians, slowing in growth, perosis, skin lesions in beaks, eyes and squirrels are seen. – In dogs, paralysis, growth retardation, skin lesions (dry, dull appearance, alopecia) are seen. – In horses, hair loss, skin lesions and dermatitis are seen. ■ Biotin, like other B-complex vitamins, is not considered to be toxic, and supplementation can Source: AnimalWellnessMagazine reverse these symptoms. 90 91

Vitamin B9 ■ Other names are Folic Acid/Folate, FA, Folacin, Pteroylglutamate and Anti-anemic Factor. ■ Overview – Thymidylate synthase is required for the synthesis of DNA. – Folic acid can be synthesized by bacteria, but not animals.

– Drugs that inhibit H2 folate reductase, or thymidylate synthase, are effective anticancer agents.

– The megaloblastic anemia associated with vitamin B12 deficiency can be partially alleviated by extra folate in the diet. – Serine becomes a methyl donor in dTMP, and thus contributes positively to DNA formation. – Thioredoxin and NADPH aid in the conversion of NDPs to their corresponding dNDPs for DNA synthesis. – Antimetabolites that inhibit ribonucleoside diphosphate reductase (RDR) reduce the number of dNTPs available for DNA synthesis. 92

Vitamin B9 ■ It is a colorless, tasteless, odorless substance which is very common in plants,low soluble in water, insoluble in alcohol, ether, and other organic solvents and in crystalline form. ■ Solvents are sensitive to heat ultraviolet rays and are affected by acids and oxidation. ■ Folic acid (FA) is found in almost all living cells as natural conjugates. ■ It consists of a molecule of para-aminobenzoic acid (PABA), with its amino end attached to a pteridine, and its carboxyl group attached to the α-amino group of glutamic acid (Glu). It can not be synthesized in mammals, whereas in some species they are synthesized by intestinal microorganisms.

■ Tetrahydrofolate (THFA or H4 folate), the active coenzyme form of FA, is formed by reduction of the pteridine ring at positions 5, 6, 7, and 8 by the enzyme, folic acid reductase. 93 94

Vitamin B9 ■ THFA is an essential molecule in both prokaryotic and eukaryotic cell metabolism. ■ It donates one-carbon units and other small molecular building blocks to the biosynthesis of purines, dTMP and selected amino acids, including methionine.

■ It is a coenzyme that carries H4 Folate (THFA) both a carbon unit and small molecular building blocks. 95

Vitamin B9 ■ Green plants, soybean, hazelnut, and most animal products (especially liver) are rich in folic acid, while grain feed, milk and eggs are poor in folic acid. 96

Absorption and Metabolism of Vitamin B9 ■ Neither animals nor bacteria can survive without a source of FA. Animals cannot synthesize FA and, therefore, they evolved a means of assimilating preformed FA from their diet. – In contrast to animals, bacteria cannot assimilate FA from their environment, and therefore they evolved enzymatic machinery to synthesize it from an appropriate pteridine, PABA, and Glu. – The difference between animals and bacteria in their capacity to assimilate and synthesize FA has been exploited to design bacteria-selective sulfonamide antibiotics. – Sulfonamides are analogs of PABA, and thus act as competitive inhibitors of PABA's participation in bacterial de novo FA biosynthesis. – Since bacteria cannot assimilate preformed FA from their environment, the competitive inhibition of PABA incorporation severely reduces their FA levels, thus inhibiting growth and replication. – In contrast, animals, which assimilate FA rather than synthesize it from PABA, generally suffer no ill effects from sulfonamides. 97

Absorption and Metabolism of Vitamin B9 ■ Dietary FA is frequently polyglutamated. Extra glutamates are removed enzymatically (γ-carboxypeptidase) in the small intestine prior to FA absorption, and most of the FA is then reduced in mucosal cells of the gut. ■ Reduction requires the presence of NADPH and FAR, and proceeds in two steps-first to dihydrofolate (DHFA or H2 folate), and then to THFA. ■ Once formed, THFA picks up the one-carbon methylene group from 5 10 serine (Ser) to form N ,N -methylene-H4 folate. This compound plays a central role in one-carbon unit metabolism. – It can be used as a source of the methyl carbon of thymine, or it can be 5 enzymatically reduced with NADH to N -methyl-H4 folate, an important coeznyme in methylcobalamin (vitamin B12)-dependent methylation of homocysteine to Met. 98

