Introduction to Human Nutrition 2nd Edition ( PDFDrive )

136 Introduction to Human Nutrition H3C CH3 CH3 CH3 H3C CH3 CH3 CH3 H3C CH3 O2 β-Carotene Carotene dioxygenase H3C CH3 CH3 CH3 H3C CH3 O O CH3 CH3 H3C CH3 H3C CH3 CH3 CH3 H CO CH3 2 × Retinaldehyde NAD(P)H H2O + O2 H2O2 NAD(P)+ Retinol Aldehyde dehydrogenase oxidase H3C CH3 CH3 CH3 H3C CH3 CH3 CH3 CH2OH COO- CH3 Retinol CH3 all-trans-Retinoic acid Figure 8.2 The oxidative cleavage of carotene to yield retinol and retinoic acid. Carotene dioxygenase (EC 1.13.11.21), retinol dehydrogenase (EC 1.1.1.105), retinaldehyde oxidase (EC 1.2.3.11). Only a proportion of carotene undergoes oxidation a molar basis, is considerably lower than that of in the intestinal mucosa, and a significant amount of retinol, not twofold higher as might be expected. In carotene enters the circulation in chylomicrons. Caro- addition to poor absorption of carotene, three factors tene in the chylomicron remnants is cleared by the may account for this. liver; some is cleaved by hepatic carotene dioxygenase, again giving rise to retinaldehyde and retinyl esters; ● The intestinal activity of carotene dioxygenase is the remainder is secreted in very low-density lipopro- relatively low, so that a relatively large proportion of teins (VLDLs), and may be taken up and cleaved by ingested β-carotene may be absorbed unchanged. carotene dioxygenase in other tissues. ● Other carotenoids in the diet may inhibit carotene Central oxidative cleavage of β-carotene, as shown dioxygenase and reduce the formation of retinol. in Figure 8.2, should yield two molecules of retinal- dehyde, which can be reduced to retinol. However, as ● The principal site of carotene dioxygenase attack is noted above, the biological activity of β-carotene, on the central bond of β-carotene, but asymmetric cleavage also occurs, leading to the formation of 8′-,

The Vitamins 137 10′- and 12′-apo-carotenals, which are oxidized to dized to 11-cis-retinaldehyde. This reacts with a lysine yield retinoic acid, but are not precursors of retinol residue in opsin, forming the holoprotein rhodopsin. or retinaldehyde. As shown in Figure 8.3, the absorption of light by rhodopsin causes isomerization of the retinaldehyde Plasma retinol binding protein bound to opsin from 11-cis to all-trans, and a confor- Retinol is released from the liver bound to an α- mational change in opsin. This results in the release of globulin, retinol binding protein (RBP); this serves to retinaldehyde from the protein and the initiation of a maintain the vitamin in aqueous solution, protects it nerve impulse. The overall process is known as bleach- against oxidation and delivers the vitamin to target ing, since it results in the loss of the color of rho- tissues. RBP is secreted from the liver as a 1:1 complex dopsin. The all-trans-retinaldehyde released from with the thyroxine-binding prealbumin, transthy- rhodopsin is reduced to all-trans-retinol, and joins the retin. This is important to prevent urinary loss of pool of retinol in the pigment epithelium for isomeri- retinol bound to the relatively small RBP, which would zation to 11-cis-retinol and regeneration of rhodop- otherwise be filtered by the kidney, with a consider- sin. The key to initiation of the visual cycle is the able loss of vitamin A from the body. availability of 11-cis-retinaldehyde, and hence vitamin A. In deficiency both the time taken to adapt to dark- Cell surface receptors on target tissues take up ness and the ability to see in poor light are impaired. retinol from the RBP–transthyretin complex, trans- ferring it on to an intracellular RBP. The receptors The excited form of rhodopsin (metarhodopsin II) also remove the carboxy-terminal arginine residue initiates a G-protein cascade leading to hyperpolar- from RBP, so inactivating it by reducing its affinity for ization of the outer section membrane of the rod or both transthyretin and retinol. As a result, apo-RBP cone, caused by the closure of sodium channels is filtered at the glomerulus; most is reabsorbed in the through the membrane, and the initiation of a nerve proximal renal tubules and hydrolyzed. The apopro- impulse. tein is not recycled. Retinoic acid and the regulation of During the development of vitamin A deficiency in experimental animals, the plasma concentration of gene expression RBP falls, whereas the liver content of apo-RBP rises. The main function of vitamin A is in the control of The administration of retinol results in release of cell differentiation and turnover. All-trans-retinoic holo-RBP from the liver. This provides the basis of acid and 9-cis-retinoic acid are active in the regulation the relative dose–response (RDR) test for liver reserves of growth, development, and tissue differentiation; of vitamin A (see below). they have different actions in different tissues. Like the steroid hormones and vitamin D, retinoic acid Metabolic functions of vitamin A interacts with nuclear receptors that bind to response and carotenes elements (control regions) of DNA, and regulate the transcription of specific genes. The first function of vitamin A to be defined was in vision. More recently, retinoic acid has been shown to There are two families of nuclear retinoid recep- have a major function in regulation of gene expres- tors: the retinoic acid receptors (RARs) bind all-trans- sion and tissue differentiation. retinoic acid or 9-cis-retinoic acid, and the retinoid X receptors (RXRs) bind 9-cis-retinoic acid, and some Vitamin A in vision of the other physiologically active retinoids as well. In the retina, retinaldehyde functions as the prosthetic RXR can form active dimers with RARs, RXRs group of the light-sensitive opsin proteins, forming (homodimers), and the receptors for calcitriol rhodopsin (in rods) and iodopsin (in cones). Any one (vitamin D), thyroid hormone, long-chain polyun- cone cell contains only one type of opsin, and hence saturated fatty acid (PUFA) derivatives [the peroxi- is sensitive to only one color of light. Color blindness some proliferators-activated receptor (PPAR)], and results from loss or mutation of one or other of the one for which the physiological ligand has not yet cone opsins. been identified (the COUP receptor). In the pigment epithelium of the retina, all-trans- The result of this is that a very large number of retinol is isomerized to 11-cis-retinol and then oxi- genes are sensitive to control by retinoic acid in

138 Introduction to Human Nutrition H3C CH3 CH3 CH3 H3C CH3 CH3 CH2OH CH3 CH3 H3C all-trans-Retinol 11-cis-Retinol CH2OH H3C CH3 CH3 CH3 H3C CO 11-cis-Retinaldehyde HC O +H3N H3C CH3 CH3 NH Lysine residue in opsin CH3 H3C CO HC N NH Rhodopsin (visual purple) Light 10–15 s H3C CH3 CH3 CH3 H CO CN CH3 NH Photorhodopsin 45 ps Bathorhodopsin 30 ns Lumirhodopsin 75 µs Metarhodopsin I 10 ms GTP Transducin-GDP Pi H3C CH3 CH3 CH3 H Metarhodopsin II GTPase CO min GDP Transducin-GTP Metarhodopsin III CH3 Inactive Active all-trans-Retinaldehyde phosphodiesterase phosphodiesterase Opsin cGMP 5'GMP Na+ channel open Na+ channel closed Figure 8.3 Role of vitamin A and the cyclic GMP cascade in the visual cycle. Retinol isomerase (EC 5.2.1.3), phosphodiesterase (EC 3.1.4.35).

The Vitamins 139 different tissues, and at different stages in develop- to green light; this is followed by impairment of the ment, and retinoic acid is essential for the normal ability to adapt to dim light, then an inability to see responses to vitamin D, thyroid hormone and long- at all in dim light: night blindness. More prolonged chain PUFA derivatives. or severe deficiency leads to the condition called xerophthalmia: keratinization of the cornea, followed Unoccupied RXRs can form dimers with calcitriol by ulceration – irreversible damage to the eye that and other receptors; these bind to hormone response causes blindness. At the same time there are changes elements on DNA, but do not lead to activation of in the skin, with excessive formation of keratinized transcription. This means that vitamin A deficiency tissue. will impair responses to vitamin D and thyroid hormone more markedly than might be expected Vitamin A also plays an important role in the dif- simply from lack of 9-cis-retinoic acid to form active ferentiation of immune system cells, and mild defi- heterodimers. ciency, not severe enough to cause any disturbance of vision, leads to increased susceptibility to a variety of Vitamin A in excess may also impair responsiveness infectious diseases. At the same time, the synthesis of to vitamin D and other hormones, since high concen- RBP is reduced in response to infection (it is a nega- trations of 9-cis-retinoic acid will lead to the forma- tive acute-phase protein), so that there is a reduction tion of RXR–RXR homodimers, leaving too few RXRs in the circulating concentration of the vitamin, and to form heterodimers with vitamin D and other hence further impairment of immune responses. receptors. There is epidemiological evidence that habitually high intakes of vitamin A are associated Signs of vitamin A deficiency also occur in protein– with poor bone health in later life as a result of energy malnutrition, regardless of whether or not the impaired responsiveness to vitamin D. intake of vitamin A is adequate. This is due to impair- ment of the synthesis of plasma RBP; functional The antioxidant function of carotenes vitamin A deficiency can occur secondary to protein– At least in vitro, and under conditions of low oxygen energy malnutrition; even if liver reserves of the availability, carotenes can act as radical-trapping anti- vitamin are adequate, it cannot be mobilized. oxidants. There is epidemiological evidence that high intakes of carotene are associated with a low incidence Vitamin A requirements and of cardiovascular disease and some forms of cancer, reference intakes although the results of intervention trials with β- carotene have been disappointing, with an increased There have been relatively few studies of vitamin A incidence of lung cancer among those taking carotene requirements in which subjects have been depleted of supplements. the vitamin for long enough to permit the develop- ment of clear deficiency signs. Current estimates of The problem is that although carotene is an anti- requirements are based on the intakes required to oxidant at a low partial pressure of oxygen, as occurs maintain a concentration in the liver of 70 μmol in most tissues, at a high partial pressure of oxygen, retinol/kg, as determined by measurement of the rate as occurs in the lungs, it is an autocatalytic pro- of metabolism of isotopically labeled vitamin A. This oxidant, acting as a source of oxygen radicals. The UK is adequate to maintain normal plasma concentra- Food Standards Agency specifically advises smokers tions of the vitamin, and people with this level of liver not to take carotene supplements. reserves can be maintained on a vitamin A-free diet for many months before they develop any detectable Vitamin A deficiency: night blindness signs of deficiency. and xerophthalmia The average requirement to maintain a concentra- Worldwide, vitamin A deficiency is a major public tion of 70 μmol/kg of liver is 6.7 μg retinol equiva- health problem and the most important preventable lents/kg body weight, and this is the basis for cause of blindness; the WHO estimates that some 256 calculation of reference intakes. million children under 5 years old show subclinical deficiency and 2.7 million have xerophthalmia. Assessment of vitamin A status The only direct assessment of vitamin A status is by The earliest signs of clinical deficiency are associ- liver biopsy and measurement of retinyl ester reserves. ated with vision. Initially, there is a loss of sensitivity

140 Introduction to Human Nutrition This is an invasive procedure that cannot be consid- needs for 4–6 months. About 1% of children so ered for routine investigations and population surveys. treated show transient signs of toxicity, but this is Status can also be assessed by clinical and functional considered an acceptable risk in view of the high tests, the plasma concentrations of retinol and RBP, prevalence and devastating effects of deficiency. and the response to a test dose of vitamin A, the RDR test. The chronic toxicity of vitamin A is a more general cause for concern; prolonged and regular intake of In field surveys, clinical signs of vitamin A defi- more than about 7.5–9 mg/day by adults (and signifi- ciency, including Bitot’s spots, corneal xerosis, corneal cantly less for children) causes signs and symptoms ulceration, and keratomalacia, can be used to identify of toxicity affecting: those suffering from vitamin A deficiency. The earliest signs of corneal damage are detected by conjunctival ● the central nervous system: headache, nausea, ataxia impression cytology (CIC); however, abnormalities and anorexia, all associated with increased cerebro- only develop when liver reserves are seriously spinal fluid pressure depleted. ● the liver: hepatomegaly with histological changes The ability to adapt to dim light is impaired early in the liver, increased collagen formation and in deficiency, and dark adaptation time is sometimes hyperlipidemia used to assess vitamin A status. However, the test is not suitable for use on children (the group most at ● bones: joint pains, thickening of the long bones, risk of deficiency) and the apparatus is not suited to hypercalcemia and calcification of soft tissues use in the field. ● the skin: excessive dryness, scaling and chapping of The fasting plasma concentration of retinol remains the skin, desquamation and alopecia. constant over a wide range of intakes and only falls significantly when liver reserves are nearly depleted. The recommended upper limits of habitual intake of Therefore, although less sensitive to subtle changes retinol, compared with reference intakes, are shown within the normal range than some methods of in Table 8.2. As discussed above, habitual high intakes assessing nutritional status, measurement of plasma of vitamin A, albeit below these prudent upper levels retinol provides a convenient and sensitive means of of intake, may be associated with impaired respon- detecting people whose intake of vitamin A is inade- siveness to vitamin D, poor mineralization of bone quate to maintain normal liver reserves. and the early development of osteoporosis. The RDR test is a test of the ability of a dose of Teratogenicity of vitamin A retinol to raise the plasma concentration several hours The synthetic retinoids (vitamin A analogues) used in after chylomicrons have been cleared from the circu- dermatology are highly teratogenic. After women lation. It depends on the fact that apo-RBP accumu- have been treated with them, it is recommended lates in the liver in vitamin A deficiency. The RDR is that contraceptive precautions be continued for 12 the ratio of the plasma concentration of retinol 5 h months, because of their retention in the body. By after the dose to that immediately before it was given. An RDR greater than 20% indicates depletion of liver Table 8.2 Prudent upper levels of habitual vitamin A intake retinol to less than 70 μmol/kg. Age group Upper limit of intake Reference intakesa Toxicity of vitamin A (μg/day) (μg/day) There is only a limited capacity to metabolize vitamin Infants 900 350–375 A. Excessively high intakes lead to accumulation in 1–3 years 1800 400 the liver and other tissues, beyond the capacity of 4–6 years 3000 400–500 normal binding proteins, so that free, unbound, 6–12 years 4500 500–700 vitamin A is present. This leads to liver and bone 13–20 years 6000 600–700 damage, hair loss, vomiting, and headaches. Single Adult men 9000 600–1000 doses of 60 mg of retinol are given to children in Adult women 7500 600–800 developing countries as a prophylactic against vitamin Pregnant women 3000–3300 700 A deficiency: an amount adequate to meet the child’s a Reference intakes show range for various national and international authorities.

The Vitamins 141 extrapolation, it has been assumed that retinol is also nuclear receptors that regulate gene expression. teratogenic, although there is little evidence. In case– Deficiency, leading to rickets in children and osteo- control studies, intakes between 2400 μg/day and malacia in adults, continues to be a problem in north- 3300 μg/day during pregnancy have been associated ern latitudes, where sunlight exposure is poor. with birth defects. Other studies have not demon- strated any teratogenic effect at this level of intake, There are relatively few sources of vitamin D, mainly and it has been suggested that the threshold plasma oily fish, with eggs, liver, and butter providing modest concentration associated with teratogenic effects is amounts; fortified milk, containing ergocalciferol, is unlikely to be reached with intakes below 7500 μg/ available in some countries. As a result, strict vegetar- day. Nevertheless, pregnant women are advised not to ians are especially at risk of deficiency, especially in consume more than 3000 μg/day (American Pediatric northern latitudes with little sunlight exposure. Association recommendation) or 3300 μg (UK Department of Health recommendation). Although meat provides apparently negligible quantities of vitamin D, it may be an important Interactions of vitamin A with drugs and source, since what is present is largely the final active other nutrients metabolite, calcitriol, which is many times more potent on a molar basis than is cholecalciferol. Historically, there was considerable confusion between vitamins A and D, and for many years it was not clear Vitamers and international units which acted in which system. By the 1950s it was believed that the problem had been solved, with The normal dietary form of vitamin D is cholecalcif- clearly defined functions of vitamin A in vision, and erol (also known as calciol). This is also the com- vitamin D in calcium homeostasis and bone develop- pound that is formed in the skin by ultraviolet (UV) ment. However, both have overlapping effects on a irradiation of 7-dehydrocholesterol. Some foods are number of systems, including bone metabolism and enriched or fortified with (synthetic) ergocalciferol, immune system function. It is now known that this is which undergoes the same metabolism as cholecalcif- the result of formation of retinoid–vitamin D recep- erol and has the same biological activity. Early studies tor heterodimers, so that in some systems both are assigned the name vitamin D1 to an impure mixture required in appropriate amounts for normal regula- of products derived from the irradiation of ergosterol; tion of gene expression. when ergocalciferol was identified it was called vitamin D2, and when the physiological compound Chlorinated hydrocarbons, as contained in agricul- was identified as cholecalciferol it was called vita- tural pesticides, deplete liver retinol. Metabolites of min D3. polychlorinated biphenyls bind to the thyroxine binding site of transthyretin, and in doing so impair Like vitamin A, vitamin D was originally measured the binding of RBP. As a result there is free RBP- in international units of biological activity before the bound retinol in plasma, which is filtered at the glom- pure compound was isolated: 1 IU = 25 ng of chole- erulus and hence lost in the urine. Habitual use of calciferol; 1 μg of cholecalciferol = 40 IU. barbiturates may also lead to deficiency as a result of induction of cytochrome P450, which catalyzes the Absorption and metabolism catabolism of retinol. Vitamin D is absorbed in lipid micelles and incorpo- 8.3 Vitamin D rated into chylomicrons; therefore, people on a low- fat diet will absorb little of such dietary vitamin D as Vitamin D is not strictly a vitamin, since it can be is available. Indeed, it is noteworthy that at the time synthesized in the skin, and indeed under most condi- that rickets was a major public health problem in tions endogenous synthesis is the major source of the Scotland, herrings (a rich source) were a significant vitamin: it is only when sunlight exposure is inade- part of the diet: it can only be assumed that the diet quate that a dietary source is required. Its main func- was so low in fat that the absorption of the vitamin tion is in the regulation of calcium absorption and was impaired. homeostasis; most of its actions are mediated by Synthesis of vitamin D in the skin As shown in Figure 8.4, the steroid 7-dehydrocholes- terol (an intermediate in the synthesis of cholesterol

HO Light 7-Dehydrocholesterol thermal OH 2 OH Kidn 24-hydr CH2 HO 24-Hydroxycalcidiol Figure 8.4 Vitamin D synthesis and metabolis

OH 142 Introduction to Human Nutrition Light CH3 Previtamin D H3C Tachysterol OH Slow l isomerization CH2 HO Vitamin D calciol (cholecalciferol) Liver, 25-hydroxylase OH OH ney, Kidney, roxylase 1-hydroxylase CH2 CH2 HO HO OH Calcidiol Calcitriol (25-hydroxycholecalciferol) sm. (1,25-dihydroxycholecalciferol)

