1.10.5 Metabolism of vitamins

5. Trimethoprim

 Allopurinol is used as drug to treat to gout, which inhibits xanthine oxidase which converts purine to uric acid.  Allopurinol is a substrate of xanthine oxidase, which converts allopurinol to oxypurinol (alloxanthine).  Oxypurinol inactivates the reduced form of the enzyme by remaining tightly bound in its active site.

1.10.5 Metabolism of vitamins

Snapshot

1. Human cells cannot perform the crucial last step of vitamin C biosynthesis, the conversion of l-gulono-g-lactone into ascorbic acid, which is catalysed by the enzyme gulonolactone oxidase. 2. The pyrimidine moiety of riboflavin is biosynthetically related to guanine. Increased concentration of purine in media increases the synthesis of riboflavin. 3. Quinolinate is the precursor to pyridine ring of NAD. 4. Higher plants, most fungi and bacteria, are phototrophic for Biotin. Others, including most vertebrates and some bacteria, rely on exogenous sources. 5. In the form of a series of tetrahydrofolate (THF) compounds, folate derivatives are substrates in a number of singlecarbon-transfer reactions, and also are involved in the synthesis of dTMP (2′-deoxythymidine-5′-phosphate) from dUMP (2′-deoxyuridine-5′-phosphate). 6. Vitamin A is required throughout life and participates in numerous cellular activities involved in reproduction, embryonic development, vision, growth, cellular differentiation and proliferation, tissue maintenance and lipid metabolism. 7. Vitamin D3 produced in the epidermis must be further metabolized to be active. The first step, 25-hydroxylation, takes place primarily in the liver, although other tissues have this enzymatic activity as well. 8. Tocopherol contain a substituted aromatic ring and a long isoprenoid side chain.

VITAMIN C

Ascorbate

 Human cells cannot perform the crucial last step of vitamin C biosynthesis, the conversion of lgulono-g-lactone into ascorbic acid, which is catalysed by the enzyme gulonolactone oxidase. (Figure 1.10.61)

Figure 1.10.61. The diversity of biosynthetic pathways f or ascorbate and its analogs Abbreviations: Ara/AraL, arabinose/arabinonolactone; Gal/GalA/GalL, galactose/galacturonic acid/galactonolactone; LGalDH, galactose dehydrogenase; L-GalLDH, galactonolactone dehydrogenase; D-GalUR, galacturonic acid reductase; Glc/GlcA/GlcL, glucose/glucuronic acid/gluconolactone; GulL, gulonolactone; L-GulO, gulonolactone oxidase; GDP, guanosine diphosphate; Man, mannose; MeGalA, methyl D-galacturonic acid; NDP, nucleoside diphosphate; UDP, uridine diphosphate.

VITAMIN B1

Thiamine diphosphate

Plants synthesize vitamin B1 (Thiamine diphosphate) in two mechanisms 1. de novo from simple precursors (Glycine, NAD+, Sulphur -donor) as seen in yeast and

2. From 5-aminoimidazole ribonucleotide (AIR) to the pyrimidine moiety of thiamine (4-amino-2methyl-5-hydroxymethylpyrimidine monophosphate, HMP-P) as seen in bacteria. (Figure

1.10.62)

Figure 1.10.62. Schematic representation of thiamine biosynthesis in plants (A) and subcellular localization of the major thiamine diphosphate-incorporating enzymes (B). The Arabidopsis genes encoding the enzymes which catalyze the biosynthetic reactions shown are specif ied. AIR, 5-aminoimidazole ribonucleotide; HET-P, 4-methyl-5-(2hydroxyethyl)thiazole phosphate; HMP-P/HMP-PP, 4-amino-2-methyl-5-hydroxymethylpyrimidine monophosphate/diphosphate; TA, thiamine; TMP, thiamine monophosphate; TDP, thiamine diphosphate; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; KGDH, α-ketoglutarate dehydrogenase; BKDH, branched-chain α-ketoacid dehydrogenase; TK, transketolase; DXPS, 1-deoxy-D-xylulose-5-phosphate synthase.

VITAMIN B2

Riboflavin

 Riboflavin (vitamin B2) is biosynthesized in plants and in many bacteria.  The pyrimidine moiety of riboflavin is biosynthetically related to guanine.

Increased concentration of purine in media increases the synthesis of riboflavin.  guanosine triphosphate (GTP) is the committed precursor of riboflavin supplying the pyrimidine ring and the nitrogen atoms of the pyrazine ring, as well as the ribityl side chain of the vitamin(Figure 1.10.63)

Figure 1.10.63. Biosynthesis of riboflavin.