Absorption and Metabolism of Vitamin B9 ■ Dietary FA is normally absorbed in the proximal part of the small intestine (jejunum), with mucosal cells reducing most of it to DHFA, and then to THFA. ■ Therefore, low plasma folate concentrations can be indicators of proximal bowel dysfunction. On the other hand, intestinal bacterial overgrowth will sometimes promote excessive folate production, thus creating high plasma folate concentrations. ■ Monoglutamate (primarily 5-methyl tetrahydrofolate) is stored after it has been converted into polyglutamates of folic acid which is transported to storage tissues. In humans, the total folic acid storage is 5-10 mg and approximately half of this amount is stored in the liver. When they are given to the blood for use, they are again hydrolyzed to monoglutamates. ■ The excretion is carried out with urine and feces. 99

Functions of Vitamin B9 ■ The active coenzyme form of Folic acid is 5,6,7,8- tetrahydropteroylglutamic acid (5,6,7,8-tetrahydrofolic acid- THFA). ■ Despite being sufficient intake and absorption of folic acid, vitamin B12 insufficiency can cause folic acid insufficiency secondarily. Because it is an importan coeznyme in methylcobalamin (vitamin B12)-dependent methylation of homocysteine to Met. ■ In cobalamin insufficiency, the increase in the amount of methyl- THFA and the decrease in the THFA are formed. THFA is required for other essential biosynthetic processes-in particular the synthesis of the essential DNA precursor. ■ In deficiency, DNA synthesis is inhibited and as a consequence, erythroblast division and maturation in bone marrow is compromised, promoting formation of megaloblasts with characteristic large, polymorphic nuclei. 100

Functions of Vitamin B9 ■ In the transfer of two carbon units, pantothenic acid acts as coenzyme, while transfer of one carbon units THFA acts (methyl CH3, methylene CH2, formyl CHO). ■ Reactions in which one-carbon unit transfer activated by folic acid is carried out include: purine and pyrimidines, conversion of serine and glycine to one another, degradation of histidine, resynthesis of methyl groups for compounds such as methionine, choline and thymine. ■ Protein and nucleic acid synthesis can not be done adequately and cell division slows down due to the interruption of these reactions mentioned in folic acid deficiency. The red blood cells do not mature and the hematopoiesis stops in the course of megaloblastic cell formation. 101

Functions of Vitamin B9 ■ Folic acid is also very important for immune system activities.

■ In folic acid deficiency, responses to T lymphocytes versus mitogens are regressed, and thymus functions are reduced.

■ In the studies performed, it is concluded that folic acid was found to be necessary for exocrine activity of the pancreas. 102

Vitamin B9 Deficiency ■ Growth rate in chickens decreases. ■ Reductions in hemoglobin concentration and leukocyte, erythrocyte and platelet counts. ■ Leukopenia, megaloblastic anemia, loss of appetite, weakness, gingivitis and diarrhea occur in animals and humans. ■ In Ruminants; Since folate is synthesized in the rumen, no signs of failure are seen in animals that have completed the development of rumen. However, calves and lambs who have not completed development of rumen is considered that it is an essential vitamin, and in cases of failure, pneumonia, diarrhea and death can be seen following the leukopenia, especially in the lambs. 103

Vitamin B9 and Cancer ■ Many types of cancer cells grow and divide more rapidly than their normal counterparts, and therefore their growth and viability are highly sensitive to antimetabolites which inhibit nucleic acid biosynthesis, in particular DNA biosynthesis. ■ There are several examples of antimetabolites that kill cancer and other proliferating cells by interfering with their ability to produce an adequate supply of dNTPs for DNA synthesis. Methotrexate (MTX) is one of them. ■ MTX is a FA structural analog that acts as a competitive inhibitor of FAR, with an affinity for the enzyme at least 100 times greater than that of FA or DHFA. MTX is widely used in the treatment of leukemias and solid tumors. 104 105 106

Vitamin B12 ■ Other names are Cobalamin and Antipernicious Anemia Factor. ■ Overview

– Vitamin B12 is generally absent from plant and vegetable foods unless they are contaminated by microbes.

– Liver is a good source of the three endogenous forms of vitamin B12 (methylcobalamin, 5'-deoxyadenosylcobalamin, and hydroxocobalamin).