The Vitamins 143 Table 8.3 Nomenclature of vitamin D metabolites Trivial name Recommended name Abbreviation Vitamin D3 Calciol – Cholecalciferol Calcidiol 25(OH)D3 25-Hydroxycholecalciferol 1(S)-Hydroxycalciol 1α(OH)D3 1α-Hydroxycholecalciferol 24(R)-Hydroxycalcidiol 24,25(OH)2D3 24,25-Dihydroxycholecalciferol Calcitriol 1,25(OH)2D3 1,25-Dihydroxycholecalciferol Calcitetrol 1,24,25(OH)3D3 1,24,25-Trihydroxycholecalciferol Ercalciol – Vitamin D2 Ercalcidiol 25(OH)D2 Ergocalciferol 24(R)-Hydroxyercalcidiol 24,25(OH)2D2 25-Hydroxyergocalciferol Ercalcitriol 1,25(OH)2D2 24,25-Dihydroxyergocalciferol Ercalcitetrol 1,24,25(OH)3D2 1,25-Dihydroxyergocalciferol 1,24,25-Trihydroxyergocalciferol The abbreviations shown in column 3 are not recommended, but are frequently used in the literature. that accumulates in the skin but not other tissues) yield ercalcitriol. The nomenclature of the vitamin D undergoes a non-enzymic reaction on exposure to metabolites is shown in Table 8.3. UV light, yielding previtamin D, which undergoes a further reaction over a period of hours to form cho- The first stage in vitamin D metabolism occurs in lecalciferol, which is absorbed into the bloodstream. the liver, where it is hydroxylated to form the 25- hydroxy derivative calcidiol. This is released into the In temperate climates there is a marked seasonal circulation bound to a vitamin D binding globulin. variation in the plasma concentration of vitamin D; There is no tissue storage of vitamin D; plasma cal- it is highest at the end of summer and lowest at the cidiol is the main storage form of the vitamin, and it end of winter. Although there may be bright sunlight is plasma calcidiol that shows the most significant in winter, beyond about 40° N or S there is very little seasonal variation in temperate climates. UV radiation of the appropriate wavelength for cho- lecalciferol synthesis when the sun is low in the sky. The second stage of vitamin D metabolism occurs By contrast, in summer, when the sun is more or less in the kidney, where calcidiol undergoes either 1- overhead, there is a considerable amount of UV light hydroxylation to yield the active metabolite 1,25- even on a moderately cloudy day, and enough can dihydroxyvitamin D (calcitriol) or 24-hydroxylation penetrate thin clothes to result in significant forma- to yield an apparently inactive metabolite, 24,25- tion of vitamin D. dihydroxyvitamin D (24-hydroxycalcidiol). Calcidiol 1-hydroxylase is also found in other tissues that are In northerly climates, and especially in polluted capable of forming calcitriol as an autocrine or para- industrial cities with little sunlight, people may well crine agent. not be exposed to enough UV light to meet their vitamin D needs, and they will be reliant on the few Regulation of vitamin D metabolism dietary sources of the vitamin. The main function of vitamin D is in the control of Metabolism to calcitriol calcium homeostasis and, in turn, vitamin D metabo- Cholecalciferol, either synthesized in the skin or from lism in the kidney is regulated, at the level of 1- or foods, undergoes two hydroxylations to yield the 24-hydroxylation, by factors that respond to plasma active metabolite, 1,25-dihydroxyvitamin D or cal- concentrations of calcium and phosphate. In tissues citriol, as shown in Figure 8.4. Ergocalciferol from other than the kidney that hydroxylate calcidiol to fortified foods undergoes similar hydroxylation to calcitriol, the enzyme is not regulated in response to plasma calcium.

144 Introduction to Human Nutrition ● Calcitriol acts to reduce its own synthesis and ● differentiation of monocyte precursor cells; increase formation of 24-hydroxycalcidiol, by regu- ● modulation of cell differentiation, proliferation and lating the expression of the genes for the two hydroxylases. apoptosis. ● Parathyroid hormone is secreted in response to a In most of its actions, the role of calcitriol seems to fall in plasma calcium. In the kidney it acts to be in the induction or maintenance of synthesis of increase the activity of calcidiol 1-hydroxylase and calcium binding proteins, and the physiological effects decrease that of 24-hydroxylase. In turn, both cal- are secondary to changes in intracellular calcium citriol and high concentrations of calcium repress concentrations. the synthesis of parathyroid hormone; calcium also inhibits the secretion of the hormone from the Calcitriol acts like a steroid hormone, binding to a parathyroid gland. nuclear receptor protein, commonly as a heterodimer with the RXR (vitamin A) receptor, then binding to ● Calcium exerts its main effect on the synthesis hormone response elements on DNA and modifying and secretion of parathyroid hormone. However, the expression of one or more genes. calcium ions also have a direct effect on the kidney, reducing the activity of calcidiol 1-hydroxylase. The best-studied actions of vitamin D are in the intestinal mucosa, where the intracellular calcium ● Phosphate also affects calcidiol metabolism; binding protein induced by vitamin D is essential for throughout the day there is an inverse fluctuation the absorption of calcium from the diet. Vitamin D of plasma phosphate and calcitriol, and feeding also acts to increase the transport of calcium across people on a low-phosphate diet results in increased the mucosal membrane by recruiting calcium trans- circulating concentrations of calcitriol. port proteins to the cell surface. Metabolic functions of vitamin D Calcitriol also raises plasma calcium by stimulating the mobilization of calcium from bone. It achieves The principal function of vitamin D is to maintain this by activating osteoclast cells. However, it acts later the plasma concentration of calcium; calcitriol to stimulate the laying down of new bone to replace achieves this in three ways: the loss, by stimulating the differentiation and recruit- ment of osteoblasts. ● increased intestinal absorption of calcium ● reduced excretion of calcium by stimulating resorp- Vitamin D deficiency: rickets and osteomalacia tion in the distal renal tubules (due to increased calbindin D synthesis) Historically, rickets is a disease of toddlers, especially ● mobilization of bone mineral. in northern industrial cities. Their bones are under- mineralized as a result of poor absorption of calcium There is a growing body of evidence that low vitamin in the absence of adequate amounts of calcitriol. D status (but not such a degree of deficiency as to When the child begins to walk, the long bones of the disturb calcium homeostasis) is associated with legs are deformed, leading to bow-legs or knock knees. impaired glucose tolerance, insulin resistance and More seriously, rickets can also lead to collapse of the non-insulin dependent diabetes mellitus, as well as ribcage and deformities of the bones of the pelvis. obesity and the low grade chronic inflammation asso- Similar problems may also occur in adolescents who ciated with (especially abdominal) obesity. There is are deficient in vitamin D during the adolescent also evidence poor vitamin D status is a factor in the growth spurt, when there is again a high demand for etiology of some cancers. Calcitriol has a variety of calcium for new bone formation. permissive or modulatory effects; it is a necessary, but not sufficient, factor, in: Osteomalacia is the adult equivalent of rickets. It results from the demineralization of bone, rather than ● synthesis and secretion of insulin, parathyroid, and the failure to mineralize it in the first place, as is the thyroid hormones; case with rickets. Women who have little exposure to sunlight are especially at risk from osteomalacia after ● inhibition of production of interleukin by activated several pregnancies, because of the strain that preg- T-lymphocytes and of immunoglobulin by acti- nancy places on their marginal reserve of calcium. vated B-lymphocytes;

The Vitamins 145 Osteomalacia also occurs in the older people. Here hypervitaminosis and hypercalcemia. Increased sun- again the problem may be inadequate exposure to light exposure will improve vitamin D status without sunlight, but there is also evidence that the capacity the risks of toxicity, but excessive sunlight exposure is to form 7-dehydrocholesterol in the skin decreases a cause of skin cancer. The main problem in trying to with advancing age, so that older people are more balance improved vitamin D status through increased reliant on the few dietary sources of vitamin D. sunlight exposure, and increased risk of skin cancer, is that there is very little information on the amount Although vitamin D is essential for prevention and of sunlight exposure required for the synthesis of a treatment of osteomalacia in older people, there is less given amount of vitamin D. evidence that it is beneficial in treating the other common degenerative bone disease of advancing age, Vitamin D toxicity osteoporosis, which is due to a loss of bone matrix, rather than enhanced release of calcium from bone During the 1950s, rickets was more or less totally with no effect on the organic matrix, as is seen in eradicated in Britain and other temperate countries. osteomalacia. The result is negative calcium balance This was due to enrichment of a large number of and loss of bone mineral, but secondary to the loss of infant foods with vitamin D. However, a small number organic matrix, owing to progressive loss of estrogens of infants suffered from vitamin D poisoning, the and androgens, rather than failure of the vitamin D most serious effect of which is an elevated plasma system. concentration of calcium. This can lead to contrac- tion of blood vessels, and hence dangerously high Vitamin D requirements and blood pressure, and calcinosis, that is the calcification reference intakes of soft tissues, including the kidney, heart, lungs, and blood vessel walls. It is difficult to determine requirements for dietary vitamin D, since the major source is synthesis in the Some infants are sensitive to intakes of vitamin D skin. Before the development of methods for mea- as low as 50 μg/day. To avoid the serious problem of surement of calcidiol the diagnosis of subclinical vitamin D poisoning in these susceptible infants, the rickets was by detection of elevated alkaline phospha- extent to which infant foods are fortified with vitamin tase in plasma; nowadays, the main criterion of ade- D has been reduced considerably. Unfortunately, this quacy is the plasma concentration of calcidiol. means that a small proportion, who have relatively high requirements, are now at risk of developing In older people with little sunlight exposure, a rickets. The problem is to identify those who have dietary intake of 10 μg of vitamin D/day results in a higher requirements and provide them with plasma calcidiol concentration of 20 nmol/l, the lower supplements. end of the reference range for younger adults at the end of winter. Therefore, the reference intake for older The toxic threshold in adults is not known, but people is 10 μg/day, whereas average intakes of vitamin those patients suffering from vitamin D intoxication D from unfortified foods are less than 4 μg/day. who have been investigated were taking supplements providing more than 250 μg/day. There is little evidence to establish what are appro- priate plasma concentrations of calcidiol; certainly Although excess dietary vitamin D is toxic, exces- the lower end of the reference range for young adults sive exposure to sunlight does not lead to vitamin D at the end of winter in a temperate climate is a mini- poisoning. There is a limited capacity to form the malist goal, and is not much higher than the level at precursor, 7-dehydrocholesterol, in the skin, and which biochemical signs of deficiency occur. However, a limited capacity to take up cholecalciferol from unfortified foods will not meet even this goal. the skin. Furthermore, prolonged exposure of previ- tamin D to UV light results in further reactions to There is increasing evidence that high vitamin D yield lumisterol and other biologically inactive status is associated with a lower incidence of various compounds. cancers, diabetes, and the metabolic syndrome, sug- gesting that desirable intakes are higher than current Interactions with drugs and other nutrients reference intakes. Widespread fortification of foods would improve vitamin D status, but might also put As discussed above, vitamin D receptors form het- a significant proportion of the population at risk of erodimers with RXR, so that vitamin D-dependent

146 Introduction to Human Nutrition functions require adequate, but not excessive, vitamin Synthetic α-tocopherol does not have the same A status. A number of drugs, including barbiturates biological potency as the naturally occurring com- and other anticonvulsants, induce cytochrome pound. This is because the side-chain of tocopherol P450, resulting in increased catabolism of calcidiol has three centers of asymmetry and when it is synthe- (and retinol), and cause drug-induced osteomalacia. sized chemically the result is a mixture of the various The antituberculosis drug isoniazid inhibits cholecal- isomers. In the naturally occurring compound all ciferol 25-hydroxylase in the liver, and prolonged three centers of asymmetry have the R-configuration, administration can lead to the development of and naturally occurring α-tocopherol is called all-R, osteomalacia. or RRR-α-tocopherol. Strontium is a potent inhibitor of the kidney 1- Absorption and metabolism hydroxylase, and strontium intoxication can lead to the development of vitamin D-resistant rickets or Tocopherols and tocotrienols are absorbed unchanged osteomalacia. Although there is normally little expo- from the small intestine, in micelles with other dietary sure to potentially toxic intakes of strontium, its lipids, and incorporated into chylomicrons. The salts are sometimes used to treat chronic lead major route of excretion is in the bile, largely as gluc- intoxication. uronides and other conjugates. 8.4 Vitamin E There are two mechanisms for tissue uptake of vitamin E. Lipoprotein lipase releases the vitamin by Although vitamin E was identified as a dietary essen- hydrolyzing the triacylglycerols in chylomicrons and tial for animals in the 1920s, it was not until 1983 that VLDLs, while separately there is uptake of low-density it was clearly demonstrated to be a dietary essential lipoprotein (LDL)-bound vitamin E by means of LDL for human beings. Unlike other vitamins, no unequiv- receptors. Retention within tissues depends on intra- ocal physiological function for vitamin E has been cellular binding proteins, and it is likely that the dif- defined; it acts as a lipid-soluble antioxidant in cell ferences in biological activity of the vitamers are due membranes, but many of its functions can be replaced to differences in the affinity of these proteins for the by synthetic antioxidants. There is epidemiological different vitamers. evidence that high intakes of vitamin E are associated with lower incidence of cardiovascular disease, Metabolic functions of vitamin E although in many intervention trials vitamin E supplements have been associated with increased The main function of vitamin E is as a radical- all-cause mortality. trapping antioxidant in cell membranes and plasma lipoproteins. It is especially important in limiting Vegetable oils are rich sources of vitamin E, but radical damage resulting from oxidation of PUFAs, by significant amounts are also found in nuts and seeds, reacting with the lipid peroxide radicals before they most green leafy vegetables, and a variety of fish. can establish a chain reaction. The tocopheroxyl radical formed from vitamin E is relatively unreactive Vitamers and units of activity and persists long enough to undergo reaction to yield non-radical products. Commonly, the vitamin E Vitamin E is the generic descriptor for two families of radical in a membrane or lipoprotein is reduced back compounds, the tocopherols and the tocotrienols to tocopherol by reaction with vitamin C in plasma. (Figure 8.5). The different vitamers have different The resultant monodehydroascorbate radical then biological potency. The most active is α-tocopherol, undergoes enzymic or non-enzymic reaction to yield and it is usual to express vitamin E intake in terms of ascorbate and dehydroascorbate, neither of which is mg α-tocopherol equivalents. This is the sum of mg a radical. α-tocopherol + 0.5 × mg β-tocopherol + 0.1 × mg γ-tocopherol + 0.3 × mg α-tocotrienol. The other The stability of the tocopheroxyl radical means that vitamers have negligible vitamin activity. it can penetrate further into cells, or deeper into plasma lipoproteins, and potentially propagate a The obsolete international unit of vitamin E activ- chain reaction. Therefore, although it is regarded ity is still sometimes used: 1 IU = 0.67 mg α- as an antioxidant, vitamin E may, like other tocopherol equivalent; 1 mg α-tocopherol = 1.49 IU. antioxidants, also have pro-oxidant actions at high

CH3 HO O CH3 α-Tocop H3C CH3 β-Tocop HO CH3 O CH3 CH3 HO γ-Tocop H3C O CH3 CH3 HO δ-Tocop O CH3 CH3 Figure 8.5 The vitamin E vitamers, tocopherols and tocotrienols.

pherol H3C O CH3 α-Tocotrienol pherol H3C CH3 β-Tocotrienol pherol γ-Tocotrienol pherol HO CH3 δ-Tocotrienol HO O CH3 H3C CH3 HO O CH3 CH3 O CH3 CH3 The Vitamins 147

148 Introduction to Human Nutrition concentrations. This may explain why, although epi- They suffer from severe damage to nerve and muscle demiological studies have shown a clear association membranes. between high blood concentrations of vitamin E and lower incidence of atherosclerosis, the results of inter- Premature infants are at risk of vitamin E defi- vention trials have generally been disappointing. In ciency, since they are often born with inadequate many trials there has been increased all-cause mortal- reserves of the vitamin. The red blood cell membranes ity among those taking vitamin E and other antioxi- of deficient infants are abnormally fragile, as a result dant supplements. of unchecked oxidative radical attack. This may lead to hemolytic anemia if they are not given supple- The tocotrienols have lower vitamin activity than ments of the vitamin. tocopherols, and indeed it is conventional to consider only γ-tocotrienol as a significant part of vitamin E Experimental animals that are depleted of vitamin intake. However, because of their unsaturated side- E become sterile. However, there is no evidence that chain, the tocotrienols also have a hypocholesterol- vitamin E nutritional status is in any way associated emic action not shared by the tocopherols. They act with human fertility, and there is certainly no evi- to reduce the activity of 3-hydroxy-3-methylglutaryl- dence that vitamin E supplements increase sexual coenzyme A (HMG CoA) reductase, the rate-limiting potency, prowess, or vigor. enzyme in the pathway for synthesis of cholesterol, by repressing synthesis of the enzyme. Vitamin E requirements Vitamin E deficiency It is difficult to establish vitamin E requirements, partly because deficiency is more or less unknown, In experimental animals vitamin E deficiency results but also because the requirement depends on the in a number of different conditions. intake of PUFAs. It is generally accepted, albeit with little experimental evidence, that an acceptable intake ● Deficient female animals suffer the death and reab- of vitamin E is 0.4 mg α-tocopherol equivalent/g sorption of the fetuses. This provided the basis of dietary PUFA. the original biological assay of vitamin E. Indices of vitamin E status ● In male animals deficiency results in testicular The plasma concentration of α-tocopherol is used to atrophy and degeneration of the germinal epithe- assess vitamin E status; since most vitamin E is trans- lium of the seminiferous tubules. ported in plasma lipoproteins, it is the concentration per gram total plasma lipid, or better per mole ● Both skeletal and cardiac muscle are affected in cholesterol, that is useful, rather than the simple deficient animals. This necrotizing myopathy is concentration. sometimes called nutritional muscular dystrophy – an unfortunate term, since there is no evidence that Erythrocytes are incapable of de novo lipid synthe- human muscular dystrophy is related to vitamin E sis, so peroxidative damage resulting from oxygen deficiency. stress has a serious effect, shortening red cell life and possibly precipitating hemolytic anemia in vitamin E ● The integrity of blood vessel walls is affected, with deficiency. This has been exploited as a method of leakage of blood plasma into subcutaneous tissues assessing status by measuring the hemolysis of red and accumulation under the skin of a green fluid: cells induced by dilute hydrogen peroxide relative to exudative diathesis. that observed on incubation in water. This gives a means of assessing the functional adequacy of vitamin ● The nervous system is affected, with the develop- E intake, albeit one that will be affected by other, unre- ment of central nervous system necrosis and axonal lated, factors. Plasma concentrations of α-tocopherol dystrophy. This is exacerbated by feeding diets rich below 2.2 mmol/mol cholesterol or 1.1 μmol/g total in PUFAs. plasma lipid are associated with increased susceptibil- ity of erythrocytes to induced hemolysis in vitro. Dietary deficiency of vitamin E in human beings is unknown, although patients with severe fat malab- An alternative method of assessing functional anti- sorption, cystic fibrosis, some forms of chronic liver oxidant status, again one that is affected by both disease or (very rare) congenital lack of plasma β- vitamin E and other antioxidants, is by measuring the lipoprotein suffer deficiency because they are unable to absorb the vitamin or transport it around the body.