VITAMIN B3:

1. Quinolinate is the precursor to pyridine ring of NAD. 2. In prokaryotes quinolinates are formed from dihydroxyacetone phosphate 3. In eukaryotes quinolinates are formed from tryptophan. 4. Humans lack an enzyme N-formylkynurenine formamidase (Figure 1.10.64)

Figure 1.10.64. Biosynthesis of vitamin B3

VITAMIN B7

 Higher plants, most fungi and bacteria, are phototrophic for Biotin. Others, including most vertebrates and some bacteria, rely on exogenous sources.  In mammals, Biotin is supplied by intestinal bacteria.  It consists of two fused rings: an Imidazol (Ureido) and a Sulfur-containing (Tetrahydrothiophene) ring; and the latter is extended via a Valeric acid side chain, which is attached in a cis-configuration with respect to the Ureido ring.  Bacterial model for Biotin metabolism includes E. coli (Escherichia coli), B. subtilis (Bacillus subtilis), B. sphaericus(Bacillus sphaericus) and R. loti (Rhizobium loti).  The Biotin biosynthetic (bio) genes in the Gram-Positive B. sphaericus are organized in two Operons located at different positions in the chromosome.  In B. sphaericus, Biotin is synthesized from Pimelic Acid involving the products of the bioW, X, F, A, D, and B genes of the bio Operon (Figure 1.10.65).

Figure 1.10.64. Biosynthesis of Biotin

KAPA (7-Keto-8-Aminopelargonic Acid) ANOS(7-Keto-8-Amino-Pelargonic Acid Synthetase). DAPA (7,8-Diaminopelargonic Acid)

Folic acid

 In the form of a series of tetrahydrofolate (THF) compounds, folate derivatives are substrates in a number of single-carbon-transfer reactions, and also are involved in the synthesis of dTMP (2′deoxythymidine-5′-phosphate) from dUMP (2′-deoxyuridine-5′-phosphate).  It is a substrate for an important reaction that involves vitamin B12 and it is necessary for the synthesis of DNA, and so required for all dividing cells.  The pathway leading to the formation of tetrahydrofolate (FH4) begins when folic acid (F) is reduced to dihydrofolate (DHF) (FH2), which is then reduced to THF. Dihydrofolate reductase catalyses the last step.  Vitamin B3 in the form of NADPH is a necessary cofactor for both steps of the synthesis. Thus, hydride molecules are transferred from NADPH to the C6 position of the pteridine ring to reduce folic acid to THF.  Methylene-THF (CH2FH4) is formed from THF by the addition of a methylene bridge from one of three carbon donors: formate, serine, or glycine. Methyl tetrahydrofolate (CH3-THF, or methyl-

THF) can be made from methylene-THF by reduction of the methylene group with NADPH.  Another form of THF, 10-formyl-THF, results from oxidation of methylene-THF or is formed from formate donating formyl group to THF. Also, histidine can donate a single carbon to THF to form methenyl-THF (Figure 1.10.66).  Vitamin B12 is the only acceptor of methyl-THF, and this reaction produces methyl-B12 (methylcobalamin).  There is also only one acceptor for methyl-B12, homocysteine, in a reaction catalyzed by homocysteine methyltransferase.  These reactions are of importance because a defect in homocysteine methyltransferase or a deficiency of B12 may lead to a so-called "methyl-trap" of

THF, in which THF is converted to a reservoir of methyl-THF which thereafter has no way of being metabolized, and serves as a sink of THF that causes a subsequent deficiency in folate.  Thus, a deficiency in B12 can generate a large pool of methyl-THF that is unable to undergo reactions and will mimic folate deficiency.  The reactions that lead to the methyl-THF reservoir can be shown in chain form:  folate → dihydrofolate → tetrahydrofolate ↔ methylene-THF → methyl-THF

Figure 1.10.65. Metabolism of f olic acid to produce methyl-vitamin B12

VITAMIN B12

Vitamin B12 normally plays a significant role in the metabolism of every cell of the body, especially affecting the DNA synthesis and regulation but also fatty acid synthesis and energy production.

 However, many (though not all) of the effects of functions of B12 can be replaced by sufficient quantities of folic acid (vitamin B9), since B12 is used to regenerate folate in the body.

 Most vitamin B12 deficiency symptoms are actually folate deficiency symptoms, since they include all the effects of pernicious anemia and megaloblastosis, which are due to poor synthesis of DNA when the body does not have a proper supply of folic acid for the production of thymine.  When sufficient folic acid is available, all known B12 related deficiency syndromes normalize, save those narrowly connected with the vitamin B12-dependent enzymes Methylmalonyl Coenzyme A mutase, and 5methyltetrahydrofolate-homocysteine methyltransferase (MTR), also known as methionine synthase; and the buildup of their respective substrates (methylmalonic acid, MMA) and homocysteine. Coenzyme B12's reactive C-Co bond participates in three main types of enzyme-catalyzed reactions. Isomerases (Figure 1.10.66)