– Ileal absorption of B12 requires intrinsic factor, which is synthesized by gastric parietal cells, as well as by pancreatic ductular cells in dogs and cats. +++ – The association of Co with B12 is the primary recognized action of this trace element in mammalian metabolism. – Entry of propionate into hepatic gluconeogenesis requires 5'deoxyadenosylcobalamin.

– The metabolism of vitamin B12 is intimately entwined with that of folic acid.

– Common symptoms of vitamin B12 deficiency include homocystinuria and methylmalonuria. – A secondary intestinal dysfunction may develop from persistent cobalamin deficiency. 107

Vitamin B12 ■ It was recognized in the early 1800's that pernicious (or megaloblastic) anemia may (in part) be due to a disorder of the digestive tract and assimilative organs. ■ It was determined in the early 1900's that this condition could be reversed and controlled by eating raw or mildly-cooked liver. ■ Therefore, investigators postulated that a gastric "intrinsic factor (IF)," combined with an "extrinsic factor" from ingested liver, would bring about absorption of an "antipernicious anemia factor." ■ The extrinsic factor was later found to be the antipernicious anemia factor, cobalamin, and the IF was determined to be an important glycoprotein, secreted into gastric or abomasal juice by parietal cells, and additionally by pancreatic ductular cells in dogs and cats. 108

■ Vitamin B12 is a dark red, water and alcohol soluble, acetone, chloroform and ether-insoluble crystalline material. ■ Cobalamin is a complex molecule, consisting of a "corrin ring," which is a more hydrogenated form of the porphyrin ring associated with heme, with differences as well in the side chains of the ring. ■ Cobalamin contains Co+++ rather than Fe++, and a 5,6- dimethylbenzimidazole grouping attached to the corrin ring through a complex linkage, involving an unusual ribose-phosphate moiety. 5,6-Dimethylbenzimidazole grouping 109

■ The 5,6-dimethylbenzimidazole grouping is also chelated to Co+++ at the active center of the corrin ring.

■ Vitamin B12 exists in four forms that differ in the nature of additional R groups attached to Co+++.

■ If R group is; – CN, so called Cyanocobalamin, – OH, so called Hydroxocobalamin,

– CH3, so called Methylcobalamin, – 5'-deoxyadenosyl, so called 5'- deoxyadenosylcobalamin 5,6-Dimethylbenzimidazole grouping 110

■ The cyano derivative, commonly known as cyanocobalamin, is the commercially available form. ■ After transport in blood, cobalamin is usually taken-up by target cells as hydroxocobalamin, where it can be converted to methylcobalamin in the cytoplasm, or 5'- deoxyadenosylcobalamin in mitochondria. ■ In the liver, it is found in all three forms. The association of Co+++ with vitamin B12 is the primary recognized function for this trace element in

5,6-Dimethylbenzimidazole mammalian metabolism grouping 111

Vitamin B12 Food Sources ■ The animal tissues are rich in vitamin B12. Especially ruminants have got it in high concentrations in the rumen (50 μg/100 g dry weight) and are from rumen microflora. In addition, ruminants have higher liver vitamin levels than monogastric animals.

■ Vitamin B12 in animal-derived foods is in a form bound to the proteins and the protein is separated by the acid pH and pepsin effect. However, the free vitamin B12 is not absorbed directly. 112

Absorption and Metabolism of Vitamin B12

■ To absorb vitamin B12 it is necessary to attach an intrinsic factor of glycoprotein structure at a molecular weight of about 50,000 daltons produced in the parietal cells of the gastric mucosa. ■ The binding of vitamin to intrinsic factor results in absorption from the distal mucosal cells. ■ It is the greatest essential molecule absorbed from the ileum. ■ Once absorbed from the ileum, the vitamin is separated from the intrinsic factor and transferred to the specific carrier protein of plasma. ■ It then binds to the receptor on the surface of the tissue cell that is to be transported and enters the cells. ■ In cytoplasm Methylcobalamin; In mitochondria, 5'- deoxyadenosylcobalamin is the form of coenzymes. Hydroxocobalamin is a transitional molecule. 113

Functions of Vitamin B12 ■ There are two important enzymatic reactions in animals that require vitamin B12: 1. Rearrangement of methylmalonyl- CoA to succinyl-CoA: Requires 5'- deoxyadenosylcobalamin. It happens in mitochondria. 2. Transfer of a methyl group from N5- methyltetrahydrofolate (N5-methyl- H4 folate) to homocysteine in the formation of methionine: Requires methylcobalamin. It happens in cytoplasm. 114

Functions of Vitamin B12 ■ The first reaction, which requires 5'-deoxyadenosylcobalamin, is important in the sequential conversion of propionate to succinyl- CoA, an intermediate of the TCA cycle. – Propionate is formed, • Microbial cellulose and starch digestion, • The terminal 3 carbons of odd-chain fatty acids during mitochondrial b-oxidation, • β-aminoisobutyrate during pyrimidine degradation, • Several amino acids during protein degradation. It is of particular significance in the process of hepatic gluconeogenesis.