The Vitamins 149 exhalation of pentane arising from the catabolism of selenium acts both to remove the cause of lipid per- the products of peroxidation of n-6 PUFAs or ethane oxidation and to recycle vitamin E. arising from n-3 PUFAs. 8.5 Vitamin K Higher levels of intake There is good epidemiological evidence that intakes Vitamin K was discovered as a result of investigations of vitamin E are associated with a lower risk of ath- into the cause of a bleeding disorder (hemorrhagic erosclerosis and ischemic heart disease. High concen- disease) of cattle fed on silage made from sweet clover trations of vitamin E will inhibit the oxidation of and of chickens fed on a fat-free diet. The missing PUFAs in plasma lipoproteins, and it is this oxidation factor in the diet of the chickens was identified as that is responsible for the development of atheroscle- vitamin K, whereas the problem in the cattle was that rosis. The plasma concentrations of α-tocopherol the feed contained dicumarol, an antagonist of the that appear to be beneficial would require an intake vitamin. of 17–40 mg/day, which is above what could be achieved by eating normal diets. Individual interven- Since the effect of an excessive intake of dicumarol tion trials of vitamin E supplements have generally was severely impaired blood clotting, it was isolated been disappointing, and metaanalysis shows a signifi- and tested in low doses as an anticoagulant, for use in cant increase in all-cause mortality among people patients at risk of thrombosis. Although it was effec- taking vitamin E (and other antioxidant) supple- tive, it had unwanted side-effects, and synthetic ments. This presumably reflects the fact that the stable vitamin K antagonists were developed for clinical use tocopheroxyl radical can penetrate deeper into tissues as anticoagulants. The most commonly used of these and plasma lipoproteins, and increase radical damage. is warfarin, which is also used, in larger amounts, to However, it is also possible that the plasma concentra- kill rodents. tion of α-tocopherol is a surrogate marker for some other protective factor in the diet. Vitamers Interactions with other nutrients Three compounds have the biological activity of vitamin K (Figure 8.6): Vitamin C in plasma and extracellular fluid is impor- tant in reducing the tocopheroxyl radical in cell ● phylloquinone, the normal dietary source, found in membranes and plasma lipoproteins back to tocoph- green leafy vegetables; erol. There is also evidence that a variety of lipid- soluble antioxidants may be important in the ● menaquinones, a family of related compounds syn- antioxidant action of vitamin E in membranes and thesized by intestinal bacteria, with differing lengths lipoproteins, including ubiquinone and synthetic of the side-chain; antioxidants used in food processing, such as butyl- ated hydroxytoluene and butylated hydroxyanisole. ● menadiol and menadiol diacetate, synthetic com- Synthetic antioxidants will prevent or cure a number pounds that can be metabolized to phylloquinone. of the signs of vitamin E deficiency in experimental animals. Dietary sources, bacterial synthesis and metabolism There is a considerable overlap between the func- tions of vitamin E and selenium. Vitamin E reduces Phylloquinone has a role in photosynthesis, and lipid peroxide radicals to unreactive fatty acids; the therefore it is found in all green leafy vegetables; the selenium-dependent enzyme glutathione peroxidase richest sources are spring (collard) greens, spinach, reduces hydrogen peroxide to water, thus lowering the and Brussels sprouts. In addition, soybean, rapeseed, intracellular concentration of potentially lipid-dam- cottonseed, and olive oils are relatively rich in vitamin aging peroxide. A membrane-specific isoenzyme of K, although other oils are not. glutathione peroxidase will also reduce the tocopher- oxyl radical back to tocopherol. Thus, vitamin E acts About 80% of dietary phylloquinone is normally to remove the products of lipid peroxidation, whereas absorbed into the lymphatic system in chylomicrons, and is then taken up by the liver from chylomicron remnants and released into the circulation in VLDLs. Intestinal bacteria synthesize a variety of menaqui- nones, which are absorbed to a limited extent from

150 Introduction to Human Nutrition O CH3 O 3 Phylloquinone (vitamin K1) O CH3 On CH3 Menaquinone (vitamin K2) CO O OH CH3 CH3 OH O Figure 8.6 The vitamin K vitamers, phylloqui- Menadiol (vitamin K3) C O Menadiol diacetate none (vitamin K1), menaquinone (vitamin K2), and CH3 (acetomenaphthone) menadiol (a synthetic compound, vitamin K3). the large intestine, again into the lymphatic system, lished. It is the cofactor for the carboxylation of cleared by the liver, and released in VLDLs. It is often glutamate residues in the postsynthetic modification suggested that about half of the requirement for of proteins to form the unusual amino acid γ- vitamin K is met by intestinal bacterial synthesis, but carboxyglutamate, abbreviated to Gla (Figure 8.7). there is little evidence for this, other than the fact that about half of the vitamin K in liver is phylloquinone In the presence of warfarin, vitamin K epoxide and the remainder a variety of menaquinones. It is cannot be reduced back to the active hydroquinone, not clear to what extent the menaquinones are bio- but accumulates and is excreted as a variety of conju- logically active. It is possible to induce signs of vitamin gates. However, if enough vitamin K is provided in K deficiency simply by feeding a phylloquinone-defi- the diet, the quinone can be reduced to the active cient diet, without inhibiting intestinal bacterial hydroquinone by the warfarin-insensitive enzyme, action. and carboxylation can continue, with stoichiometric utilization of vitamin K and excretion of the epoxide. The synthetic compound menadiol is absorbed High doses of vitamin K are used to treat patients largely into the hepatic portal system, and undergoes who have received an overdose of warfarin, and at alkylation in the liver to yield menaquinone-4, which least part of the resistance of some populations of rats is released together with phylloquinone and other to the action of warfarin is due to a high consumption menaquinones in VLDLs. of vitamin K from maram grass, although there are also genetically resistant populations of rodents. Metabolic functions of vitamin K Prothrombin and several other proteins of the Although it has been known since the 1920s that blood clotting system (factors VII, IX and X, and vitamin K was required for blood clotting, it was not proteins C and S) each contain between four and until the 1970s that its precise function was estab- six γ-carboxyglutamate residues per mole. γ-Carboxy-

The Vitamins 151 H2C COO– HC COO– –OOC CHCOO– CH2 CH2 O2 CO2 CH2 NH HC C O Vitamin K NH HC C O Nonenzymic HN CHC O epoxidase Glutamate residue Glutamate carbanion γ-Carboxyglutamate residue OH O CH3 CH3 O R R OH Vitamin K hydroquinone O Vitamin K epoxide NADP+ Disulphide Sulphydryl Vitamin K quinone quinone Vitamin K epoxide reductase reductase reductase NADPH Sulphydryl Disulphide O CH3 R O Vitamin K quinone Figure 8.7 Role of vitamin K in the carboxylation of glutamate. Vitamin K epoxidase (EC 1.14.99.20), warfarin-sensitive epoxide/quinone reduc- tase (EC 1.1.4.1), warfarin-insensitive quinone reductase (EC 1.1.4.2). glutamate chelates calcium ions, and so permits the acterized protein simply known as bone matrix Gla binding of the blood clotting proteins to lipid mem- protein. Osteocalcin is interesting in that as well as γ- branes. In vitamin K deficiency, or in the presence of carboxyglutamate, it also contains hydroxyproline, so an antagonist such as warfarin, an abnormal precur- its synthesis is dependent on both vitamins K and C; sor of prothrombin (preprothrombin) containing in addition, its synthesis is induced by vitamin D, and little or no γ-carboxyglutamate is released into the the release into the circulation of osteocalcin provides circulation. Preprothrombin cannot chelate calcium a sensitive index of vitamin D action. It constitutes or bind to phospholipid membranes, and so is unable some 1–2% of total bone protein, and modifies the to initiate blood clotting. Preprothrombin is some- crystallization of bone mineral. The matrix Gla times known as PIVKA: the protein induced by protein is found in a variety of tissues, and acts to vitamin K absence. prevent mineralization of soft connective tissue. Other vitamin K-dependent proteins The fetal warfarin syndrome involves neurological It has long been known that treatment of pregnant as well as bone abnormalities. The vitamin K- women with warfarin or other anticoagulants can dependent carboxylase is expressed in different brain lead to bone abnormalities in the child: the fetal war- regions at different times during embryological devel- farin syndrome. Two proteins in bone matrix contain opment, and the product of the growth arrest-specific γ-carboxyglutamate: osteocalcin and a less well char- gene 6 (Gas6) is a Gla-containing growth factor that is important in the regulation of growth and

152 Introduction to Human Nutrition development, and the regulation of apoptosis and cell 8.6 Vitamin B1 (thiamin) survival. Historically, thiamin deficiency affecting the periph- Vitamin K deficiency and requirements eral nervous system (beriberi) was a major public health problem in south-east Asia following the intro- Apart from deliberate experimental manipulation, duction of the steam-powered mill that made highly vitamin K deficiency is unknown, and determination polished (and therefore thiamin-depleted) rice widely of requirements is complicated by a lack of informa- available. There are still sporadic outbreaks of defi- tion on the importance of menaquinones synthesized ciency among people whose diet is rich in carbohy- by intestinal bacteria. drate and poor in thiamin. More commonly, thiamin deficiency affecting the heart and central nervous The classical way of determining vitamin K status, system is a problem in people with an excessive con- and monitoring the efficacy of anticoagulant therapy, sumption of alcohol, to the extent that there was a is by measuring the time required for the formation serious suggestion in Australia at one time that of a fibrin clot in citrated blood plasma after the addi- thiamin should be added to beer. tion of calcium ions and thromboplastin: the pro- thrombin time. A more sensitive index is provided by The structures of thiamin and the coenzyme direct measurement of preprothrombin in plasma, thiamin diphosphate are shown in Figure 8.8. most commonly by immunoassay using antisera against preprothrombin that do not react with Thiamin is widely distributed in foods, with pork prothrombin. being an especially rich source; potatoes, whole-grain cereals, meat, and fish are the major sources in most Based on determination of clotting time, and direct diets. Like other water-soluble vitamins, thiamin is measurement of prothrombin and preprothrombin, readily lost by leaching into cooking water; further- an intake of 1 μg/kg body weight per day is consid- more, it is unstable to light, and although bread and ered adequate; this forms the basis of reference intakes flour contain significant amounts of thiamin, much of between 65 and 80 μg/day for adults. of this can be lost when baked goods are exposed to sunlight in a shop window. A small number of newborn infants have very low reserves of vitamin K and are at risk of potentially Thiamin is also destroyed by sulfites, and in potato fatal hemorrhagic disease. It is therefore generally rec- products that have been blanched by immersion in ommended that all neonates should be given a single sulfite solution there is little or no thiamin remaining. prophylactic dose of vitamin K. Polyphenols, including tannic acid in tea and betel nuts, also destroy thiamin, and have been associated Toxicity and drug interactions with thiamin deficiency. There is no evidence that phylloquinone has any sig- Thiamin NH2 CH3 nificant toxicity. However, high intakes can overcome H3C N CH2 CH2OH the effects of warfarin and other anticoagulants. This means that patients who are being treated with war- N CN farin could overcome the beneficial effects of their H2 S medication if they took supplements of vitamin K. The danger is that if their dose of warfarin is increased Thiamin diphosphate to counteract the effects of the vitamin supplements and they then stop taking the supplements, they H3C N NH2 CH3 H2 O O O O- would be receiving considerably too much warfarin N CH2 C O P P and would be at risk of hemorrhage. CN O- O- It is unlikely that a normal diet could provide a H2 S sufficient excess of vitamin K to lead to problems, but habitual consumption of especially rich sources could Figure 8.8 Thiamin (vitamin B1) and the coenzyme thiamin result in intakes close to those that antagonize thera- diphosphate. peutic warfarin. A diet containing relatively large amounts of foods prepared with vitamin K-rich oils may pose a risk.

The Vitamins 153 Thiaminases that catalyze base exchange or Thiamin triphosphate has a role in nerve conduc- hydrolysis of thiamin are found in microorganisms tion, as the phosphate donor for phosphorylation of (including some that colonize the gut), a variety of a nerve membrane sodium transport protein. plants, and raw fish. The presence of thiaminase in fermented fish is believed to be a significant factor in Thiamin deficiency the etiology of thiamin deficiency in parts of south- east Asia. Thiamin deficiency can result in three distinct syndromes: Absorption and metabolism of thiamin ● a chronic peripheral neuritis, beriberi, which may Thiamin is absorbed in the duodenum and proximal or may not be associated with heart failure and jejunum, and then transferred to the portal circula- edema tion by an active transport process that is inhibited by alcohol. This may explain why alcoholics are espe- ● acute pernicious (fulminating) beriberi (shoshin cially susceptible to thiamin deficiency. beriberi), in which heart failure and metabolic abnormalities predominate, with little evidence of Tissues take up both free thiamin and thiamin peripheral neuritis monophosphate, then phosphorylate them further to yield thiamin diphosphate (the active coenzyme) and, ● Wernicke’s encephalopathy with Korsakoff ’s psy- in the nervous system, thiamin triphosphate. chosis, a thiamin-responsive condition associated especially with alcohol and narcotic abuse. Some free thiamin is excreted in the urine, increas- ing with diuresis, and a significant amount may also In general, a relatively acute deficiency is involved be lost in sweat. Most urinary excretion is as thio- in the central nervous system lesions of the Wernicke– chrome, the result of non-enzymic cyclization, as well Korsakoff syndrome, and a high energy intake, as in as a variety of products of side-chain oxidation and alcoholics, is also a predisposing factor. Dry beriberi ring cleavage. is associated with a more prolonged, and presumably less severe, deficiency, and a generally low food intake, There is little storage of thiamin in the body, and whereas higher carbohydrate intake and physical biochemical signs of deficiency can be observed activity predispose to wet beriberi. within a few days of initiating a thiamin-free diet. The role of thiamin diphosphate in pyruvate dehy- Metabolic functions of thiamin drogenase means that in deficiency there is impaired conversion of pyruvate to acetyl-CoA, and hence Thiamin has a central role in energy-yielding metabo- impaired entry of pyruvate into the citric acid cycle. lism, and especially the metabolism of carbohydrates. Especially in subjects on a relatively high carbohy- Thiamin diphosphate (also known as thiamin pyro- drate diet, this results in increased plasma concentra- phosphate, see Figure 8.8) is the coenzyme for three tions of lactate and pyruvate, which may lead to oxidative decarboxylation reactions: pyruvate dehy- life-threatening lactic acidosis. The increase in plasma drogenase in carbohydrate metabolism, α-keto- lactate and pyruvate after a test dose of glucose has glutarate dehydrogenase in the citric acid cycle, and been used as a means of assessing thiamin nutritional the branched-chain keto-acid dehydrogenase involved status. in the metabolism of leucine, isoleucine, and valine. These three enzymes are multienzyme complexes that Dry beriberi catalyze oxidative decarboxylation of the substrate Chronic deficiency of thiamin, especially associated linked to reduction of enzyme-bound lipoamide, and with a high carbohydrate diet, results in beriberi, eventually reduction of NAD+ to NADH. which is a symmetrical ascending peripheral neuritis. Initially, the patient complains of weakness, stiffness Thiamin diphosphate is also the coenzyme for and cramps in the legs, and is unable to walk more transketolase, in the pentose phosphate pathway of than a short distance. There may be numbness of the carbohydrate metabolism. This is the major pathway dorsum of the feet and ankles, and vibration sense of carbohydrate metabolism in some tissues, and an may be diminished. As the disease progresses, the important alternative to glycolysis in all tissues, being ankle jerk reflex is lost, and the muscular weakness the source of half of the NADPH required for fatty spreads upwards, involving first the extensor muscles acid synthesis.