 Rearrangements in which a hydrogen atom is directly transferred between two adjacent atoms with concomitant exchange of the second substituent, X, which may be a carbon atom with substituents, an oxygen atom of an alcohol, or an amine. These use the adoB12 (adenosylcobalamin) form of the vitamin. Methyltransferases

 Methyl (-CH3) group transfers between two molecules. These use MeB12 (methylcobalamin) form of the vitamin. Dehalogenases

 Reactions in which a halogen atom is removed from an organic molecule. Enzymes in this class have not been identified in humans.  In humans, two major coenzyme B12-dependent enzyme families corresponding to the first two reaction types, are known. These are typified by the following two enzymes: o MUT is an isomerase which uses the AdoB12 form and reaction type 1 to catalyze a carbon skeleton rearrangement (the X group is -COSCoA). o MUT's reaction converts MMl-CoA to Su-CoA, an important step in the extraction of energy from proteins and fats (for more see MUT's reaction mechanism). This functionality is lost in vitamin B12 deficiency, and can be measured clinically as an increased methylmalonic acid (MMA) level. o Unfortunately, an elevated MMA, though sensitive to B12 deficiency, is probably overly sensitive, and not all who have it actually have B12 deficiency. o For example, MMA is elevated in 90–98% of patients with B12 deficiency; however 20–25% of patients over the age of 70 have elevated levels of MMA, yet 25–33% of them do not have B12 deficiency. o For this reason, assessment of MMA levels is not routinely recommended in the elderly. There is no "gold standard" test for B12 deficiency because as a B12 deficiency occurs, serum values may be maintained while tissue B12 stores become depleted. o Therefore, serum B12 values above the cut-off point of deficiency do not necessarily indicate adequate B12 status. o The MUT function cannot be affected by folate supplementation, which is necessary for myelin synthesis (see mechanism below) and certain other functions of the central nervous system. o Other functions of B12 related to DNA synthesis related to MTR dysfunction (see below) can often be corrected with supplementation with the vitamin folic acid, but not the elevated levels of homocysteine, which is normally converted to methionine by MTR.

 MTR, also known as methionine synthase, is a methyltransferase enzyme, which uses the MeB12 and reaction type 2 to catalyze the conversion of the amino acid homocysteine (Hcy) back into methionine (Met) (for more see MTR's reaction mechanism). o This functionality is lost in vitamin B12 deficiency, and can be measured clinically as an increased homocysteine level in vitro. Increased homocysteine can also be caused by a folic acid deficiency, since B12 helps to regenerate the tetrahydrofolate (THF) active form of folic acid. Without B12, folate is trapped as 5-methyl-folate, from which THF cannot be recovered unless a MTR process reacts the 5-methyl-folate with homocysteine to produce methionine and THF, thus decreasing the need for fresh sources of THF from the diet. o THF may be produced in the conversion of homocysteine to methionine, or may be obtained in the diet. It is converted by a non-B12-dependent process to 5,10-methyleneTHF, which is involved in the synthesis of thymine. o Reduced availability of 5,10-methylene-THF results in problems with DNA synthesis, and ultimately in ineffective production cells with rapid turnover, in particular blood cells, and also intestinal wall cells which are responsible for absorption. o The failure of blood cell production results in the oncedreaded and fatal disease, pernicious anemia. All of the DNA synthetic effects, including the megaloblastic anemia of pernicious anemia, resolve if sufficient folate is present (since levels of 5,10-methyleneTHF still remain adequate with Figure 1.10.66. Metabolism of f olic acid. The role of Vitamin B12 enough dietary folate). o Thus the best-known "function" of B12 (that which is involved with DNA synthesis, celldivision, and anemia) is actually a facultative function which is mediated by B12conservation of an active form of folate which is needed for efficient DNA production. o Other cobalamin-requiring methyltransferase enzymes are also known in bacteria, such as Me-H4-MPT, coenzyme M methyl transferase.

Absorption and distribution

 The human physiology of vitamin B12 is complex, and therefore is prone to mishaps leading to vitamin B12 deficiency.  Protein-bound vitamin B12 must be released from the proteins by the action of digestive proteases in both the stomach and small intestine.  Gastric acid releases the vitamin from food particles; therefore antacid and acid-blocking medications (especially proton-pump inhibitors) may inhibit absorption of B12. In addition some elderly people produce less stomach acid as they age thereby increasing their probability of B12 deficiencies.  B12 taken in a low-solubility, non-chewable supplement pill form may bypass the mouth and stomach and not mix with gastric acids, but these are not necessary for the absorption of free B12 not bound to protein.  R-proteins (such as haptocorrins and cobalaphilin) are B12 binding proteins that are produced in the salivary glands. They must wait until B12 has been freed from proteins in food by pepsin in the