■ Through the second reaction, which requires methylcobalamin, H4 folate is made available to participate in purine, pyrimidine, and nucleic acid biosynthesis.

– The metabolism of vitamin B12 is thus intimately entwined with that of another water-soluble vitamin, folic acid, and both are fundamental to one-carbon metabolism. 115

Functions of Vitamin B12 ■ Other reactions involving this vitamin, as a coenzyme or otherwise, cannot be excluded at this time. It has proposed involvement in the synthesis of either the lipid or protein components of myelin, independent of the methylmalonyl-CoA reaction. This would help to explain the demyelination (or lack of myelination), and nerve degeneration observed in B12 deficiency. ■ A deficiency of this vitamin also appears to result in a loss of tissue carnitine, perhaps in the form of a propionic acid adduct entering blood and urine. However, this could occur because of methylmalonate and propionate accumulation due to their lack of conversion to succinyl-CoA via methylmalonyl-CoA isomerase (or mutase). ■ Less carnitine would have consequences for the shuttling of long- chain fatty acids across mitochondrial membranes 116

Vitamin B12 Deficiency ■ In humans;

– Diet-related factors: Vegetables do not contain vitamin B12, so vitamin B12 deficiency can occur in vegetarians. – It is often seen in vegetarians. Complete or partial malnutrition from meat, milk, dairy products and egg causes the formation of vitamin B12 deficiency. – At the same time, infants who are dependent on their parents who are in such a state of incapability are also affected by the same disability. – Clinical manifestations are anorexia, irritability, inadequate brain and intelligence development in childhood. – Due to disorder of absorption and transport disorder and/or intrinsic factor insufficiency leads to pernicious anemia (megaloblastic anemia). – Although the underlying causes of deficiency are disorders that inhibit absorption in the small intestine, overdose of alcohol and certain drugs, chronic pancreatitis, ileal B12 receptor deficiency and inadequacy of B12 transport proteins may also result in insufficiency. 117

Megaloblastic Anemia (Pernicious Anemia)

• Characteristic signs are, • Poikilocytosis • Anisocytosis, • Anemia, • Atrophy in gastrointestinal mucosa • Degenerative disorders in the spinal cord.

Source: Routh and Koeing, 2014 118

Vitamin B12 Deficiency ■ In humans;

– Storage: Vitamin B12 stores are about half the daily requirement in humans. In addition to dietary sources, the B12 is synthesized in significant amounts by microflora in the intestine, which is responsible for the continuity of the deposits. – Hereditary factors: Pernicious anemia is an autosomal dominant condition that occurs in individuals as intrinsic factor deficiency.

■ The inadequacy of vitamin B12 in pregnancy with folic acid significantly increases the risk of neural tube defect (NTD). – At the same time, the risk of cardiovascular and cerebrovascular disease increases with the development of hyperhomocysteinemia. ■ Decreased growth rate in chicks, reduced laying yield in adults are seen. 119 120 121 122 Vitamin Like Compounds

Lipoic Acid (Thioctic acid), Myo-inositol, Carnitine (Vitamin BT),

Essential Fatty Acids (Vitamin F), Flavonoids (Vitamin P), Choline 124 Lipoic Acid (Thioctic acid) ■ Lipoic acid is also consider a B-complex vitamin. ■ Also known as α-lipoic acid (ALA). ■ It’s only known function is to participate in the oxidative decarboxylations of a-ketoacids. Conversion of; 1.Pyruvate to acetyl-CoA, (Pyruvate dehidrogenase enzyme complex) 2. α-ketoglutarate to succinyl-CoA. ■ They are two similar dehydrogenase complexes of the TCA cycle. ■ However, it has not been shown to be essential as a dietary component of animals. 125 126 127 128 129 Myo-Inositol ■ It is found in plant and animal tissues. ■ It plays a role in the metabolism of mitochondria as a building block of phospholipids and in the stimulation of nerves. ■ Prevents liver to get fatty. ■ It is synthesized by animals. 130

Carnitine (formerly Vitamin BT) ■ It is found in different amounts in plant and animal tissues.