154 Introduction to Human Nutrition of the foot, then the muscles of the calf, and finally ated with alcohol misuse, the more usual presentation the extensors and flexors of the thigh. At this stage is as the Wernicke–Korsakoff syndrome, due to central there is pronounced toe and foot drop: the patient is nervous system lesions. unable to keep either the toe or the whole foot extended off the ground. When the arms are affected Initially, there is a confused state, Korsakoff ’s psy- there is a similar inability to keep the hand extended: chosis, which is characterized by confabulation and wrist drop. loss of recent memory, although memory for past events may be unimpaired. Later, clear neurological The affected muscles become tender, numb, and signs develop: Wernicke’s encephalopathy. This is hyperesthetic. The hyperesthesia extends in the form characterized by nystagmus and extraocular palsy. of a band around the limb, the so-called stocking and Post-mortem examination shows characteristic brain glove distribution, and is followed by anesthesia. lesions. There is deep muscle pain, and in the terminal stages, when the patient is bed-ridden, even slight pressure, Like shoshin beriberi, Wernicke’s encephalopathy as from bedclothes, causes considerable pain. can develop acutely, without the more gradual development of Korsakoff ’s psychosis, among Wet beriberi previously starved patients given intravenous glu- The heart may also be affected in beriberi, with dilata- cose and seriously ill patients given parenteral tion of arterioles, rapid blood flow, and increased hyperalimentation. pulse rate leading to right-sided heart failure and edema, so-called wet beriberi. The signs of chronic Thiamin requirements heart failure may be seen without peripheral neuritis. The arteriolar dilatation probably results from high Because thiamin has a central role in energy-yielding, circulating concentrations of lactate and pyruvate and especially carbohydrate, metabolism, require- as a result of impaired activity of pyruvate ments depend mainly on carbohydrate intake, and dehydrogenase. have been related to “non-fat calories.” In practice, requirements and reference intakes are calculated on Acute pernicious (fulminating) beriberi: the basis of total energy intake, assuming that the shoshin beriberi average diet provides 40% of energy from fat. For Heart failure without increased cardiac output, and diets that are lower in fat, and hence higher in carbo- no peripheral edema, may also occur acutely, associ- hydrate, thiamin requirements may be somewhat ated with severe lactic acidosis. This was a common higher. presentation of deficiency in Japan, where it was called shoshin (meaning acute) beriberi; in the 1920s From depletion/repletion studies, an intake of at some 26 000 deaths a year were recorded. least 0.2 mg of thiamin/1000 kcal is required to prevent the development of deficiency signs and With improved knowledge of the cause and maintain normal urinary excretion, but an intake of improved nutritional status, the disease has become 0.23 mg/1000 kcal is required for a normal transketo- more or less unknown, although in the 1980s it lase activation coefficient (see below). reappeared among Japanese adolescents consuming a diet based largely on such high-carbohydrate, low- Reference intakes are calculated on the basis of nutrient, foods as sweet carbonated drinks, “instant” 100 μg/MJ (0.5 mg/1000 kcal) for adults consuming noodles, and polished rice. It also occurs among more than 2000 kcal/day, with a minimum require- alcoholics, when the lactic acidosis may be life- ment for people with a low energy intake of 0.8– threatening, without clear signs of heart failure. Acute 1.0 mg/day to allow for metabolism of endogenous beriberi has also been reported when previously substrates. starved subjects are given intravenous glucose. Assessment of thiamin status Wernicke–Korsakoff syndrome Whereas peripheral neuritis, acute cardiac beriberi The impairment of pyruvate dehydrogenase in and lactic acidosis occur in thiamin deficiency associ- thiamin deficiency results in a considerable increase in the plasma concentrations of lactate and pyruvate. This has been exploited as a means of assessing thiamin status, by measuring changes in the plasma concentrations of lactate and pyruvate after an oral

The Vitamins 155 dose of glucose and mild exercise. The test is not intense yellow color, riboflavin is widely used as a specific for thiamin deficiency since a variety of other food color. conditions can also result in metabolic acidosis. Although it may be useful in depletion/repletion Photolytic destruction studies, it is little used nowadays in assessment of nutritional status. Photolysis of riboflavin leads to the formation of lumiflavin (in alkaline solution) and lumichrome (in Whole blood total thiamin below 150 nmol/l is acidic or neutral solution), both of which are biologi- considered to indicate deficiency. However, the cally inactive. Exposure of milk in clear glass bottles changes observed in depletion studies are small. Even to sunlight or fluorescent light can result in the loss in patients with frank beriberi the total thiamin con- of significant amounts of riboflavin. This is poten- centration in erythrocytes is only 20% lower than tially nutritionally important. Lumiflavin and lumi- normal, so whole blood thiamin is not a sensitive chrome catalyze oxidation of lipids (to lipid perox- index of status. ides) and methionine (to methional), resulting in the development of an unpleasant flavor, known as the Although there are several urinary metabolites of “sunlight” flavor. thiamin, a significant proportion is excreted either unchanged or as thiochrome, and therefore the Absorption and metabolism urinary excretion of the vitamin (measured as thio- chrome) can provide information on nutritional Apart from milk and eggs, which contain relatively status. Excretion decreases proportionally with intake large amounts of free riboflavin bound to specific in adequately nourished subjects, but at low intakes binding proteins, most of the vitamin in foods is as there is a threshold below which further reduction in flavin coenzymes bound to enzymes, which are intake has little effect on excretion. released when the protein is hydrolyzed. Intestinal phosphatases then hydrolyze the coenzymes to liber- The activation of apo-transketolase in erythrocyte ate riboflavin, which is absorbed in the upper small lysate by thiamin diphosphate added in vitro has intestine. The absorption of riboflavin is limited and become the most widely used and accepted index of after moderately high doses only a small proportion thiamin nutritional status. Apo-transketolase is is absorbed. unstable both in vivo and in vitro, so problems may arise in the interpretation of results, especially if Much of the absorbed riboflavin is phosphorylated samples have been stored for any appreciable time. An in the intestinal mucosa and enters the bloodstream activation coefficient >1.25 is indicative of deficiency, as riboflavin phosphate, although this does not seem and <1.15 is considered to reflect adequate thiamin to be essential for absorption of the vitamin. status. About 50% of plasma riboflavin is free riboflavin, 8.7 Vitamin B2 (riboflavin) which is the main transport form, with 44% as flavin adenine dinucleotide (FAD) and the remainder as Riboflavin deficiency is a significant public health riboflavin phosphate. The vitamin is largely protein- problem in many areas of the world. The vitamin has bound in plasma; free riboflavin binds to both a central role as a coenzyme in energy-yielding albumin and α- and β-globulins; both riboflavin and metabolism, yet deficiency is rarely, if ever, fatal, since the coenzymes also bind to immunoglobulins. there is very efficient conservation and recycling of riboflavin in deficiency. Uptake into tissues is by passive carrier-mediated transport of free riboflavin, followed by metabolic The structures of riboflavin and the riboflavin- trapping by phosphorylation to riboflavin phosphate, derived coenzymes are shown in Figure 8.9. and onward metabolism to FAD. Milk and dairy products are important sources, Riboflavin phosphate and FAD that are not bound providing 25% or more of total riboflavin intake in to proteins are rapidly hydrolyzed to riboflavin, which most diets, and it is noteworthy that average ribofla- diffuses out of tissues into the bloodstream. Ribofla- vin status in different countries reflects milk con- vin and riboflavin phosphate that are not bound to sumption to a considerable extent. Other rich sources plasma proteins are filtered at the glomerulus; renal are eggs, meat, and fish. In addition, because of its tubular resorption is saturated at normal plasma con- centrations. There is also active tubular secretion of

156 Introduction to Human Nutrition OH OH OH CH2 C CC CH2OH H HH H3C N N O NH Riboflavin H3C N O OH OH OH OH CH2 C CC CH2 O P O H HH OH H3C N N O H3C N NH Riboflavin monophosphate (flavin mononucleotide) O NH2 OH OH OH OH OH NN CH2 C CC CH2 O P O P O CH2 O N N H HH OH OH H3C N N O H3C N NH OH OH Flavin adenine dinucleotide O Figure 8.9 Riboflavin (vitamin B2) and the flavin coenzymes, riboflavin monophosphate and flavin adenine dinucleotide. the vitamin; urinary excretion of riboflavin after at which there is quantitative excretion of the moderately high doses can be two- to threefold greater vitamin. than the glomerular filtration rate. There is very efficient conservation of riboflavin in Under normal conditions about 25% of the urinary deficiency, and almost the only loss from tissues will excretion of riboflavin is as the unchanged vitamin, be the small amount that is covalently bound to with a small amount as glycosides of riboflavin and enzymes and cannot be salvaged for reuse. There is its metabolites. only a fourfold difference between the minimum con- centration of flavins in the liver in deficiency and the Riboflavin balance level at which saturation occurs. In the central nervous There is no significant storage of riboflavin; apart system there is only a 35% difference between defi- from the limitation on absorption, any surplus ciency and saturation. intake is excreted rapidly, so that once metabolic requirements have been met urinary excretion of Metabolic functions of the flavin riboflavin and its metabolites reflects intake until coenzymes intestinal absorption is saturated. In depleted animals, the maximum growth response is achieved with The metabolic function of the flavin coenzymes is as intakes that give about 75% saturation of tissues, electron carriers in a wide variety of oxidation and and the intake to achieve tissue saturation is that reduction reactions central to all metabolic processes, including the mitochondrial electron transport chain,

The Vitamins 157 and key enzymes in fatty acid and amino acid oxida- mouth (angular stomatitis), a painful desquamation tion, and the citric acid cycle. The flavin coenzymes of the tongue, so that it is red, dry, and atrophic remain bound to the enzyme throughout the catalytic (magenta tongue), and a seborrheic dermatitis, with cycle. The majority of flavoproteins have FAD as the filiform excrescences, affecting especially the nasola- prosthetic group rather than riboflavin phosphate; bial folds, eyelids, and ears. some have both flavin coenzymes and some have other prosthetic groups as well. There may also be conjunctivitis with vasculariza- tion of the cornea and opacity of the lens. This last is Flavins can undergo a one-electron reduction to the only lesion of ariboflavinosis for which the bio- the semiquinone radical or a two-electron reduction chemical basis is known: glutathione is important in to dihydroflavin. In some enzymes formation of dihy- maintaining the normal clarity of crystallin in the droflavin occurs by two single-electron steps, with lens, and glutathione reductase is a flavoprotein that intermediate formation of the semiquinone radical. is particularly sensitive to riboflavin depletion. Dihydroflavin can be oxidized by reaction with a substrate, NAD(P)+, or cytochromes in a variety of The main metabolic effect of riboflavin deficiency dehydrogenases, or can react with molecular oxygen is on lipid metabolism. Riboflavin-deficient animals in oxygenases and mixed function oxidases have a lower metabolic rate than controls and require (hydroxylases). a 15–20% higher food intake to maintain body weight. Feeding a high-fat diet leads to more marked impair- Flavins and oxidative stress ment of growth and a higher requirement for ribofla- Reoxidation of the reduced flavin in oxygenases and vin to restore growth. mixed function oxidases proceeds by way of formation of the flavin radical and flavin hydroperoxide, with the Resistance to malaria in riboflavin deficiency intermediate generation of superoxide and perhy- Several studies have noted that in areas where malaria droxyl radicals and hydrogen peroxide. Because of this, is endemic, riboflavin-deficient subjects are relatively flavin oxidases make a significant contribution to the resistant and have a lower parasite burden than ade- total oxidant stress of the body. Overall, some 3–5% of quately nourished subjects. The biochemical basis of the daily consumption of about 30 mol of oxygen by this resistance to malaria in riboflavin deficiency is an adult is converted to singlet oxygen, hydrogen not known, but two possible mechanisms have been peroxide, and superoxide, perhydroxyl, and hydroxyl proposed. radicals, rather than undergoing complete reduction to water in the electron transport chain. There is thus ● The malarial parasites may have a particularly high a total production of some 1.5 mol of reactive oxygen requirement for riboflavin. Some flavin analogues species daily, potentially capable of causing damage to have antimalarial action. membrane lipids, proteins, and nucleic acids. ● As a result of impaired antioxidant activity in Riboflavin deficiency erythrocytes, there may be increased fragility of erythrocyte membranes or reduced membrane flu- Although riboflavin is involved in all areas of metabo- idity. As in sickle cell trait, which also protects lism, and deficiency is widespread on a global scale, against malaria, this may result in exposure of the deficiency is not fatal. There seem to be two reasons parasites to the host’s immune system at a vulner- for this. One is that, although deficiency is common, able stage in their development, resulting in the the vitamin is widespread in foods and most diets will production of protective antibodies. provide minimally adequate amounts to permit main- tenance of central metabolic pathways. The second, Riboflavin requirements more important, reason is that in deficiency there is extremely efficient reutilization of the riboflavin that Estimates of riboflavin requirements are based on is released by the turnover of flavoproteins, so that depletion/repletion studies to determine the minimum only a very small amount is metabolized or excreted. intake at which there is significant excretion of the vitamin. In deficiency there is virtually no excretion Riboflavin deficiency is characterized by lesions of of the vitamin; as requirements are met, so any excess the margin of the lips (cheilosis) and corners of the is excreted in the urine. On this basis the minimum adult requirement for riboflavin is 0.5–0.8 mg/day.

158 Introduction to Human Nutrition At intakes of 1.1–1.6 mg/day urinary excretion rises an increase in the EGR activation coefficient and sharply, suggesting that tissue reserves are saturated. increased urinary excretion of riboflavin, with reduced tissue concentrations of riboflavin phosphate and A more generous estimate of requirements, and the FAD, despite feeding diets providing more riboflavin basis of reference intakes, is the level of intake at than is needed to meet requirements. Although there which there is normalization of the activity of the red is no evidence that patients treated with these drugs cell enzyme glutathione reductase; the activity of this for a prolonged period develop clinical signs of ribo- flavoprotein is especially sensitive to riboflavin nutri- flavin deficiency, long-term use of chlorpromazine is tional status. Normal values of the activation coeffi- associated with a reduction in metabolic rate. cient are seen in subjects whose habitual intake of riboflavin is between 1.2 mg/day and 1.5 mg/day. Riboflavin deficiency is sometimes associated with hypochromic anemia as a result of impaired Because of the central role of flavin coenzymes in iron absorption. A greater proportion of a test dose energy-yielding metabolism, reference intakes are of iron is retained in the intestinal mucosal cells sometimes calculated on the basis of energy intake: bound to ferritin, and hence lost in the feces, rather 0.14–0.19 mg/MJ (0.6–0.8 mg/1000 kcal). However, than being absorbed, because the mobilization of iron in view of the wide range of riboflavin-dependent bound to ferritin in mucosal cells for transfer to reactions, other than those of energy-yielding metab- transferrin requires oxidation by a flavin-dependent olism, it is difficult to justify this basis for the calcula- enzyme. tion of requirements. Riboflavin depletion decreases the oxidation of Assessment of riboflavin nutritional status dietary vitamin B6 to pyridoxal; pyridoxine oxidase is a flavoprotein and is very sensitive to riboflavin deple- The urinary excretion of riboflavin and its metabo- tion. It is not clear to what extent there is functional lites (either basal excretion or after a test dose) can be vitamin B6 deficiency in riboflavin deficiency. This is used as an index of status. However, riboflavin excre- partly because vitamin B6 nutritional status is gener- tion is only correlated with intake in subjects who are ally assessed by the metabolism of a test dose of tryp- in nitrogen balance. In subjects in negative nitrogen tophan, and kynurenine hydroxylase in the tryptophan balance there may be more urinary excretion than oxidative pathway is a flavoprotein; riboflavin defi- would be expected, as a result of the catabolism of ciency can therefore disturb tryptophan metabolism tissue flavoproteins, and loss of their prosthetic quite separately from its effects on vitamin B6 nutri- groups. Higher intakes of protein than are required tional status. to maintain nitrogen balance do not affect the require- ment for riboflavin or indices of riboflavin nutri- The disturbance of tryptophan metabolism in tional status. riboflavin deficiency, due to impairment of kynuren- ine hydroxylase, can also result in reduced synthesis Glutathione reductase is especially sensitive to of NAD from tryptophan, and may therefore be a riboflavin depletion. The activity of the enzyme in factor in the etiology of pellagra. erythrocytes can therefore be used as an index of riboflavin status. Interpretation of the results can be 8.8 Niacin complicated by anemia, and it is more usual to use the activation of erythrocyte glutathione reductase Niacin is not strictly a vitamin, since it can be synthe- (EGR) by FAD added in vitro. An activation coeffi- sized in the body from the essential amino acid tryp- cient of 1.0–1.4 reflects adequate nutritional status, tophan. Indeed, it is only when tryptophan metabo- whereas >1.7 indicates deficiency. lism is deranged that dietary preformed niacin becomes important. Nevertheless, niacin was discov- Interactions with drugs and other nutrients ered as a nutrient during studies of the deficiency disease pellagra, which was a major public health The phenothiazines such as chlorpromazine, used in problem in the southern USA throughout the first the treatment of schizophrenia, and the tricyclic anti- half of the twentieth century, and continued to be a depressant drugs such as imipramine, are structural problem in parts of India and sub-Saharan Africa analogues of riboflavin, and inhibit flavokinase. In until the 1990s. experimental animals, administration of these drugs at doses equivalent to those used clinically results in

The Vitamins 159 Vitamers and niacin equivalents Several studies have investigated the equivalence of dietary tryptophan and preformed niacin as precur- Two compounds, nicotinic acid and nicotinamide, sors of the nicotinamide nucleotides, generally by have the biological activity of niacin. When nicotinic determining the excretion of niacin metabolites in acid was discovered as the curative and preventive response to test doses of the precursors, in subjects factor for pellagra, it was already known as a chemical maintained on deficient diets. The most extensive compound, and was therefore never assigned a such study was that of Horwitt et al. in 1956. They number among the B vitamins. The name niacin was found that there was a considerable variation between coined in the USA when it was decided to enrich subjects in the response to tryptophan and niacin, maize meal with the vitamin to prevent pellagra; it and in order to allow for this individual variation they was considered that the name nicotinic acid was not proposed the ratio of 60 mg of tryptophan equivalent desirable because of its similarity to nicotine. In the to 1 mg of preformed niacin. Changes in hormonal USA the term niacin is commonly used to mean spe- status may result in considerable changes in this ratio, cifically nicotinic acid, and nicotinamide is known as with between 7 and 30 mg of dietary tryptophan niacinamide; elsewhere “niacin” is used as a generic being equivalent to 1 mg of preformed niacin in late descriptor for both vitamers. Figure 8.10 shows the pregnancy. structures of nicotinic acid and niacin, as well as the nicotinamide nucleotide coenzymes, NAD and The niacin content of foods is generally expressed NADP. as mg niacin equivalents; 1 mg niacin equivalent = mg preformed niacin + 1/60 × mg tryptophan. Because The nicotinamide ring of NAD can be synthesized most of the niacin in cereals is biologically unavail- in the body from the essential amino acid tryptophan. able (see below), it is conventional to ignore pre- In adults almost all of the dietary intake of trypto- formed niacin in cereal products. phan, apart from the small amount that is used for net new protein synthesis, and synthesis of the neu- Because endogenous synthesis from tryptophan is rotransmitter serotonin, is metabolized by this more important than preformed dietary niacin, the pathway, and hence is potentially available for NAD main dietary sources of niacin are generally those that synthesis. are also rich sources of protein. It is only when the dietary staple is a cereal such as maize, which is COOH CONH2 N N Nicotinic acid Nicotinamide CONH2 OO NH2 NN N CH2 O P O P O CH2 O N N OH OH OH OH OH OH O Figure 8.10 The niacin vitamers, nicotinic acid and Nicotinamide adenine dinucleotide nicotinamide, and the coenzyme nicotinamide adenine dinucleotide. Phosphorylated in NADP