stomach. B12 then binds to the R-Proteins to avoid degradation of it in the acidic environment of the stomach.  This pattern of secretion of a binding protein secreted in a previous digestive step, is repeated once more before absorption. The next binding protein is intrinsic factor (IF), a protein synthesized by gastric parietal cells that is secreted in response to histamine, gastrin and pentagastrin, as well as the presence of food. In the duodenum, proteases digest R-proteins and release B12, which then binds to IF, to form a complex (IF/B12). B12 must be attached to IF for it to be absorbed, as receptors on the enterocytes in the terminal ileum of the small bowel only recognize the B12-IF complex; in addition, intrinsic factor protects the vitamin from catabolism by intestinal bacteria.  Absorption of food vitamin B12 thus requires an intact and functioning stomach, exocrine pancreas, intrinsic factor, and small bowel. Problems with any one of these organs makes a vitamin B12 deficiency possible. Individuals who lack intrinsic factor have a decreased ability to absorb B12. In pernicious anemia, there is a lack of IF due to autoimmune atrophic gastritis, in which antibodies form against parietal cells. Antibodies may alternately form against and bind to IF, inhibiting it from carrying out its B12 protective function. Due to the complexity of B12 absorption, geriatric patients, many of whom are hypoacidic due to reduced parietal cell function, have an increased risk of B12 deficiency.

This results in 80–100% excretion of oral doses in the feces versus 30–60% excretion in feces as seen in individuals with adequate IF.  Once the IF/B12 complex is recognized by specialized ileal receptors, it is transported into the portal circulation. The vitamin is then transferred to transcobalamin II (TC-II/B12), which serves as the plasma transporter. Hereditary defects in production of the transcobalamins and their receptors may produce functional deficiencies in B12 and infantile megaloblastic anemia, and abnormal B12 related biochemistry, even in some cases with normal blood B12 levels.[62] For the vitamin to serve inside cells, the TC-II/B12 complex must bind to a cell receptor, and be endocytosed. The transcobalamin-

II is degraded within a lysosome, and free B12 is finally released into the cytoplasm, where it may be transformed into the proper coenzyme, by certain cellular enzymes .  It's important to note that investigations into the intestinal absorption of B12 point out that the upper limit per single dose, under normal conditions, is about 1.5 µg: "Studies in normal persons indicated that about 1.5 µg is assimilated when a single dose varying from 5 to 50 µg is administered by mouth.

In a similar study Swendseid et al. stated that the average maximum absorption was 1.6 µg [...]" [63]  The total amount of vitamin B12 stored in body is about 2–5 mg in adults. Around 50% of this is stored in the liver.  Approximately 0.1% of this is lost per day by secretions into the gut, as not all these secretions are reabsorbed. Bile is the main form of B12 excretion; however, most of the B12 secreted in the bile is recycled via enterohepatic circulation. Excess B12 beyond the blood's binding capacity is typically excreted in urine.  Owing to the extremely efficient enterohepatic circulation of B12, the liver can store several years’ worth of vitamin B12; therefore, nutritional deficiency of this vitamin is rare. How fast B12 levels change depends on the balance between how much B12 is obtained from the diet, how much is secreted and how much is absorbed. B12 deficiency may arise in a year if initial stores are low and genetic factors unfavourable, or may not appear for decades. In infants, B12 deficiency can appear much more quickly.

FAT SOLUBLE VITAMINS

VITAMIN A

Retinol : Vitamin A has three active forms (retinal, retinol and retinoic acid) and a storage form (retinyl ester): Retinyl ester ß à Retinol ß à Retinal à Retinoic acid