■ It was originally labeled vitamin BT; however, because carnitine is synthesized in the human body, it is no longer considered a vitamin. ■ Carnitine is involved in transporting fatty acids across the mitochondrial membrane, by forming a long chain acetylcarnitine ester and being transported by carnitine palmitoyltransferase I and carnitine palmitoyltransferase II. Carnitine also plays a role in stabilizing Acetyl-CoA and coenzyme A levels through the ability to receive or give an acetyl group. ■ It is also a growth factor for some insects. 131 Essential Fatty Acids (EFA- formerly Vitamin F)

■ It is found in different amounts in plant and animal tissues. ■ They participate in the construction of the membrane structure as the building block of phospholipids. ■ They are used to synthesize eicosanoids. ■ Dermatitis and eczema in the case of insufficiency in rats and humans. 132 Flavonoids (formerly Vitamin P) ■ They are a class of plant and fungus secondary metabolites. ■ It is thought to have enhanced resistance of capillaries, functions as anti-histaminic, anti-inflammatory, anti-tumoral and anti- hyalurinidase activity. 133 Choline ■ Choline, essential for all organisms, is not fully included in the vitamin classification, but it is mostly studied in B complex vitamins. Unlike group B vitamins, the choline synthesized in the liver is physiologically active in its own chemical structure, not in the form of coenzymes. ■ Chemically defined as β-hydroxyethyl trimethyl ammonium hydroxide, the choline functions as a strong methyl group donor in the metabolic reactions. Soluble in water, formaldehyde and alcohol, gives a neutral reaction in water. ■ Many foods include it as sphingomyelin and lecithin. – Egg yolk (1.7%), glandular organs(0.6%), brain and fish (0.2%) are the richest animal sources in terms of the choline. In Plant choline sources, legumes (0.2- 0.35%) and fatty seeds take the first order. Soybeans, cotton seeds, peanuts are also good sources of choline. Corn is insufficien. 134 Choline ■ Absorption and Metabolism – Choline found in the food are largely in form of lecithin, less than 10% free or in the form of sphingomyelin. 50% of received lecithin is involved in circulation through the lymphatic system, while the rest is degraded into glycerophosphatidylcholine in the intestinal mucosa. ■ Functions – Phosphatidylcholine (lecithin), a phospholipid, is of great importance because it is a fundamental building block of the cell membrane and is involved in the transport of lipid molecules. Lecithin is also a major component of VLDLs, the carrier molecule of triglycerides. In the prevention of perosis, choline is important as a phospholipid component for the maturation of cartilage matrices of bones. – Choline plays a role in fat metabolism in the liver. This function is accomplished by increasing the use of fatty acids in the liver or by preventing abnormal fat accumulation. For this reason, it is also called lipotropic factor. – Acetylcholine, which is responsible for the transmission of nerve impulses, is a neurotransmitter that can be synthesized from choline. 135 Choline ■ The choline acts as a methyl group donor in conversion of homocysteine to methionine and guanidoacetic acid to creatine. ■ Synthesis of purine and pyrimidine bases, which are important in DNA synthesis, also occurs with the presence of methyl groups.

■ In the metabolism of folic acid, methionine and vitamin B12 methyl groups, methionine is a substituent that is effective in choline synthesis. ■ Studies in the rats have shown that severe folic acid deficiency causes secondary choline insufficiency of the liver. 136 References

■ Ası. T. 1999. Tablolarla Biyokimya, Cilt 2 ■ Combs GF, McClung JP. The Vitamins. 5th ed. Academic Press. ■ Engelking LR. 2014. Textbook of Veterinary Physiological Chemistry. 3rd ed. Academic Press. ■ Prof.Dr. Meryem EREN. Ders Notları (Teşekkürlerimle). ■ Sözbilir Bayşu N, Bayşu N (2008). Biyokimya, Güneş Kitabevi. ■ Traber MG. 2007. Vitamin E regulatory mechanisms. Annu Rev Nutr., 27: 347-362 137 Question 1

■ Which of the following diseases occur in Niacin deficiency?

a) Scurvy

b) Beriberi

c) Adrenal insufficiency

d) Pellegra

e) Pernicious anemia Cevap: d Cevap: 138

Questions ? Next chapter; Hormones 140

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