160 Introduction to Human Nutrition remarkably lacking in tryptophan, that problems of Catabolism of NAD(P) deficiency occur. Trigonelline in coffee beans is The catabolism of NAD+ is catalyzed by four demethylated to nicotinic acid during roasting, and enzymes: moderate coffee consumption may meet a significant proportion of niacin requirements. ● NAD glycohydrolase, which releases nicotinamide and ADP-ribose; Unavailable niacin in cereals Chemical analysis reveals niacin in cereals (largely in ● NAD pyrophosphatase, which releases nicotin- the bran), but this is biologically unavailable, since it amide mononucleotide; this can be either hydro- is bound as niacytin – nicotinoyl esters to a variety of lyzed by NAD glycohydrolase to release nicotin- macromolecules. In wheat bran some 60% is esteri- amide, or reutilized to form NAD; fied to polysaccharides, and the remainder to poly- peptides and glycopeptides. ● ADP-ribosyltransferases; ● poly(ADP-ribose) polymerase. Treatment of cereals with alkali (e.g., by soaking overnight in calcium hydroxide solution, as is the tra- The activation of ADP-ribosyltransferase and ditional method for the preparation of tortillas in poly(ADP-ribose) polymerase by toxins, oxidative Mexico) and baking with alkaline baking powder stress or DNA damage may result in considerable releases much of the nicotinic acid. This may explain depletion of intracellular NAD(P), and may indeed why pellagra has always been rare in Mexico, despite provide a protective mechanism to ensure that cells the fact that maize is the dietary staple. that have suffered very severe DNA damage die as a result of NAD(P) depletion. The administration of Up to 10% of the niacin in niacytin may be biologi- DNA-breaking carcinogens to experimental animals cally available as a result of hydrolysis by gastric results in the excretion of large amounts of nicotin- acid. amide metabolites and depletion of tissue NAD(P); addition of the compounds to cells in culture has a Absorption and metabolism similar effect. Chronic exposure to such carcinogens and mycotoxins may be a contributory factor in the Niacin is present in tissues, and therefore in foods, etiology of pellagra when dietary intakes of trypto- largely as the nicotinamide nucleotides. The post- phan and niacin are marginal. mortem hydrolysis of NAD(P) is extremely rapid in animal tissues, so it is likely that much of the niacin Urinary excretion of niacin and metabolites of meat (a major dietary source of the preformed Under normal conditions there is little or no urinary vitamin) is free nicotinamide. excretion of either nicotinamide or nicotinic acid. This is because both vitamers are actively reabsorbed Nicotinamide nucleotides present in the intestinal from the glomerular filtrate. It is only when the con- lumen are not absorbed as such, but are hydrolyzed centration is so high that the reabsorption mecha- to free nicotinamide. Many intestinal bacteria nism is saturated that there is any significant excre- have high nicotinamide deamidase activity, and tion of niacin. a significant proportion of dietary nicotinamide may be deamidated in the intestinal lumen. Both Nicotinamide in excess of requirements for nicotinic acid and nicotinamide are absorbed from NAD synthesis is methylated by nicotinamide N- the small intestine by a sodium-dependent saturable methyltransferase. N1-Methylnicotinamide is actively process. secreted into the urine by the proximal renal tubules. N1-Methylnicotinamide can also be meta- The nicotinamide nucleotide coenzymes can be bolized further, to yield methylpyridone-2- and synthesized from either of the niacin vitamers and 4-carboxamides. from quinolinic acid, an intermediate in the metabo- lism of tryptophan. In the liver, synthesis of the coen- Nicotinamide can also undergo oxidation to nico- zymes increases with increasing intake of tryptophan, tinamide N-oxide when large amounts are ingested. but not preformed niacin. The liver exports nicotin- Nicotinic acid can be conjugated with glycine to form amide, derived from turnover of coenzymes, for nicotinuric acid (nicotinoyl-glycine) or may be meth- uptake by other tissues. ylated to trigonelline (N1-methylnicotinic acid). It is

The Vitamins 161 not clear to what extent urinary excretion of trigonel- calcium concentrations by releasing calcium from line reflects endogenous methylation of nicotinic intracellular stores, acting as second messengers in acid, since there is a significant amount of trigonelline response to nitric oxide, acetylcholine, and other in foods, which may be absorbed, but cannot be neurotransmitters. utilized as a source of niacin, and is excreted unchanged. Pellagra: a disease of tryptophan and niacin deficiency Metabolic functions of niacin Pellagra became common in Europe when maize The best-defined role of niacin is in the metabolism of was introduced from the New World as a convenient metabolic fuels, as the functional nicotinamide part of high-yielding dietary staple, and by the late the coenzymes NAD and NADP, which play a major nineteenth century it was widespread throughout role in oxidation and reduction reactions. The oxi- southern Europe, north and south Africa, and the dized coenzymes have a positive charge on the nico- southern USA. The proteins of maize are particularly tinamide ring nitrogen and undergo a two-electron lacking in tryptophan, and as with other cereals little reduction. The oxidized forms are conventionally or none of the preformed niacin is biologically shown as NAD(P)+ and the reduced forms either as available. NAD(P)H2 or, more correctly, as NAD(P)H + H+, since although it is a two-electron reduction, only one Pellagra is characterized by a photosensitive der- proton is incorporated into the ring, the other remain- matitis, like severe sunburn, typically with a butterfly- ing associated with the coenzyme. like pattern of distribution over the face, affecting all parts of the skin that are exposed to sunlight. Similar In general, NAD+ acts as an electron acceptor in skin lesions may also occur in areas not exposed to energy-yielding metabolism, being oxidized by the sunlight, but subject to pressure, such as the knees, mitochondrial electron transport chain, while the elbows, wrists, and ankles. Advanced pellagra is also major coenzyme for reductive synthetic reactions is accompanied by dementia (more correctly a depres- NADPH. An exception to this general rule is the sive psychosis), and there may be diarrhea. Untreated pentose phosphate pathway of glucose metabolism, pellagra is fatal. which results in the reduction of NADP+ to NADPH, and is the source of half the reductant for fatty acid The depressive psychosis is superficially similar to synthesis. schizophrenia and the organic psychoses, but clini- cally distinguishable by sudden lucid phases that In addition to its coenzyme role, NAD is the source alternate with the most florid psychiatric signs. It is of ADP-ribose for the ADP-ribosylation of a variety probable that these mental symptoms can be explained of proteins and poly(ADP-ribosylation) and hence by a relative deficit of the essential amino acid tryp- activation of nucleoproteins involved in the DNA tophan, and hence reduced synthesis of the neuro- repair mechanism. transmitter 5-hydroxytryptamine (serotonin), and not to a deficiency of niacin per se. In the nucleus, poly(ADP-ribose)polymerase is activated by binding to breakage points in DNA. The Additional factors in the etiology of pellagra enzyme is involved in activation of the DNA repair Pellagra also occurs in India among people whose mechanism in response to strand breakage caused by dietary staple is jowar (Sorghum vulgare), even though radical attack or UV radiation. In cells that have suf- the protein in this cereal contains enough tryptophan fered considerable DNA damage, the activation of to permit adequate synthesis of NAD. Here the poly (ADP-ribose) polymerase may deplete intracel- problem seems to be the relative excess of leucine in lular NAD to such an extent that ATP formation is the protein, which can inhibit the synthesis of NAD impaired, leading to cell death. from tryptophan. It is likely that leucine is a factor in the etiology of pellagra only when the dietary intakes ADP-ribose cyclase catalyzes the formation of of both tryptophan and niacin are low, a condition cyclic ADP-ribose from NAD, and of nicotinic acid that may occur when sorghum is the dietary staple, adenine dinucleotide phosphate from NADP (by cat- especially at times of food shortage. alyzing the exchange of nicotinamide for nicotinic acid). Both of these compounds act to raise cytosolic

162 Introduction to Human Nutrition Although the nutritional etiology of pellagra is well niacin metabolites, neither of which is wholly established, and tryptophan or niacin will prevent or satisfactory. cure the disease, additional factors, including defi- ciency of riboflavin or vitamin B6, both of which are Niacin toxicity required for synthesis of NAD from tryptophan, may be important when intakes of tryptophan and niacin Nicotinic acid has been used to lower blood triacyl- are only marginally adequate. glycerol and cholesterol in patients with hyperlipid- emia. However, relatively large amounts are required During the first half of the twentieth century, of the (of the order of 1–6 g/day, compared with reference 87 000 people who died from pellagra in the USA intakes of 18–20 mg/day). At this level of intake, there were twice as many women as men. Reports of nicotinic acid causes dilatation of blood vessels and individual outbreaks of pellagra, both in the USA and flushing, with skin irritation, itching, and a burning more recently elsewhere, show a similar gender ratio. sensation. This effect wears off after a few days. This may well be the result of inhibition of trypto- phan metabolism by estrogen metabolites, and hence High intakes of both nicotinic acid and nicotin- reduced synthesis of NAD from tryptophan. amide, in excess of 500 mg/day, also cause liver damage, and prolonged use can result in liver failure. Several bacterial, fungal and environmental toxins This is especially a problem with sustained-release activate ADP-ribosyltransferase or poly(ADP-ribose) preparations of niacin, which permit a high blood polymerase, and it is possible that chronic exposure level to be maintained for a relatively long time. to such toxins will deplete tissue NAD(P) and hence be a contributory factor in the development of pella- 8.9 Vitamin B6 gra when intakes of tryptophan and niacin are marginal. Apart from a single outbreak in the 1950s, due to overheated infant milk formula, vitamin B6 deficiency Niacin requirements is unknown except under experimental conditions. Nevertheless, there is a considerable body of evidence On the basis of depletion/repletion studies in which that marginal status and biochemical deficiency may the urinary excretion of niacin metabolites was mea- be relatively widespread in developed countries. sured after feeding tryptophan or preformed niacin, the average requirement for niacin is 1.3 mg of niacin Vitamin B6 is widely distributed in a variety of equivalents/MJ energy expenditure, and reference foods. However, a considerable proportion of the intakes are based on 1.6 mg/MJ. vitamin in plant foods may be present as glucosides, which are probably not biologically available, although Average intakes of tryptophan in Western diets will a proportion may be hydrolyzed by intestinal more than meet requirements without the need for a bacteria. dietary source of preformed niacin. When foods are heated, pyridoxal and pyridoxal Assessment of niacin status phosphate can react with the ε-amino groups of lysine to form a Schiff base (aldimine). This renders both Although the nicotinamide nucleotide coenzymes the vitamin B6 and the lysine biologically unavailable; function in a large number of oxidation and reduc- more importantly, the pyridoxyl-lysine released tion reactions, this cannot be exploited as a means of during digestion is absorbed and has antivitamin B6 assessing the state of the body’s niacin reserves, antimetabolic activity. Overall, it is estimated that because the coenzymes are not firmly attached to some 70–80% of dietary vitamin B6 is available. their apoenzymes, as are thiamin pyrophosphate, riboflavin, and pyridoxal phosphate, but act as cosub- Vitamers strates of the reactions, binding to and leaving the enzyme as the reaction proceeds. No specific meta- The generic descriptor vitamin B6 includes six vita- bolic lesions associated with NAD(P) depletion have mers: the alcohol pyridoxine, the aldehyde pyridoxal, been identified. the amine pyridoxamine, and their 5′-phosphates. There is some confusion in the older literature, The two methods of assessing niacin nutritional because at one time “pyridoxine,” which is now used status are measurement of the ratio of NAD/ specifically for the alcohol, was used as a generic NADP in red blood cells and the urinary excretion of

The Vitamins 163 descriptor, with “pyridoxol” as the specific name for metabolic trapping. Much of the ingested pyridoxine the alcohol. The vitamers are metabolically intercon- is released into the portal circulation as pyridoxal, vertible and, as far as is known, they have equal bio- after dephosphorylation at the serosal surface. Unlike logical activity; they are all converted in the body to other B vitamins, there seems to be no limit on the the metabolically active form, pyridoxal phosphate. amount of vitamin B6 that is absorbed. 4-Pyridoxic acid is a biologically inactive end-product of vitamin B6 metabolism. Most of the absorbed vitamin is taken up by the liver by passive diffusion, followed by metabolic trap- Absorption and metabolism ping as phosphate esters, which do not cross cell membranes, then oxidation to pyridoxal phosphate. The phosphorylated vitamers are dephosphorylated The liver exports both pyridoxal phosphate (bound by membrane-bound alkaline phosphatase in the to albumin) and pyridoxal (which binds to both intestinal mucosa; pyridoxal, pyridoxamine, and pyri- albumin and hemoglobin). Free pyridoxal remaining doxine are all absorbed rapidly by passive diffusion. in the liver is rapidly oxidized to 4-pyridoxic acid, Intestinal mucosal cells have pyridoxine kinase and which is the main excretory product. pyridoxine phosphate oxidase (Figure 8.11), so that there is net accumulation of pyridoxal phosphate by Extrahepatic tissues take up pyridoxal and pyri- doxal phosphate from the plasma. The phosphate is H2 CH2OH O- H2 CH2OH HO C OH OPOC OH Kinase O- N CH3 Phosphatase N CH3 Pyridoxine Pyridoxine phosphate Oxidase H2 COO– H2 HC O OH O- H2 HC O OH HO C OH HO C Kinase O P O C Oxidase O- N CH3 N CH3 Phosphatase N CH3 4-Pyridoxic acid Pyridoxal Pyridoxal phosphate Transaminases Oxidase H2 H2C NH2 O- H2 CH2NH2 HO C OH Kinase O P O C OH N CH3 O- N CH3 Phosphatase Pyridoxamine Pyridoxamine phosphate Figure 8.11 Interconversion of the vitamin B6 vitamers. Pyridoxal kinase (EC 2.7.1.38), pyridoxine phosphate oxidase (EC 1.1.1.65), pyridoxamine phosphate oxidase (EC 1.4.3.5).

164 Introduction to Human Nutrition hydrolyzed to pyridoxal, which can cross cell mem- The cause of the convulsions was severe impair- branes, by extracellular alkaline phosphatase, then ment of the activity of the pyridoxal phosphate- trapped intracellularly by phosphorylation. Tissue dependent enzyme glutamate decarboxylase, which concentrations of pyridoxal phosphate are controlled catalyzes the synthesis of the inhibitory neurotrans- by the balance between phosphorylation and mitter γ-aminobutyric acid (GABA), together with dephosphorylation. accumulation of hydroxykynurenine as a result of impaired activity of kynureninase, which is also pyri- Some 80% of the body’s total vitamin B6 is pyri- doxal phosphate dependent. doxal phosphate in muscle, mostly associated with glycogen phosphorylase. This does not function as a Moderate vitamin B6 deficiency results in a number reserve of the vitamin and is not released from muscle of abnormalities of amino acid metabolism, espe- in times of deficiency; it is released into the circula- cially of tryptophan and methionine. In experimental tion (as pyridoxal) in starvation, when glycogen animals, a moderate degree of deficiency leads to reserves are exhausted and there is less requirement increased sensitivity of target tissues to steroid for phosphorylase activity. Under these conditions it hormone action. This may be important in the devel- is available for redistribution to other tissues, and opment of hormone-dependent cancer of the breast, especially the liver and kidneys, to meet the increased uterus, and prostate, and may therefore affect the need for transamination of amino acids to provide prognosis. Vitamin B6 supplementation may be a substrates for gluconeogenesis. useful adjunct to other therapy in these common cancers; certainly, there is evidence that poor vitamin Metabolic functions of vitamin B6 B6 nutritional status is associated with a poor prog- nosis in women with breast cancer. Pyridoxal phosphate is a coenzyme in three main areas of metabolism: Vitamin B6 requirements ● in a wide variety of reactions of amino acids, espe- Most studies of vitamin B6 requirements have fol- cially transamination, in which it functions as the lowed the development of abnormalities of trypto- intermediate carrier of the amino group, and decar- phan and methionine metabolism during depletion boxylation to form amines and normalization during repletion with graded intakes of the vitamin. Although the tryptophan load ● as the cofactor of glycogen phosphorylase in muscle test is unreliable as an index of vitamin B6 nutritional and liver, where it is the phosphate group that is status in field studies, under the controlled conditions catalytically important of depletion/repletion studies it gives a useful indica- tion of the state of vitamin B6 nutrition. ● in the regulation of the action of steroid hormones. Pyridoxal phosphate acts to remove the hormone– Since the major role of vitamin B6 is in amino acid receptor complex from DNA binding, and so ter- metabolism it is likely that protein intake will affect minate the action of the hormones. In vitamin B6 vitamin B6 requirements. Adults maintained on deficiency there is increased sensitivity and respon- vitamin B6-deficient diets develop abnormalities of siveness of target tissues to low concentrations of tryptophan and methionine metabolism more quickly, steroid hormones, including estrogens, androgens, and their blood vitamin B6 falls more rapidly, when cortisol, and vitamin D. their protein intake is relatively high (80–160 g/day in various studies) than on low protein intakes (30–50 g/ Vitamin B6 deficiency day). Similarly, during repletion of deficient subjects, tryptophan and methionine metabolism and blood Deficiency of vitamin B6 severe enough to lead to vitamin B6 are normalized more rapidly at low than clinical signs is extremely rare, and unequivocal defi- at high levels of protein intake. ciency has only been reported in one outbreak, during the 1950s, when babies were fed on a milk preparation From such studies the average requirement for that had been severely overheated during manufac- vitamin B6 is estimated to be 13 μg/g dietary protein, ture. Many of the affected infants suffered convul- and reference intakes are based on 15–16 μg/g dietary sions, which ceased rapidly following the administra- protein. tion of vitamin B6.

The Vitamins 165 Requirements of infants The tryptophan load test Estimation of the vitamin B6 requirements of infants The tryptophan load test for vitamin B6 nutritional presents a problem, and there is a clear need for status (the ability to metabolize a test dose of trypto- further research. Human milk, which must be assumed phan) is one of the oldest metabolic tests for func- to be adequate for infant nutrition, provides only tional vitamin nutritional status. It was developed as some 2.5–3 μg of vitamin B6/g protein. This is very a result of observation of the excretion of an abnor- much lower than the requirement for adults, although mal colored compound, later identified as the trypto- there is no reason why infants should have a lower phan metabolite xanthurenic acid, in the urine of requirement. deficient animals. Based on the body content of 3.7 μg (15 nmol) Kynureninase (see Figure 8.12) is a pyridoxal phos- of vitamin B6/g body weight, and the rate of weight phate-dependent enzyme, and its activity falls mark- gain, a minimum requirement for infants over the edly in vitamin B6 deficiency, at least partly because it first 6 months of life is 100 μg/day to establish undergoes a slow mechanism-dependent inactivation tissue reserves, and an additional 20% to allow for that leaves catalytically inactive pyridoxamine phos- metabolic turnover. Even if the mother receives phate at the active site of the enzyme. The enzyme can daily supplements of 2.5 mg of vitamin B6 through- only be reactivated if there is an adequate supply of out lactation, thus more than doubling her normal pyridoxal phosphate. This means that in vitamin B6 intake, the infant’s intake ranges from 100 μg/day to deficiency there is a considerable accumulation of 300 μg/day over the first 6 months of life. At 1 month both hydroxykynurenine and kynurenine, sufficient this is only 8.5 μg/g protein, rising to 15 μg/g by to permit greater metabolic flux than usual through 2 months. kynurenine transaminase, resulting in increased for- mation of kynurenic and xanthurenic acids. Assessment of vitamin B6 status Xanthurenic and kynurenic acids, and kynurenine Fasting plasma total vitamin B6 (measured microbio- and hydroxykynurenine, are easy to measure in urine, logically), or more specifically pyridoxal phosphate, is so the tryptophan load test [the ability to metabolize widely used as an index of vitamin B6 nutritional a test dose of 2–5 g (150–380 μmol/kg body weight) status. Despite the fall in plasma pyridoxal phosphate of tryptophan] has been widely adopted as a con- in pregnancy, which has been widely interpreted as venient and very sensitive index of vitamin B6 indicating vitamin B6 depletion or an increased nutritional status. However, because glucocorticoid requirement, the plasma concentration of pyridoxal hormones increase tryptophan dioxygenase activity, phosphate plus pyridoxal is unchanged. This suggests abnormal results of the tryptophan load test must be that determination of plasma pyridoxal phosphate regarded with caution, and cannot necessarily be alone may not be a reliable index of vitamin B6 nutri- interpreted as indicating vitamin B6 deficiency. tional status. Increased entry of tryptophan into the pathway will overwhelm the capacity of kynureninase, leading to About half of the normal dietary intake of vitamin increased formation of xanthurenic and kynurenic B6 is excreted as 4-pyridoxic acid. Urinary excretion acids. Similarly, estrogen metabolites inhibit kyn- of 4-pyridoxic acid will largely reflect the recent intake ureninase, leading to results that have been misinter- of the vitamin rather than the underlying nutritional preted as vitamin B6 deficiency. status. The methionine load test Coenzyme saturation of transaminases The metabolism of methionine includes two pyri- The most widely used method of assessing vitamin B6 doxal phosphate-dependent steps: cystathionine status is by the activation of erythrocyte transami- synthetase and cystathionase (see Figure 8.16). nases by pyridoxal phosphate added in vitro. An acti- Cystathionase activity falls markedly in vitamin B6 vation coefficient for alanine transaminase >1.25, or deficiency, and as a result there is an increase in the for aspartate transaminase >1.8, is considered to indi- urinary excretion of homocysteine and cystathionine, cate deficiency. both after a loading dose of methionine and under