 Circulating retinol is primarily bound to retinol-binding protein (RBP), and can enter and leave the liver several times per day in a process known as retinol recycling, which acts to relate the amount of retinol in circulation and protects cells from the damaging effects of free retinol or retinoic acid. Retinol bound to a cellular RBP (CRBP or CRBP-II) can be esterified by the enzyme lecithin:retinol acyltransferase (LCAT), the resulting retinyl ester being stored primarily in liver stellate cells. LCAT provides a readily retrievable storage form of vitamin A, as well as regulating its availability for other pathways.  Vitamin A is required throughout life and participates in numerous cellular activities involved in reproduction, embryonic development, vision, growth, cellular differentiation and proliferation, tissue maintenance and lipid metabolism. The three active forms of vitamin A each serve different overlapping functions. For instance, retinal is required for rhodopsin formation and vision, while retinoic acid is the principal hormonal metabolite required for proper growth and differentiation of epithelial cells. Some of the major roles of vitamin A are discussed below:  Vision and Vitamin A o Vitamin A is required for the formation of the photoreceptor rhodopsin, which is a complex of retinal and the vision protein opsin, where retinal functions as the chromophore. Rhodopsins are found in animals and green algae where they act as regulators of light-activated photochannels, and in archaea where they act as light-driven ion pumps. In animals, the light-sensitive pigment rhodopsin occurs embedded in the membrane of rod cells in the retina at the back of the eye. When light passes through the lens, it is sensed in the retina by both rod cells (black and white vision) and cone cells (colour vision). In rod cells, the exposure of rhodopsin to light causes 11-cis-retinal to be released from opsin, resulting in a conformational change in the photoreceptor that activates the G-protein transducin. Transducin activation leads to the closure of the sodium channel in the membrane and the hyperpolarisation of the rod cell, which propagates a nerve impulse to the brain that is perceived as light. Rod cells are especially important for night vision as they can detect very small amounts of light. Inadequate amounts of retinol can led to Night Blindness and corneal malformations, therefore eating carrots does let you see better in the dark! Gene Expression and Vitamin A o Retinol and retinoic acid are important signalling molecules in vertebrates that act to alter the transcriptional activation or repression of numerous genes. Several of these retinoidcontrolled genes are involved in growth and differentiation, such as those involved in the differentiation of the three germ layers, organogenesis and limb development during embryogenesis. Retinoic acid exerts its effect through its binding to retinoic acid receptors (members of the steroid hormone superfamily of proteins), where the vitamin-receptor complex interacts with the genes. Two families of receptors interact with vitamin A: the retinoic acid receptor (RAR) family that bind all-trans-retinoic acid (and 9-cis-retinoic acid), and the retinoic acid X receptor (RXR) family that bind only 9-cis retinoic acid. Together these receptors can regulate the rate of gene expression. Both vitamin A deficiency and excess can cause birth defects.  Immunity and Vitamin A o Vitamin A is required for the normal functioning of the immune system. Retinol and its derivatives are required for the maintenance of the skin and mucosal cells that function as a barrier against infection, and are also required for the development of white blood cells that play a critical role in mounding an immune response. For example, the activation of T-cell lymphocytes requires the binding of the RAR receptor to retinoic acid. A deficiency in vitamin A can cause the mucosal membranes to atrophy, decreasing resistance to infection, and can increase the severity of infection. As such, vitamin A deficiency can be regarded as a nutritionally acquired immunodeficiency disease.  Cancer and Vitamin A

o Vitamin A intake has a complex relationship with cancer prevention: while small doses of vitamin A or beta-carotene appear to help prevent cancer, higher doses seem to have the reverse effect. The anti-cancer effects of beta-carotene appear to stem from its antioxidative ability to scavenge for reactive oxygen species, as well as through its conversion to vitamin A, which can improve immune function in addition to eliciting an anti-proliferative effect through the RAR and RXR receptors, thereby acting to block certain carcinogenic processes and inhibit tumour cell growth. However, an excessive intake of beta-carotene appears to have carcinogen effects, possibly through its promotion of the eccentric (or asymmetric) pathway of beta-carotene cleavage, which produces breakdown products that might lead to the destruction of retinoic acid through the activation of the P450 enzyme, which in turn could decrease retinoid signalling leading to enhanced cell proliferation. Therefore dosage seems to be an important factor in beta-carotene action.  Red Blood Cell Development and Vitamin A o Vitamin A is also involved in the production of red blood cells, which are derived from stem cells that are dependent upon retinoids for their proper differentiation. In addition, vitamin A appears to facilitate the mobilisation of iron stores to developing red blood cells, where it is incorporated into the oxygen carrier haemoglobin.

VITAMIN D

Calciferol

Metabolism

 Vitamin D3 produced in the epidermis must be further metabolized to be active. The first step, 25-hydroxylation, takes place primarily in the liver, although other tissues have this enzymatic activity as well. As will be discussed below, there are several 25hydroxylases. 25OHD is the major circulating form of vitamin D. However, in order for vitamin D metabolites to achieve maximum biologic activity they must be further hydroxylated in the 1α position by the enzyme CYP27B1; 1,25(OH)2D is the most potent metabolite of vitamin D and accounts for most of its biologic actions. The 1α hydroxylation occurs primarily in the kidney, although as for the 25-hydroxylase, other tissues have this enzyme. Vitamin

D and its metabolites, 25OHD and 1,25(OH)2D, can also be hydroxylated in the 24 position. In the absence of 25-hydroxylation this may serve to activate the metabolite or analog as 1,25(OH)2D and 1,24(OH)2D have similar biologic potency. However, 24-hydroxylation of metabolites with an existing 25OH group reduces their activity and leads to further catabolism. The details of these reactions are described below.  Cutaneous production of vitamin D3. Although irradiation of 7-DHC was known to produce pre-D3 (which subsequently undergoes a temperature rearrangement of the triene structure to form D3), lumisterol, and tachysterol (figure 1), the physiologic regulation of this pathway was not well understood until the studies of Holick and his colleagues (9-11). They demonstrated that the formation of pre-D3 under the influence of solar or UV irradiation (maximal effective wavelength between 290310) is relatively rapid and reaches a maximum within hours. UV irradiation further converts pre-D3 to lumisterol and tachysterol. Both the degree of epidermal pigmentation and the intensity of exposure correlate with the time required to achieve this maximal concentration of pre-D3, but do not alter the maximal level achieved. Although pre-D3 levels reach a maximum level, the biologically inactive lumisterol continues to accumulate with continued UV exposure. Tachysterol is also formed, but like pre-D3, does not accumulate with extended UV exposure. The formation of lumisterol is reversible and can be converted back to pre-D3 as pre-D3 levels fall. At 0oC, no D3 is formed; however, at 37oC

pre-D3 is slowly converted to D3. Thus, short exposure to sunlight would be expected to lead to a prolonged production of D3 in the exposed skin because of the slow thermal conversion of pre-D3 to