166 Introduction to Human Nutrition H2C COO– O2 C NH3+ H Kynurenine aminotransferase Kynurenine Kynurenic acid N H Tryptophan dioxygenase and O2 Tryptophan formylkynurenine formamidase NADPH Kynurenine hydroxylase NADP+ Hydroxykynurenine Xanthurenic acid Kynurenine H2O aminotransferase Kynureninase Alanine 3-Hydroxyanthranilic acid 3-Hydroxyanthranilic acid oxidase Aminocarboxymuconic semialdehyde Total oxidation Quinolinic acid via acetyl CoA for NAD synthesis Figure 8.12 Oxidative pathway of tryptophan: the basis of the tryptophan load test. Tryptophan dioxygenase (EC 1.13.11.11), formylkynurenine formamidase (EC 3.5.1.9), kynurenine hydroxylase (EC 1.14.13.9), kynureninase (EC 3.7.1.3). basal conditions. However, as discussed below, homo- All of the studies that suggested that oral contra- cysteine metabolism is more affected by folate status ceptives cause vitamin B6 deficiency used the metabo- than by vitamin B6 status and, like the tryptophan load lism of tryptophan as a means of assessing vitamin B6 test, the methionine load test is probably not reliable nutritional status. When other biochemical markers as an index of vitamin B6 status in field studies. of status were also assessed, they were not affected by oral contraceptive use. Furthermore, most of these Non-nutritional uses of vitamin B6 studies were performed using the now obsolete high- dose contraceptive pills. Several studies have suggested that oral contraceptives cause vitamin B6 deficiency. As a result of this, supple- Oral contraceptives do not cause vitamin B6 defi- ments of vitamin B6 of 50–100 mg/day, and some- ciency. The problem is that estrogen metabolites times higher, have been used to overcome the side- inhibit kynureninase and reduce the activity of kyn- effects of oral contraceptives. Similar supplements urenine hydroxylase. This results in the excretion of have also been recommended for the treatment of the abnormal amounts of tryptophan metabolites, similar premenstrual syndrome, although there is little evi- to what is seen in vitamin B6 deficiency, but for a dif- dence of efficacy from placebo-controlled trials. ferent reason.

The Vitamins 167 Doses of 50–200 mg of vitamin B6/day have an Structure and vitamers antiemetic effect, and the vitamin is widely used, alone or in conjunction with other antiemetics, to The structure of vitamin B12 is shown in Figure 8.13. minimize the nausea associated with radiotherapy The term corrinoid is used as a generic descriptor for and to treat pregnancy sickness. There is no evidence cobalt-containing compounds of this general struc- that vitamin B6 has any beneficial effect in pregnancy ture that, depending on the substituents in the pyrrole sickness, or that women who suffer from morning rings, may or may not have vitamin activity. The term sickness have lower vitamin B6 nutritional status than “vitamin B12” is used as a generic descriptor for the other pregnant women. cobalamins, that is, those corrinoids having the bio- logical activity of the vitamin. Some of the corrinoids Doses of vitamin B6 of 100 mg/day have been that are growth factors for microorganisms not only reported to be beneficial in the treatment of the carpal have no vitamin B12 activity, but may be antimetabo- tunnel syndrome or tenosynovitis. However, most of lites of the vitamin. the reports originate from one centre and there appears to be little independent confirmation of the Although cyanocobalamin was the first form in usefulness of the vitamin in this condition. which vitamin B12 was isolated, it is not an important naturally occurring vitamer, but rather an artifact due Vitamin B6 toxicity to the presence of cyanide in the charcoal used in the extraction procedure. It is more stable to light than In experimental animals, doses of vitamin B6 of the other vitamers, and hence is used in pharmaceuti- 50 mg/kg body weight cause histological damage to cal preparations. Photolysis of cyanocobalamin in dorsal nerve roots, and doses of 200 mg/kg body solution leads to the formation of aquocobalamin or weight lead to the development of signs of peripheral hydroxocobalamin, depending on pH. Hydroxoco- neuropathy, with ataxia, muscle weakness, and loss of balamin is also used in pharmaceutical preparations, balance. The clinical signs of vitamin B6 toxicity in and is better retained after parenteral administration animals regress within 3 months after withdrawal of than is cyanocobalamin. these massive doses, but sensory nerve conduction velocity, which decreases during the development of Vitamin B12 is found only in foods of animal origin, the neuropathy, does not recover fully. although it is also formed by bacteria. There are no plant sources of this vitamin. This means that Sensory neuropathy has been reported in seven strict vegetarians (vegans), who eat no foods of patients taking 2–7 g of pyridoxine/day. Although animal origin, are at risk of developing dietary vitamin there was some residual damage, withdrawal of these B12 deficiency, although the small amounts of extremely high doses resulted in a considerable vitamin B12 formed by bacteria on the surface of recovery of sensory nerve function. Other reports fruits may be adequate to meet requirements. Prepa- have suggested that intakes as low as 50 mg/day rations of vitamin B12 made by bacterial fermentation are associated with neurological damage, although that are ethically acceptable to vegans are readily these studies were based on patients reporting symp- available. toms rather than objective neurological examination. There have been no reports of nerve damage in chil- There are claims that yeast and some plants (espe- dren with vitamin B6-dependent homocystinuria, cially some algae) contain vitamin B12. This seems to or other inborn errors of metabolism, who take be incorrect. The problem is that the officially recog- 200–300 mg/day. nized, and legally required, method of determining vitamin B12 in food analysis is a microbiological assay 8.10 Vitamin B12 using organisms for which vitamin B12 is an essential growth factor. However, these organisms can also use Dietary deficiency of vitamin B12 occurs only in strict some corrinoids that have no vitamin activity. There- vegans, since the vitamin is found almost exclusively fore, analysis reveals the presence of something that in animal foods. However, functional deficiency (per- appears to be vitamin B12, but in fact is not the active nicious anemia, with spinal cord degeneration) as a vitamin and is useless in human nutrition. Biologi- result of impaired absorption is relatively common, cally active vitamin B12 has been identified in some especially in older people with atrophic gastritis. preparations of algae, but this seems to be the result

168 Introduction to Human Nutrition O O H2C C NH2 H2 H2 O H3C H2N C CH2 H3C C C C NH2 H2N O H2C N H2 Co N CC N CH3 N CH2CH3 CH3 H3C H2 O H3C H2C C C CH3 NH2 H2N C C O H2 CH2 CO NH CH2 O CH3 CH3 H3C C OPO N Figure 8.13 Vitamin B12. Four coordination sites on H N the central cobalt atom are occupied by nitrogen O OH atoms of the ring, and one by the nitrogen of the dimethylbenzimidazole side-chain. The sixth HO CH2 O coordination site may be occupied by cyanide (cyanocobalamin), a hydroxyl ion (hydroxocobala- min), water (aquocobalamin), or a methyl group (methylcobalamin). of bacterial contamination of the lakes where the Gastric acid and pepsin play a role in vitamin B12 algae were harvested. nutrition, serving to release the vitamin from protein binding, so making it available. Atrophic gastritis is a Absorption and metabolism of vitamin B12 relatively common problem of advancing age; in the early stages there is failure of acid secretion but more Absorption or less normal secretion of intrinsic factor. This can Very small amounts of vitamin B12 can be absorbed result in vitamin B12 depletion due to failure to release by passive diffusion across the intestinal mucosa, but the vitamin from dietary proteins, although the under normal conditions this is insignificant; the absorption of free vitamin B12 (as in supplements or major route of vitamin B12 absorption is by attach- fortified foods) is unaffected. In the stomach, vitamin ment to a specific binding protein in the intestinal B12 binds to cobalophilin, a binding protein secreted lumen. in the saliva. This binding protein is intrinsic factor, so called In the duodenum cobalophilin is hydrolyzed, because in the early studies of pernicious anemia it releasing vitamin B12 to bind to intrinsic factor. Pan- was found that two curative factors were involved: an creatic insufficiency can therefore be a factor in the extrinsic or dietary factor, which is now known to be development of vitamin B12 deficiency, since failure to vitamin B12, and an intrinsic or endogenously pro- hydrolyze cobalophilin will result in the excretion of duced factor. Intrinsic factor is a small glycoprotein cobalophilin-bound vitamin B12 rather than transfer secreted by the parietal cells of the gastric mucosa, to intrinsic factor. Intrinsic factor binds the various which also secrete hydrochloric acid. vitamin B12 vitamers, but not other corrinoids.

The Vitamins 169 Vitamin B12 is absorbed from the distal third of the The other clinical feature of vitamin B12 deficiency, ileum. There are intrinsic factor–vitamin B12 binding which is rarely seen in folic acid deficiency, is degen- sites on the brush border of the mucosal cells in this eration of the spinal cord; hence the name “perni- region; neither free intrinsic factor nor free vitamin cious” for the anemia of vitamin B12 deficiency. The B12 interacts with these receptors. spinal cord degeneration is due to a failure of the methylation of one arginine residue in myelin basic In plasma, vitamin B12 circulates bound to transco- protein. About one-third of patients who present with balamin I, which is required for tissue uptake of the megaloblastic anemia due to vitamin B12 deficiency vitamin, and transcobalamin II, which seems to be a also have spinal cord degeneration, and about one- storage form of the vitamin. third of deficient subjects present with neurological signs but no anemia. Enterohepatic circulation of vitamin B12 There is a considerable enterohepatic circulation of The most common cause of pernicious anemia is vitamin B12. A third plasma vitamin B12 binding failure of the absorption of vitamin B12, rather than protein, transcobalamin III, is rapidly cleared by the dietary deficiency. Classical pernicious anemia is due liver, with a plasma half-life of the order of 5 min. to failure of intrinsic factor secretion, commonly the This provides a mechanism for returning vitamin B12 result of autoimmune disease, with production of anti- and its metabolites from peripheral tissues to the liver, bodies against either the gastric parietal cells or intrin- as well as for clearance of other corrinoids without sic factor. Atrophic gastritis with increasing age also vitamin activity, which may arise from either foods or leads to progressive failure of vitamin B12 absorption. the products of intestinal bacterial action, and be absorbed passively across the lower gut. Dietary deficiency of vitamin B12 does occur, rarely, in strict vegetarians (vegans). The rarity of vitamin These corrinoids are then secreted into the bile, B12 deficiency among people who have no apparent bound to cobalophilins; 3–8 μg (2.25–6 nmol) of dietary source of the vitamin suggests that bacterial vitamin B12 may be secreted in the bile each day, about contamination of water and foods with vitamin B12- the same as the dietary intake. Like dietary vitamin producing organisms will provide minimally adequate B12 bound to salivary cobalophilin, the biliary coba- amounts of the vitamin. The fruit bat develops lophilins are hydrolyzed in the duodenum, and the vitamin B12 deficiency when fed on washed fruit vitamin binds to intrinsic factor, so permitting reab- under laboratory conditions, but in the wild micro- sorption in the ileum. Although cobalophilins and bial contamination of the outside of the fruit provides transcorrin III have low specificity, and will bind a an adequate intake of the vitamin. variety of corrinoids, intrinsic factor binds only cobalamins, and so only the biologically active vitamin Vitamin B12 requirements is reabsorbed. Most estimates of vitamin B12 requirements are based Metabolic functions of vitamin B12 on the amounts given parenterally to maintain normal health in patients with pernicious anemia due to a There are three vitamin B12-dependent enzymes in failure of vitamin B12 absorption. This overestimates human tissues: methylmalonyl-CoA mutase (dis- normal requirements, because of the enterohepatic cussed below under methylmalonic aciduria), leucine circulation of vitamin B12; in people lacking intrinsic amino-mutase, and methionine synthetase (discussed factor, or secreting anti-intrinsic factor antibodies, in Section 8.11). the vitamin that is excreted in the bile will be lost in the feces, whereas normally it is almost completely Vitamin B12 deficiency: pernicious anemia reabsorbed. Vitamin B12 deficiency causes pernicious anemia; the The total body pool of vitamin B12 is of the order release into the bloodstream of immature precursors of 2.5 mg (1.8 μmol), with a minimum desirable body of red blood cells (megaloblastic anemia). As dis- pool of about 1 mg (0.3 μmol). The daily loss is about cussed below, vitamin B12 deficiency causes functional 0.1% of the body pool in subjects with normal entero- folate deficiency; this is what disturbs the rapid mul- hepatic circulation of the vitamin; on this basis tiplication of red blood cells, causing immature pre- requirements are about 1–2.5 μg/day and reference cursors to be released into the circulation. intakes for adults range between 1.4 μg and 2.0 μg.

170 Introduction to Human Nutrition Assessment of vitamin B12 status bolic reactions; it is therefore metabolically closely related to vitamin B12. Deficiency of either causes Measurement of plasma concentrations of vitamin megaloblastic anemia, and the hematological effects B12 is the method of choice, and several simple and of vitamin B12 deficiency are due to disturbance of reliable radioligand binding assays have been devel- folate metabolism. oped. A serum concentration of vitamin B12 below 110 pmol/l is associated with megaloblastic bone Apart from liver, the main dietary sources of folate marrow, incipient anemia, and myelin damage. Below are fruits and vegetables. Although folate is widely 150 pmol/l there are early bone marrow changes, distributed in foods, dietary deficiency is not uncom- abnormalities of the deoxyuridine monophosphate mon, and a number of commonly used drugs can (dUMP) suppression test (see Section 8.11) and cause folate depletion. More importantly, there is methylmalonic aciduria after a valine load. good evidence that intakes of folate considerably higher than normal dietary levels reduce the risk of The Schilling test for vitamin B12 absorption neural tube defects, and, where cereal products are The absorption of vitamin B12 can be determined by not fortified with folate by law, pregnant women are the Schilling test. An oral dose of [57Co] or [58Co]- recommended to take supplements. There is also evi- vitamin B12 is given with a parenteral flushing dose of dence that high intakes of folate may be effective in 1 mg of non-radioactive vitamin to saturate body reducing plasma homocysteine in subjects genetically reserves, and the urinary excretion of radioactivity is at risk of hyperhomocystinemia (some 10–20% of the followed as an index of absorption of the oral mate- population), which may reduce the risk of ischemic rial. Normal subjects excrete 16–45% of the radioac- heart disease and stroke. tivity over 24 h, whereas patients lacking the intrinsic factor excrete less than 5%. Vitamers and dietary equivalence The test can be repeated, giving the intrinsic factor As shown in Figure 8.14, folic acid consists of a orally together with the radioactive vitamin B12; if the reduced pterin linked to p-aminobenzoic acid, impaired absorption was due to a simple lack of forming pteroic acid. The carboxyl group of the p- intrinsic factor, and not to anti-intrinsic factor anti- aminobenzoic acid moiety is linked by a peptide bond bodies in the saliva or gastric juice, then a normal to the α-amino group of glutamate, forming pteroyl- amount of the radioactive material should be absorbed glutamate (PteGlu). The coenzymes may have up to and excreted. seven additional glutamate residues linked by γ- peptide bonds, forming pteroyldiglutamate (PteGlu2), Methylmalonic aciduria pteroyltriglutamate (PteGlu3), etc., collectively Methylmalonyl-CoA is formed as an intermediate in known as folate or pteroyl polyglutamate conjugates the catabolism of valine and by the carboxylation of (PteGlun). propionyl-CoA arising in the catabolism of isoleu- cine, cholesterol, and (rare) fatty acids with an odd “Folate” is the preferred trivial name for pteroyl- number of carbon atoms. Normally, it undergoes glutamate, although both “folate” and “folic acid” may vitamin B12-dependent rearrangement to succinyl- be used as a generic descriptor to include various CoA, catalyzed by methylmalonyl-CoA mutase. polyglutamates. PteGlu2 is sometimes referred to as Vitamin B12 deficiency leads to an accumulation of folic acid diglutamate, PteGlu3 as folic acid trigluta- methylmalonyl-CoA, which is hydrolyzed to methyl- mate, and so on. malonic acid, which is excreted in the urine. Urinary excretion of methylmalonic acid, especially after a O H H2 H H2 H COO– loading dose of valine, provides a means of assessing N CN C N CH vitamin B12 nutritional status. HN CH2 8.11 Folic acid CH2 H2N N N Tetrahydrofolate CO Folic acid functions in the transfer of one-carbon H (Glu)n fragments in a wide variety of biosynthetic and cata- Figure 8.14 Tetrahydrofolate (folic acid).