D3 and the conversion of lumisterol to pre-D3. Prolonged exposure to sunlight would not produce toxic amounts of D3 because of the photoconversion of pre-D3 to lumisterol and tachysterol as well as the photoconversion of D3 itself to suprasterols I and II and 5,6 transvitamin D3 .  Melanin in the epidermis, by absorbing UV irradiation, can reduce the effectiveness of sunlight in producing D3 in the skin. This may be one important reason for the lower 25OHD levels (a well documented surrogate measure for vitamin D levels in the body) in Blacks and Hispanics living in temperate latitudes. Sunlight exposure increases melanin production, and so provides another mechanism by which excess D3 production can be prevented. The intensity of UV irradiation is also important for effective D3 production. The seasonal variation of 25OHD levels can be quite pronounced with higher levels during the summer months and lower levels during the winter. The extent of this seasonal variation depends on the latitude, and thus the intensity of the sunlight striking the exposed skin. In Edmonton, Canada (52oN) very little D3 is produced in exposed skin from mid-

October to mid-April; Boston (42oN) has a somewhat longer period for effective D3 production; whereas in Los Angeles (34oN) and San Juan (18oN) the skin is able to produce D3 all year long .

Peak D3 production occurs around noon, with a larger portion of the day being capable of producing

D3 in the skin during the summer than other times of the year. Clothing and sunscreens effectively prevent D3 production in the covered areas. This is one likely explanation for the observation that the

Bedouins in the Middle East, who totally cover their bodies with clothing, are more prone to develop rickets and osteomalacia than the Israeli Jews with comparable sunlight exposure.  Hepatic production of 25OHD. The next step in the bioactivation of D2 and D3, hydroxylation to 25OHD, takes place primarily in the liver although a number of other tissues express this enzymatic activity. 25OHD is the major circulating form of vitamin D and provides a clinically useful marker for vitamin D status. DeLuca and colleagues were the first to identify 25OHD and demonstrate its production in the liver over 30 years ago, but ambiguity remains as to the actual enzyme(s) responsible for this activity. 25-hydroxylase activity has been found in both the liver mitochondria and endoplasmic reticulum, and the enzymatic activities appear to differ suggesting different proteins. At this point most attention has been paid to the mitochondrial CYP27A1 and the microsomal CYP2R1.

However, in mouse knockout studies and in human with mutations in these enzymes, only CYP2R1 loss is associated with changes in vitamin D metabolite production. These are mixed function oxidases, but differ in apparent Kms and substrate specificities.  The mitochondrial 25-hydroxylase is now well accepted as CYP27A1, an enzyme first identified as catalyzing a critical step in the bile acid synthesis pathway. This is a high capacity, low affinity enzyme consistent with the observation that 25-hydroxylation is not generally rate limiting in vitamin D metabolism. Although initial studies suggested that the vitamin D3-25-hydroxylase and cholestane triol 27-hydroxyase activities in liver mitochondria were due to distinct enzymes with differential regulation, the cloning of CYP27A1 and the demonstration that it contained both activities has put this issue to rest. CYP27A1 is widely distributed throughout different tissues with highest levels in liver and muscle, but also in kidney, intestine, lung, skin, and bone. Mutations in CYP27A1 lead to cerebrotendinous xanthomatosis , and is associated with abnormal vitamin D and/or calcium metabolism in some but not all of these patients . However, it is not clear that mutations in or even absence of CYP27A1 necessarily lead to cessation of vitamin D 25-hydroxylase activity despite leading to abnormalities in bile acid synthesis. CYP27A1 can hydroxylate vitamin D and related compounds at the 24, 25, and 27 positions. However, D2 appears to be preferentially 24-hydroxylated, whereas D3 is preferentially 25-hydroxylated. The 1αOH derivatives of D are more rapidly hydroxylated than the parent compounds. These differences between D2 and D3 and their 1αOH derivatives may explain the differences in biologic activity between D2 and D3 or between 1αOHD2 and 1αOHD3.  The major microsomal 25-hydroxylase is CYP2R1, although other enzymes have been shown in in vitro studies to have 25-hydroxylase activity. This enzyme like that of CYP27A1 is widely distributed, although it is most abundantly expressed in liver, skin and testes. The skin expresses less and the

testes lack CYP27A1 expression. Unlike CYP27A1, CYP2R1 25-hydroxylates D2 and D3 equally (27). A patient with an inactivating mutation in CYP2R1 has been described with rickets and reduced 25OHD levels, reduced serum calcium and phosphate, but normal 1,25(OH)2D levels. The subject responded to D2 therapy. No other phenotype was reported in this subject, in particular no abnormalities in bile acid synthesis. Thus neither CYP27A1 nor CYP2R1 by themselves account for all 25-hydroxyase activity in the body, but each most likely contributes and together may account for most if not all of the 25-hydroxylase activity in humans.  Studies of the regulation of 25-hydroxylation have not been completely consistent, most likely because of the initial failure to appreciate that at least two enzymatic activities were involved and because of species differences. In general, 25-hydroxylation in the liver is little affected by vitamin D status.

However, CYP27A1 expression in the intestine and kidney is reduced by 1,25(OH)2D. Not surprisingly bile acids decrease CYP27A1 expression as does insulin through an unknown mechanism.

Dexamethasone, on the other hand, increases CYP27A1 expression. The regulation of CYP2R1 has been less well studied. Whether any of these manipulations alters 25-hydroxylase activity in the liver remains unknown. Estrogen given to male rats increases 25-hydroxylase activity, whereas testosterone given to female rats has the opposite effect. However, evidence for such sex steroid hormone regulation of 25-hydroxylase activity in humans is lacking.  Renal production of 1,25(OH)2D. 1,25(OH)2D is the most potent metabolite of vitamin D, and mediates most of its hormonal actions especially those involving the vitamin D receptor (VDR), a transcription factor that will be discussed in the section on mechanism of action. 1,25(OH)2D is produced from 25OHD by the enzyme 25OHD-1αhydroxylase (CYP27B1). The recent cloning of CYP27B1 by four independent groups ended a long effort to determine the structure of this critical enzyme in vitamin D metabolism. Mutations in this gene are responsible for the rare autosomal disease of pseudovitamin

D deficiency. An animal model in which the gene is knocked out by homologous recombination reproduces the clinical features of this disease including retarded growth, rickets, hypocalcemia, hyperparathyroidism, and undetectable 1,25(OH)2D.  CYP27B1 is a mitochondrial mixed function oxidase with significant homology to other mitochondrial steroid hydroxylases including CYP27A1 (39%), CYP24A1 (30%), CYPscc (32%), and CYP11β (33%) (35). However, within the heme-binding domain the homology is much greater with 73% and 65% sequence identity with CYP27A1 and CYP24A1. These mitochondrial P450 enzymes are located in the inner membrane of the mitochondrion, and serve as the terminal acceptor for electrons transferred from NADPH through ferrodoxin reductase and ferrodoxin. Expression of CYP27B1 is highest in epidermal keratinocytes (35) cells that previously had been shown to contain high levels of this enzymatic activity. However, the kidney also expresses this enzyme in the renal tubules as does the brain, placenta, testes, intestine, macrophages, lymph nodes, bone and cartilage.  more effectively than PTH and may be less inhibited by calcium, phosphate, and 1,25(OH)2D depending on the tissue. Administration of PTH in vivo or in vitro stimulates renal production of 1,25(OH)2D. This action of PTH can be mimicked by cAMP and forskolin indicating that at least part of the effect of PTH is mediated via its activation of adenylate cyclase. However, PTH activation of protein kinase C (PKC) also appears to be involved in that concentrations of PTH sufficient to stimulate PKC activation and 1,25(OH)2D production are below that required to increase cAMP levels . Furthermore, synthetic fragments of PTH lacking the ability to activate adenylate cyclase but which stimulate PKC activity were found to increase 1,25(OH)2D production. Direct activation of PKC with phorbol esters results in increased 1,25(OH)2D production. Although the promoter of

CYP27B1contains several AP-1 (PKC activated) and cAMP response elements, it is not yet clear how

PTH regulates CYP27B1 gene expression. Calcium modulates the ability of PTH to increase 1,25(OH)2D production. Calcium by itself can decrease CYP27B1activity and block the stimulation by PTH. Given in vivo, calcium can exert its effect in part by reducing PTH secretion, but this does not explain its direct actions in vitro or its effects in parathyroidectomized or PTH infused animals.