The Vitamins 171 Tetrahydrofolate can carry one-carbon fragments Despite this, there is very little fecal loss of folate; attached to N-5 (formyl, formimino, or methyl jejunal absorption of methyl-tetrahydrofolate is a groups), N-10 (formyl), or bridging N-5–N-10 (methy- very efficient process, and the fecal excretion of some lene or methenyl groups). 5-Formyl-tetrahydrofolate 450 nmol (200 μg) of folates per day represents syn- is more stable to atmospheric oxidation than is folate, thesis by intestinal flora and does not reflect intake to and is therefore commonly used in pharmaceutical any significant extent. preparations; it is also known as folinic acid, and the synthetic (racemic) compound as leucovorin. Tissue uptake of folate Methyl-tetrahydrofolate circulates bound to albumin, The extent to which the different forms of folate and is available for uptake by extrahepatic tissues, can be absorbed varies; on average only about half of where it is trapped by formation of polyglutamates, the folate in the diet is available, compared with more which do not cross cell membranes. or less complete availability of the monoglutamate. To permit calculation of folate intakes, the dietary folate The main circulating folate is methyl-tetrahydro- equivalent has been defined as 1 μg mixed food folates folate, which is a poor substrate for polyglutamylation; or 0.6 μg free folic acid. On this basis, total dietary demethylation by the action of methionine synthetase folate equivalents = μg food folate + 1.7 × synthetic (see below) is required for effective metabolic trap- (free) folic acid. ping of folate. In vitamin B12 deficiency, when methio- nine synthetase activity is impaired, there will Absorption and metabolism of folate therefore be impairment of the uptake of folate into tissues. About 80% of dietary folate is as polyglutamates; a variable amount may be substituted with various Folate excretion one-carbon fragments or be present as dihydrofolate There is very little urinary loss of folate, only some derivatives. Folate conjugates are hydrolyzed in the 5–10 nmol/day. Not only is most folate in plasma small intestine by conjugase (pteroylpolyglutamate bound to proteins (either folate binding protein hydrolase), a zinc-dependent enzyme of the pancre- for unsubstituted folate or albumin for methyl- atic juice, bile, and mucosal brush border; zinc defi- tetrahydrofolate), and thus protected from glomeru- ciency can impair folate absorption. lar filtration, but the renal brush border has a high concentration of folate binding protein, which acts to Free folate, released by conjugase action, is absorbed reabsorb any filtered in the urine. by active transport in the jejunum. The folate in milk is mainly bound to a specific binding protein; the The catabolism of folate is largely by cleavage of the protein–tetrahydrofolate complex is absorbed intact, C-9–N-10 bond, catalyzed by carboxypeptidase G. mainly in the ileum, by a mechanism that is distinct The p-aminobenzoic acid moiety is amidated and from the active transport system for the absorption excreted in the urine as p-acetamidobenzoate and p- of free folate. The biological availability of folate from acetamidobenzoyl-glutamate; pterin is excreted either milk, or of folate from diets to which milk has been unchanged or as a variety of biologically inactive added, is considerably greater than that of unbound compounds. folate. Metabolic functions of folate Much of the dietary folate undergoes methylation and reduction within the intestinal mucosa, so that The metabolic role of folate is as a carrier of one- what enters the portal bloodstream is largely 5- carbon fragments, both in catabolism and in biosyn- methyl-tetrahydrofolate. Other substituted and thetic reactions. These may be carried as formyl, unsubstituted folate monoglutamates, and dihydrofo- formimino, methyl or methylene residues. The major late, are also absorbed; they are reduced and methyl- sources of these one-carbon fragments and their ated in the liver, then secreted in the bile. The liver major uses, as well as the interconversions of the sub- also takes up various folates released by tissues; again, stituted folates, are shown in Figure 8.15. these are reduced, methylated and secreted in the bile. The major point of entry for one-carbon fragments into substituted folates is methylene-tetrahydrofolate, The total daily enterohepatic circulation of folate is which is formed by the catabolism of glycine, serine, equivalent to about one-third of the dietary intake.

172 Introduction to Human Nutrition Sources of one-carbon units Synthesis using one-carbon units Serine Serine Glycine Methylene-THF (a) Methyl-THF (b) Methionine Choline TMP + Dihydrofolate Formimino-THF Histidine Methenyl-THF Formate Formyl-THF Purines CO2 Figure 8.15 Interconversion of the principal one-carbon substituted folates; sources of one-carbon fragments are shown on the left, and pathways in which one-carbon units are used and free tetrahydrofolate is regenerated on the right. (a) Methylene-tetrahydrofolate reductase (EC 1.5.1.20); (b) methionine synthetase (EC 2.1.1.13). and choline. Serine is the most important source of Thymidylate synthetase and substituted folates for biosynthetic reactions, and the activity of serine hydroxymethyltransferase is dihydrofolate reductase regulated by the state of folate substitution and the The methylation of dUMP to thymidine monophos- availability of folate. The reaction is freely reversible, phate (TMP), catalyzed by thymidylate synthetase, is and under appropriate conditions in liver it functions essential for the synthesis of DNA, although pre- to form serine from glycine as a substrate for formed TMP arising from the catabolism of DNA can gluconeogenesis. be reutilized. Methylene-, methenyl-, and 10-formyl-tetrahydro- The methyl donor for thymidylate synthetase is folates are freely interconvertible. This means that methylene-tetrahydrofolate; the reaction involves when one-carbon folates are not required for syn- reduction of the one-carbon fragment to a methyl thetic reactions, the oxidation of formyl-tetrahydro- group at the expense of the folate, which is oxidized folate to carbon dioxide and folate provides a means to dihydrofolate. Dihydrofolate is then reduced to of maintaining an adequate tissue pool of free tetrahydrofolate by dihydrofolate reductase. folate. Thymidylate synthase and dihydrofolate reductase By contrast, the reduction of methylene-tetra- are especially active in tissues with a high rate of cell hydrofolate to methyl-tetrahydrofolate is irreversible, division, and hence a high rate of DNA replication and the only way in which free folate can be formed and a high requirement for thymidylate. Because of from methyl-tetrahydrofolate is by the reaction of this, inhibitors of dihydrofolate reductase have been methionine synthetase (see below). exploited as anticancer drugs (e.g. methotrexate). Chemotherapy consists of alternating periods of

The Vitamins 173 administration of methotrexate to inhibit tumor ● Methionine synthetase is a vitamin B12-dependent growth, and folate (normally as 5-formyl-tetrahydro- enzyme, for which the methyl donor is folate, leucovorin) to replete tissues and avoid folate methyl-tetrahydrofolate. deficiency; this is known as leucovorin rescue. ● Homocysteine methyltransferase utilizes betaine Methionine synthetase and (an intermediate in the catabolism of choline) as the methyl-folate trap the methyl donor, and is not vitamin B12 In addition to its role in the synthesis of proteins, dependent. methionine, as the S-adenosyl derivative, acts as a methyl donor in a wide variety of biosynthetic reac- Both enzymes are found in most tissues, but only the tions. As shown in Figure 8.16, the resultant homo- vitamin B12-dependent methionine synthetase is cysteine may be either metabolized to yield cysteine found in the central nervous system. or remethylated to yield methionine. The reduction of methylene-tetrahydrofolate to Two enzymes catalyze the methylation of homo- methyl-tetrahydrofolate is irreversible, and the major cysteine to methionine: source of folate for tissues is methyl-tetrahydrofolate. The only metabolic role of methyl-tetrahydrofolate is the methylation of homocysteine to methionine, and Methylated product Acceptor S-Adenosylmethionine S-Adenosylhomocysteine Methyltransferases Adenosine PPi + Pi Methionine adenosyltransferase ATP SH CH3 CH2 S CH2 HC NH3+ CH2 Methionine synthetase COO– CH2 Homocysteine Homocysteine HC NH3+ COO– Methionine Methyl THF Tetrahydrofolate Serine Cystathionine b -synthetase H2O Cystathionine H2O g -Cystathionase α-Ketobutyrate + NH4+ Cysteine Figure 8.16 Methionine metabolism. Methionine synthetase (EC 2.1.1.13), methionine adenosyltransferase (EC 2.5.1.6), cystathionine synthetase (EC 4.2.1.22), cystathionase (EC 4.4.1.1).

174 Introduction to Human Nutrition this is the only way in which methyl-tetrahydrofolate disease. However, a number of intervention trials with can be demethylated to yield free folate in tissues. folate supplements have shown no reduction in death Methionine synthetase thus provides the link between from myocardial infarction, nor any decrease in all- the physiological functions of folate and vitamin B12. cause mortality, despite a significant decrease in Impairment of methionine synthetase activity in plasma homocysteine. Similarly, in countries where vitamin B12 deficiency will result in the accumulation there has been mandatory enrichment of flour with of methyl-tetrahydrofolate, which can neither be uti- folate for some years, there is no evidence of reduced lized for any other one-carbon transfer reactions nor mortality from cardiovascular disease. It is possible be demethylated to provide free folate. that elevated plasma homocysteine is not so much a cause of atherosclerosis (although there are good This functional deficiency of folate is exacerbated mechanisms to explain why it might be atherogenic) by low tissue concentrations of methionine and an as the result of impaired kidney function due to early accumulation of homocysteine, since the transulfura- atherosclerosis. If this is so, the lowering of plasma tion pathway to form cysteine from homocysteine is homocysteine by increasing folate intake would regulated by the availability of cysteine: it is a biosyn- not be expected to affect the development of thetic pathway rather than a pathway for disposal of atherosclerosis. methionine and homocysteine. Methylene-tetrahydrofolate reductase Folate in pregnancy During the 1980s a considerable body of evidence and hyperhomocysteinemia accumulated that spina bifida and other neural tube Elevated blood homocysteine is a significant risk defects (which occur in about 0.75–1% of pregnan- factor for atherosclerosis, thrombosis, and hyperten- cies) were associated with low intakes of folate, and sion, independent of factors such as dietary lipids and that increased intake during pregnancy might be pro- plasma lipoproteins. About 10–15% of the popula- tective. It is now established that supplements of tion, and almost 30% of people with ischemic heart folate begun periconceptually result in a significant disease, have an abnormal variant of methylene- reduction in the incidence of neural tube defects, and tetrahydrofolate reductase, which is unstable, and it is recommended that intakes be increased by loses activity more quickly than normal. As a 400 μg/day before conception. (Closure of the neural result, people with the abnormal form of the enzyme tube occurs by day 28 of pregnancy, which is before have an impaired ability to form methyl-tetrahydro- the woman knows she is pregnant.) The studies were folate (the main form in which folate is taken up by conducted using folate monoglutamate and it is tissues) and suffer from functional folate deficiency. unlikely that an equivalent increase in intake could be Therefore, they are unable to remethylate homocys- achieved from unfortified foods. In many countries teine to methionine adequately and develop there is mandatory enrichment of flour with folate, hyperhomocysteinemia. and there has been a 25–50% decrease in the number of infants born with neural tube defects since the People with the abnormal variant of methylene- introduction of fortification. The true benefit is tetrahydrofolate reductase do not develop hyperho- greater than this, since some affected fetuses abort mocysteinemia if they have a relatively high intake of spontaneously and there are few data on the number folate. This seems to be due to the methylation of therapeutic terminations of pregnancy for neural of folate in the intestinal mucosa during absorption; tube defects detected by antenatal screening; there- intestinal mucosal cells have a rapid turnover fore, supplements are recommended. Where folate (some 48 h between proliferation in the crypts and enrichment is not mandatory, the advice is that all shedding at the tip of the villus), and therefore it is women who are, or may be about to become, preg- not important that methylene-tetrahydrofolate nant, should take supplements of 400 μg/day. reductase is less stable than normal, as there is still an adequate activity of the enzyme in the intestinal Folate and cancer mucosa to maintain a normal circulating level of Much of the regulation and silencing of gene expres- methyl-tetrahydrofolate. sion that underlies tissue differentiation involves This has led to the suggestion that supplements of folate will reduce the incidence of cardiovascular

The Vitamins 175 methylation of CpG islands in DNA, and there is Assessment of folate status evidence that some cancers (and especially colorectal cancer) are associated with under-methylation of Measurement of the serum or red blood cell concen- CpG islands as a result of low folate status. A number tration of folate is the method of choice, and several of small studies have suggested that folate supple- simple and reliable radioligand binding assays have ments may be protective against colorectal cancer, but been developed. There are problems involved in no results from large-scale randomized controlled radioligand binding assays for folate, and in some trials have yet been reported, and to date there is no centers microbiological determination of plasma or evidence of a decrease in colorectal cancer in coun- whole blood folates is the preferred technique. Serum tries where folate enrichment of flour is mandatory. folate below 7 nmol/l or erythrocyte folate below 320 nmol/l indicates negative folate balance and early Folate deficiency: megaloblastic anemia depletion of body reserves. At this stage the first bone marrow changes are detectable. Dietary deficiency of folic acid is not uncommon and, as noted above, deficiency of vitamin B12 also leads to Histidine metabolism: functional folic acid deficiency. In either case, it is cells the formiminoglutamate test that are dividing rapidly, and therefore have a large The ability to metabolize a test dose of histidine pro- requirement for thymidine for DNA synthesis, that vides a sensitive functional test of folate nutritional are most severely affected. These are the cells of the status; formiminoglutamate (FIGLU) is an interme- bone marrow that form red blood cells, the cells of diate in histidine catabolism, and is metabolized by the intestinal mucosa and the hair follicles. Clinically, the folate-dependent enzyme formiminoglutamate folate deficiency leads to megaloblastic anemia, the formiminotransferase. In folate deficiency the activity release into the circulation of immature precursors of of this enzyme is impaired, and FIGLU accumulates red blood cells. and is excreted in the urine, especially after a test dose of histidine: the so-called FIGLU test. Megaloblastic anemia is also seen in vitamin B12 deficiency, where it is due to functional folate defi- Although the FIGLU test depends on folate nutri- ciency as a result of trapping folate as methyl-tetra- tional status, the metabolism of histidine will also be hydrofolate. However, the neurological degeneration impaired, and hence a positive result obtained, in of pernicious anemia is rarely seen in folate deficiency, vitamin B12 deficiency, because of the secondary and indeed a high intake of folate can mask the devel- deficiency of free folate. About 60% of vitamin B12- opment of megaloblastic anemia in vitamin B12 defi- deficient subjects show increased FIGLU excretion ciency, so that the presenting sign is irreversible nerve after a histidine load. damage. The dUMP suppression test Folate requirements Rapidly dividing cells can either use preformed TMP for DNA synthesis, or synthesize it de novo from Depletion/repletion studies to determine folate dUMP. Stimulated lymphocytes incubated with [3H]- requirements using folate monoglutamate suggest TMP will incorporate the label into DNA. In the a requirement of the order of 80–100 μg (170– presence of adequate amounts of methylene-tetrahy- 220 nmol)/day. The total body pool of folate in adults drofolate, the addition of dUMP as a substrate for is some 17 μmol (7.5 mg), with a biological half-life thymidylate synthetase reduces the incorporation of of 101 days. This suggests a minimum requirement [3H]-TMP as a result of dilution of the pool of labeled for replacement of 37 μg (85 nmol)/day. Studies of material by newly synthesized TMP and inhibition of the urinary excretion of folate metabolites in subjects thymidylate kinase by thymidine triphosphate. maintained on folate-free diets suggest that there is catabolism of some 80 μg (170 nmol) of folate/day. In normal cells the incorporation of [3H]-thymi- dine into DNA after preincubation with dUMP is Because of the problems in determining the bio- 1.4–1.8% of that without preincubation. By contrast, logical availability of the various folate polyglutamate cells that are deficient in folate form little or no thy- conjugates found in foods, reference intakes allow a midine from dUMP, and hence incorporate nearly as wide margin of safety, and are based on an allowance of 3 μg (6.8 nmol)/kg body weight.

176 Introduction to Human Nutrition much of the [3H]-thymidine after incubation with come by adding vitamin B12 to foods as well as dUMP as they do without preincubation. folate. Whereas gastric acid is essential for the release of vitamin B12 bound to dietary proteins, Either a primary deficiency of folic acid or func- crystalline vitamin B12 used in food enrichment is tional deficiency secondary to vitamin B12 deficiency free to bind to cobalophilin without the need for will have the same effect. In folate deficiency, addition gastric acid. of any biologically active form of folate, but not ● Antagonism between folic acid and the anticonvul- vitamin B12, will normalize the dUMP suppression of sants used in the treatment of epilepsy is part of [3H]-thymidine incorporation. In vitamin B12 defi- their mechanism of action; about 2% of the popu- ciency, addition of vitamin B12 or methylene- lation have (drug-controlled) epilepsy. Relatively tetrahydrofolate, but not methyl-tetrahydrofolate, large supplements of folic acid (in excess of 1000 μg/ will normalize dUMP suppression. day) may antagonize the beneficial effects of some anticonvulsants and may lead to an increase in the Drug–nutrient interactions of folate frequency of epileptic attacks. If enrichment of a food such as bread with folate is to provide 400 μg/ Several folate antimetabolites are used clinically, as day to those who eat little bread, those who eat a cancer chemotherapy (e.g., methotrexate), and as relatively large amount may well have an intake in antibacterial (trimethoprim) and antimalarial (pyri- excess of 1000 μg/day. There is, however, no evi- methamine) agents. Drugs such as trimethoprim and dence of a significant problem in countries where pyrimethamine act by inhibiting dihydrofolate reduc- enrichment of flour has been mandatory for some tase, and they owe their clinical usefulness to a con- years. siderably higher affinity for the dihydrofolate reduc- tase of the target organism than the human enzyme; 8.12 Biotin nevertheless, prolonged use can result in folate deficiency. Biotin was originally discovered as part of the complex called bios, which promoted the growth of yeast and, A number of anticonvulsants used in the treatment separately, as vitamin H, the protective or curative of epilepsy, including diphenylhydantoin (phenytoin), factor in “egg white injury,” the disease caused in and sometimes phenobarbital and primidone, can humans and experimental animals being fed diets also cause folate deficiency. Although overt megalo- containing large amounts of uncooked egg white. The blastic anemia affects only some 0.75% of treated structures of biotin, biocytin, and carboxy-biocytin epileptics, there is some degree of macrocytosis in (the active metabolic intermediate) are shown in 40%. The megaloblastosis responds to folic acid sup- Figure 8.17. plements, but in about 50% of such patients treated with relatively high supplements for 1–3 years there Biotin is widely distributed in many foods. It is is an increase in the frequency of epileptic attacks. synthesized by intestinal flora, and in balance studies the total output of biotin in urine plus feces is three Folate toxicity to six times greater than the intake, reflecting bacterial synthesis. It is not known to what extent this is avail- There is some evidence that folate supplements in able to the host. excess of 400 μg/day may impair zinc absorption. In addition, there are two potential problems that have Absorption and metabolism of biotin to be considered when advocating either widespread use of folate supplements or enrichment of foods Most biotin in foods is present as biocytin (ε-amino- with folate for protection against neural tube defect biotinyllysine), which is released on proteolysis, then and possibly cardiovascular disease and cancer. hydrolyzed by biotinidase in the pancreatic juice and intestinal mucosal secretions, to yield free biotin. The ● Folate supplements will mask the megaloblastic extent to which bound biotin in foods is biologically anemia of vitamin B12 deficiency, so that the pre- available is not known. senting sign is irreversible nerve damage. This is especially a problem for older people, who may Free biotin is absorbed from the small intestine by suffer impaired absorption of vitamin B12 as a result active transport. Biotin circulates in the bloodstream of atrophic gastritis. This problem might be over-

The Vitamins 177 O C O- onyl-CoA carboxylase, and methylcrotonyl-CoA HN NH O carboxylase to form the active holoenzymes from (inactive) apoenzymes and free biotin. S Biotin Biotin also has a role in the control of the cell cycle, and acts via cell surface receptors to regulate the O expression of key enzymes involved in glucose metab- HN NH olism. In response to mitogenic stimuli there is a con- siderable increase in the tissue uptake of biotin, much of which is used to biotinylate histones and other nuclear proteins. H CO Biotin deficiency and requirements S CN CH NH Biotin is widely distributed in foods and deficiency is O unknown, except among people maintained for many Biotinyl lysine (biocytin) months on total parenteral nutrition, and a very small number of people who eat large amounts of uncooked O egg. Avidin, a protein in egg white, binds biotin extremely tightly and renders it unavailable for -OOC N NH absorption. Avidin is denatured by cooking and then loses its ability to bind biotin. The amount of avidin S H CO in uncooked egg white is relatively small, and prob- Carboxybiocytin CN CH lems of biotin deficiency have only occurred in people O NH eating a dozen or more raw eggs a day, for some years. Figure 8.17 Biotin, biotinyl-lysine (biocytin) and the role of biocytin as a carbon dioxide carrier. The few early reports of human biotin deficiency are all of people who consumed large amounts of both free and bound to a serum glycoprotein that uncooked eggs. They developed a fine scaly dermatitis has biotinidase activity, catalyzing the hydrolysis of and hair loss (alopecia). Histology of the skin biocytin. showed an absence of sebaceous glands and atrophy of the hair follicles. Provision of biotin supplements Biotin enters tissues by a saturable transport system of 200–1000 μg/day resulted in cure of the skin and is then incorporated into biotin-dependent lesions and regrowth of hair, despite continuing the enzymes as the ε-amino-lysine peptide, biocytin. abnormal diet providing large amounts of avidin. Unlike other B vitamins, where concentrative uptake There have been no studies of providing modest into tissues can be achieved by facilitated diffusion doses of biotin to such patients, and none in followed by metabolic trapping, the incorporation of which their high intake of uncooked eggs was biotin into enzymes is relatively slow, and cannot be not either replaced by an equivalent intake of cooked considered part of the uptake process. On catabolism eggs (in which avidin has been denatured by heat, and of the enzymes, biocytin is hydrolyzed by biotinidase, the yolks of which are a good source of biotin) or permitting reutilization. continued unchanged, so there is no information from these case reports of the amounts of biotin Metabolic functions of biotin required for normal health. More recently, similar signs of biotin deficiency have been observed in Biotin functions to transfer carbon dioxide in a small patients receiving total parenteral nutrition for pro- number of carboxylation reactions. The reactive longed periods after major resection of the gut. The intermediate is 1-N-carboxy-biocytin (Figure 8.17), signs resolve following the provision of biotin, but formed from bicarbonate in an ATP-dependent reac- again there have been no studies of the amounts of tion. A single enzyme acts on the apoenzymes of biotin required; intakes have ranged between 60 μg/ acetyl-CoA carboxylase, pyruvate carboxylase, propi- day and 200 μg/day.