Phosphate deprivation can stimulate CYP27B1 activity in vivo and in vitro. The in vivo actions of phosphate deprivation can be blocked by hypophysectomy and partially restored by growth hormone (GH) (64, 65) and insulin-like growth factor (IGF-I). However, like PTH, the exact mechanism by which

GH and/or IG and TNF-The principal regulators of CYP27B1 activity in the kidney are parathyroid hormone (PTH), FGF23, calcium, phosphate, and 1,25(OH)2D. Extrarenal production tends to be stimulated by cytokines such as IFN-,25(OH)2D administration leads to an apparent reduction in

CYP27B1 activity . It was initially thought that this feedback inhibition was mediated at the level of gene expression. However, no vitamin D response element has been identified in the promoter of the 1α-hydroxylase gene. In keratinocytes, 1,25(OH)2D has little or no effect on CYP27B1 mRNA and protein levels when given in vitro. When 24-hydroxylase activity is blocked, 1,25(OH)2D administration fails to reduce the levels of 1,25(OH)2D produced. Thus the apparent feedback regulation of CYP27B1 activity by 1,25(OH)2D appears to be due to its stimulation of CYP24A1 and subsequent catabolism, not to a direct effect on CYP27B1 expression or activity at least in keratinocytes. However, in one renal cell line a chromatin remodeling complex (WINAC) has been described that mediates the ability of the vitamin D receptor to regulate CYP27B1 gene expression in a non classic manner enabling 1,25(OH)2D suppression of this gene; whether this mechanism is operative in other cells including normal kidney remains to be demonstrated.F-I mediates the effects of phosphate on CYP27B1 expression remains unclear. More recently FGF23 has been shown to inhibit CYP27B1 activity in vivo and in vitro. FGF23 has been implicated as at least one of the factors responsible for impaired phosphate reabsorption and 1,25(OH)2D production in conditions such as

X-linked and autosomal dominant hypophosphatemic rickets and oncogenic osteomalacia .  Renal production of 24,25(OH)2D. The kidney is also the major producer of a second important metabolite of 25OHD, namely 24,25(OH)2D, and the enzyme responsible is 25OHD-24 hydroxylase (CYP24A1). CYP24A1 and CYP27B1 are homologous enzymes that coexist in the mitochondria of tissues where both are found, such as the kidney tubule. However, they are located on different chromosomes (chromosome 20q13 and chromosome 12q14 for CYP24A1 and CYP27B1, respectively). They are likely to share the same ferrodoxin and ferrodoxin reductase components, although this has not been clearly established. While CYP27B1 activates the parent molecule, 25OHD, CYP24A1 initiates a series of catabolic steps that lead to its inactivation. However, in some tissues 24,25(OH)2D has been shown to have biologic effects different from 1,25(OH)2D as will be described subsequently. CYP24A1 requires a 25OH group, but can 24-hydroxylate both 25OHD and 1,25(OH)2D. The 24-hydroxylation is then followed by oxidation of 24OH to a 24-keto group, 23hydroxylation, cleavage between C23-24, and the eventual production of calcitroic acid, a metabolite with no biologic activity. CYP24A1 appears to catalyze all the steps in this catabolic pathway. Although

CYP24A1 is highly expressed in the kidney tubule, its tissue distribution is quite broad including the intestine, osteoblasts, placenta, keratinocytes, and prostate. In general, CYP24A1 can be found wherever the VDR is found. The affinity for 1,25(OH)2D is higher than that for 25OHD, making this enzyme an efficient means for eliminating 1,25(OH)2D. Thus, CYP24A1 is likely to play the important role of protecting the body against excess 1,25(OH)2D. There are no known diseases due to

CYP24A1 deficiency. However, an animal model in which CYP24A1 has been knocked out showed very high levels of 1,25(OH)2D and impaired mineralization of intramembranous bone. The skeletal abnormalities could be corrected by crossing this mouse to one lacking the VDR suggesting that excess 1,25(OH)2D (which acts through the VDR) rather than deficient 24,25(OH)2D (which does not) is to blame.

VITAMIN E

tocopherol

o Tocopherol contain a substituted aromatic ring and a long isoprenoid side chain. o Because they are hydrophobic tocopherol associate with cell membranes, lipid droplets and lipoprotein. o This vitamin is a natural antioxidant.

Unit 1 o The aromatic ring reacts with and destroys the most reactive forms of oxygen radicals and free radicals. o Vitamin E deficiency is very rare; the principal symptom is fragile erythrocytes. o Tyrosine is the precursor of tocopherol synthesis, which by converting to homogentisic acid is converted to tocopherol. (Figure 1.10.67)

Figure 1.10.67. Metabolism of f olic acid. The role of Vitamin D

Critical thinking Questions 1. When we digest food and use it to produce energy, we convert a few large, complex molecules into many small, simple molecules. Under these circumstances, what is the effect on entropy? 2. In addition to the regulators of enzyme activity within the citric acid cycle, two enzymes outside the cycle profoundly affect its activity. Which ones? 3. If you were handed a sample of a white, greasy substance and asked to determine whether it were a triacylglycerol or a fatty acid, how would you do it? 4. Why are Arachidonic acid and EPA are classified as essential? 5. Fatty acid oxidation occurs mostly within mitochondria, but fatty acids themselves cannot easily cross the mitochondrial membrane. How do they pass?