178 Introduction to Human Nutrition Glucose metabolism in biotin deficiency day and 200 μg/day. Since dietary deficiency does not Biotin is the coenzyme for one of the key enzymes of occur, such intakes are obviously more than adequate gluconeogenesis, pyruvate carboxylase, and deficiency to meet requirements. can lead to fasting hypoglycemia. In addition, biotin acts via cell surface receptors to induce the synthesis 8.13 Pantothenic acid of phosphofructokinase and pyruvate kinase (key enzymes of glycolysis), phospho-enolpyruvate Pantothenic acid (sometimes known as vitamin B5, carboxykinase (a key enzyme of gluconeogenesis) and and at one time called vitamin B3) has a central role glucokinase. in energy-yielding metabolism as the functional moiety of coenzyme A (CoA) and in the biosynthesis Rather than the expected hypoglycemia, biotin of fatty acids as the prosthetic group of acyl carrier deficiency may sometimes be associated with hyper- protein. The structures of pantothenic acid and CoA glycemia as a result of the reduced synthesis of gluco- are shown in Figure 8.18. kinase. Glucokinase is the high Km isoenzyme of hexokinase that is responsible for uptake of glucose Pantothenic acid is widely distributed in all food- into the liver for glycogen synthesis when blood con- stuffs; the name derives from the Greek for “from centrations are high. It also acts as the sensor for everywhere,” as opposed to other vitamins that were hyperglycemia in the β-islet cells of the pancreas; originally isolated from individual especially rich metabolism of the increased glucose 6-phosphate sources. As a result, deficiency has not been unequivo- formed by glucokinase leads to the secretion of cally reported in human beings except in specific insulin. There is some evidence that biotin supple- depletion studies, most of which have used the antag- ments can improve glucose tolerance in diabetes. onist ω-methyl-pantothenic acid. Lipid metabolism in biotin deficiency Absorption, metabolism, and metabolic The skin lesions of biotin deficiency are similar to functions of pantothenic acid those seen in deficiency of essential fatty acids, and serum linoleic acid is lower than normal in biotin- About 85% of dietary pantothenic acid is as CoA and deficient patients owing to impairment of the phosphopantetheine. In the intestinal lumen these are elongation of PUFAs as a result of reduced activity of hydrolyzed to pantetheine; intestinal mucosal cells acetyl-CoA carboxylase. have a high pantetheinase activity and rapidly hydro- lyze pantetheine to pantothenic acid. The intestinal The impairment of lipogenesis also affects the absorption of pantothenic acid seems to be by simple tissue fatty acid composition, with an increase in the diffusion and occurs at a constant rate throughout the proportion of palmitoleic acid, mainly at the expense length of the small intestine; bacterial synthesis may of stearic acid, apparently as a result of increased fatty contribute to pantothenic acid nutrition. acid desaturase activity in biotin deficiency. Although dietary protein and fat intake also affect tissue fatty The first step in pantothenic acid utilization is phos- acid composition, the ratio of palmitoleic to stearic phorylation. Pantothenate kinase is rate limiting, so acid may provide a useful index of biotin nutritional that, unlike vitamins that are accumulated by meta- status in some circumstances. bolic trapping, there can be significant accumulation of free pantothenic acid in tissues. It is then used for Biotin deficiency also results in an increase in the synthesis of CoA and the prosthetic group of acyl normally small amounts of odd-chain fatty acids carrier protein. Pantothenic acid arising from the turn- (mainly C15:0 and C17:0) in triacylglycerols, phos- over of CoA and acyl carrier protein may be either pholipids, and cholesterol esters. This is a result of reused or excreted unchanged in the urine. impaired activity of propionyl-CoA carboxylase, leading to an accumulation of propionyl-CoA, which Coenzyme A and acyl carrier protein can be incorporated into lipids in competition with All tissues are capable of forming CoA from panto- acetyl-CoA. thenic acid. CoA functions as the carrier of fatty acids, as thioesters, in mitochondrial β-oxidation. The Safe and adequate levels of intake resultant two-carbon fragments, as acetyl-CoA, then There is no evidence on which to estimate require- undergo oxidation in the citric acid cycle. CoA also ments for biotin. Average intakes are between 10 μg/ functions as a carrier in the transfer of acetyl (and

The Vitamins 179 Pantothenic acid Coenzyme A (CoASH) O C OH H2 H2 CH2 O C NH C C SH CH2 NH CH2 -SH group forms thioesters CO CH2 with fatty acids CHOH NH H3C C CH3 CH2 CO OH CHOH H3C C CH3 NH2 CH2 NN O O- -O P O P O CH2 O N N OO O OH -O P O Figure 8.18 Pantothenic acid O- and coenzyme A. other fatty acyl) moieties in a variety of biosynthetic rather the subjects were given yeast extract and other and catabolic reactions, including: rich sources of all vitamins as part of an urgent program of nutritional rehabilitation. ● cholesterol and steroid hormone synthesis ● long-chain fatty acid synthesis from palmitate and Experimental pantothenic acid depletion, together with the administration of ω-methyl-pantothenic elongation of PUFAs in mitochondria acid, results in the following signs and symptoms after ● acylation of serine, threonine and cysteine residues 2–3 weeks: on proteolipids, and acetylation of neuraminic ● neuromotor disorders, including paresthesia of the acid. hands and feet, hyperactive deep tendon reflexes, and muscle weakness. These can be explained Fatty acid synthesis is catalyzed by a cytosolic multi- by the role of acetyl-CoA in the synthesis of the enzyme complex in which the growing fatty acyl neurotransmitter acetylcholine, and impaired chain is bound by thioester linkage to an enzyme- formation of threonine acyl esters in myelin. bound 4′-phosphopantetheine residue, rather than to Dysmyelination may explain the persistence and free CoA, as in β-oxidation. This component of the recurrence of neurological problems many years fatty acid synthetase complex is the acyl carrier after nutritional rehabilitation in people who had protein. suffered from burning foot syndrome Pantothenic acid deficiency and safe and ● mental depression, which again may be related to adequate levels of intake either acetylcholine deficit or impaired myelin synthesis Prisoners of war in the Far East in the 1940s, who were severely malnourished, showed, among other ● gastrointestinal complaints, including severe vom- signs and symptoms of vitamin deficiency diseases, a iting and pain, with depressed gastric acid secretion new condition of paresthesia and severe pain in the in response to gastrin feet and toes, which was called the “burning foot syn- drome” or nutritional melalgia. Although it was ten- ● increased insulin sensitivity and a flattened glucose tatively attributed to pantothenic acid deficiency, no tolerance curve, which may reflect decreased antag- specific trials of pantothenic acid were conducted, onism by glucocorticoids

180 Introduction to Human Nutrition ● decreased serum cholesterol and decreased urinary guinea pig, bats, the passeriform birds, and most fishes. excretion of 17-ketosteroids, reflecting the impair- Ascorbate is synthesized as an intermediate in the gulo- ment of steroidogenesis nolactone pathway of glucose metabolism; in those vertebrate species for which it is a vitamin, one enzyme ● decreased acetylation of p-aminobenzoic acid, sul- of the pathway, gulonolactone oxidase, is absent. fonamides and other drugs, reflecting reduced availability of acetyl-CoA for these reactions The vitamin C deficiency disease, scurvy, has been known for many centuries and was described in the ● increased susceptibility to upper respiratory tract Ebers papyrus of 1500 bc and by Hippocrates. The infections. Crusaders are said to have lost more men through scurvy than were killed in battle, while in some of the There is no evidence on which to estimate panto- long voyages of exploration of the fourteenth and thenic acid requirements. Average intakes are between fifteenth centuries up to 90% of the crew died from 3 mg/day and 7 mg/day, and since deficiency does not scurvy. Cartier’s expedition to Quebec in 1535 was occur, such intakes are obviously more than adequate struck by scurvy; the local native Americans taught to meet requirements. him to use an infusion of swamp spruce leaves to prevent or cure the condition. Non-nutritional uses of pantothenic acid Recognition that scurvy was due to a dietary defi- Blood levels of pantothenic acid have been reported ciency came relatively early. James Lind demonstrated to be low in patients with rheumatoid arthritis; some in 1757 that orange juice and lemon juice were pro- workers have reported apparently beneficial effects of tective, and Cook kept his crew in good health during supplementation, but these reports remain uncon- his circumnavigation of the globe (1772–1775) by firmed and there are no established pharmacological stopping frequently to take on fresh fruit and vegeta- uses of the vitamin. bles. In 1804 the British Navy decreed a daily ration of lemon or lime juice for all ratings, a requirement Pantothenic acid deficiency in rats leads to a loss of that was extended to the merchant navy in 1865. fur color and at one time pantothenic acid was known as the “anti-grey hair factor.” There is no evidence that The structure of vitamin C is shown in Figure 8.19; the normal graying of hair with age is related to pan- both ascorbic acid and dehydroascorbic acid have tothenic acid nutrition, or that pantothenic acid sup- vitamin activity. Monodehydroascorbate is a stable plements have any effect on hair color. Its use in radical formed by reaction of ascorbate with reactive shampoo is not based on any evidence of efficacy. oxygen species, and can be reduced back to ascorbate by monodehydroascorbate reductase. Alternatively, 2 Pantothenic acid has very low toxicity; intakes of up mol of monodehydroascorbate can react together to to 10 g/day of calcium pantothenate (compared with a yield 1 mol each of ascorbate and dehydroascorbate. normal dietary intake of 2–7 mg/day) have been given Dehydroascorbate may either be reduced to ascorbate for up to 6 weeks with no apparent ill-effects. or undergo hydration to diketogulonate and onward metabolism. 8.14 Vitamin C (ascorbic acid) Vitamin C is found in fruits and vegetables. Very Vitamin C is a vitamin for only a limited number of significant losses occur as vegetables wilt, or when vertebrate species: humans and the other primates, the CH2OH CH2OH CH2OH HO CH O HO CH O HO CH O O O OO O OH OH .O OH Ascorbate Monodehydroascorbate Dehydroascorbate Figure 8.19 Vitamin C (ascor- (semidehydroascorbate) bic acid, monodehydroascor- bate and dehydroascorbate).

The Vitamins 181 they are cut, as a result of the release of ascorbate is recovered from subjects receiving high intakes of oxidase from the plant tissue. Significant losses of the the vitamin, this is the result of bacterial metabolism vitamin also occur in cooking, both through leaching of unabsorbed vitamin in the intestinal lumen. into the cooking water and also atmospheric oxida- tion, which continues when foods are left to stand The fate of the greater part of ascorbic acid is excre- before serving. tion in the urine, either unchanged or as dehydro- ascorbate and diketogulonate. Both ascorbate and Absorption and metabolism of vitamin C dehydroascorbate are filtered at the glomerulus then reabsorbed. When glomerular filtration of ascorbate There is active transport of the vitamin at the intesti- and dehydroascorbate exceeds the capacity of the nal mucosal brush border membrane. Both ascorbate transport systems, at a plasma concentration of and dehydroascorbate are absorbed across the buccal ascorbate between 70 and 85 μmol/l, the vitamin is mucosa by carrier-mediated passive processes. excreted in the urine in amounts proportional to Intestinal absorption of dehydroascorbate is carrier intake. mediated, followed by reduction to ascorbate before transport across the basolateral membrane. Metabolic functions of vitamin C Some 80–95% of dietary ascorbate is absorbed at Ascorbic acid has specific roles in two groups of usual intakes (up to about 100 mg/day). The frac- enzymes: the copper-containing hydroxylases and the tional absorption of larger amounts of the vitamin is 2-oxoglutarate-linked iron-containing hydroxylases. lower, and unabsorbed ascorbate from very high It also increases the activity of a number of other doses is a substrate for intestinal bacterial meta- enzymes in vitro, although this is a non-specific bolism, causing gastrointestinal discomfort and reducing action rather than reflecting any metabolic diarrhea. function of the vitamin. In addition, it has a number of non-enzymic effects due to its action as a reducing About 70% of blood ascorbate is in plasma and agent and oxygen radical quencher. erythrocytes, which do not concentrate the vitamin from plasma. The remainder is in white cells, which Copper-containing hydroxylases have a marked ability to concentrate it. Dopamine β-hydroxylase is a copper-containing enzyme involved in the synthesis of the catechol- Both ascorbate and dehydroascorbate circulate in amines norepinephrine (noradrenaline) and epi- free solution, and also bound to albumin. About 5% nephrine (adrenaline) from tyrosine in the adrenal of plasma vitamin C is normally dehydroascorbate. medulla and central nervous system. The enzyme Both vitamers are transported into cells by glucose contains Cu+, which is oxidized to Cu2+ during the transporters, and concentrations of glucose of the hydroxylation of the substrate; reduction back to Cu+ order of those seen in diabetic hyperglycemia inhibit specifically requires ascorbate, which is oxidized to tissue uptake of ascorbate. monodehydroascorbate. There is no specific storage organ for ascorbate; Some peptide hormones have a carboxy-terminal apart from leukocytes (which account for only 10% amide that is hydroxylated on the α-carbon by a of total blood ascorbate), the only tissues showing a copper-containing enzyme, peptidylglycine hydroxy- significant concentration of the vitamin are the lase. The α-hydroxyglycine residue then decomposes adrenal and pituitary glands. Although the concentra- non-enzymically to yield the amidated peptide and tion of ascorbate in muscle is relatively low, skeletal glyoxylate. The copper prosthetic group is oxidized in muscle contains much of the body’s pool of 900– the reaction, and, as in dopamine β-hydroxylase, 1500 mg (5–8.5 mmol). ascorbate is specifically required for reduction back to Cu+. Diketogulonate arising from dehydroascorbate can undergo metabolism to xylose, thus providing a route Oxoglutarate-linked iron-containing for entry into central carbohydrate metabolic path- hydroxylases ways via the pentose phosphate pathway. However, Several iron-containing hydroxylases share a common oxidation to carbon dioxide is only a minor fate of reaction mechanism, in which hydroxylation of the ascorbate in humans. At usual intakes of the vitamin, less than 1% of the radioactivity from [14C]-ascorbate is recovered as carbon dioxide. Although more 14CO2

182 Introduction to Human Nutrition substrate is linked to decarboxylation of 2- monodehydroascorbate. At physiological concentra- oxoglutarate. Many of these enzymes are involved in tions of ascorbate, both Fe3+ and Cu2+ ions are reduced the modification of precursor proteins to yield the by ascorbate, yielding monodehydroascorbate. Fe2+ final, mature, protein. This is a process of postsyn- and Cu+ are readily reoxidized by reaction with hydro- thetic modification of an amino acid residue after it gen peroxide to yield hydroxide ions and hydroxyl has been incorporated into the protein during synthe- radicals. Cu+ also reacts with molecular oxygen to sis on the ribosome. yield superoxide. Thus, as well as its antioxidant role, ascorbate has potential pro-oxidant activity. However, ● Proline and lysine hydroxylases are required for the because at high levels of intake the vitamin is excreted postsynthetic modification of procollagen in the quantitatively, is it unlikely that tissue concentrations formation of mature, insoluble, collagen, and will rise high enough for there to be significant for- proline hydroxylase is also required for the post- mation of oxygen radicals. synthetic modification of the precursor proteins of osteocalcin and the C1q component of Vitamin C deficiency: scurvy complement. The vitamin C deficiency disease scurvy was formerly ● Aspartate β-hydroxylase is required for the postsyn- a common problem at the end of winter, when there thetic modification of the precursor of protein C, had been no fresh fruit and vegetables for many the vitamin K-dependent protease that hydrolyzes months. activated factor V in the blood-clotting cascade. Although there is no specific organ for storage of ● Trimethyl-lysine and γ-butyrobetaine hydroxylases vitamin C in the body, signs of deficiency do not are required for the synthesis of carnitine. develop in previously adequately nourished subjects until they have been deprived of the vitamin for 4–6 Ascorbate is oxidized during the reaction of these months, by which time plasma and tissue concentra- enzymes, but not stoichiometrically with the decar- tions have fallen considerably. The earliest signs of boxylation of 2-oxoglutarate and hydroxylation of the scurvy in volunteers maintained on a vitamin C-free substrate. The purified enzyme is active in the absence diet are skin changes, beginning with plugging of hair of ascorbate, but after some 5–10 s (about 15–30 cycles follicles by horny material, followed by enlargement of enzyme action) the rate of reaction begins to fall. At of the hyperkeratotic follicles, and petechial hemor- this stage the iron in the catalytic site has been oxi- rhage with significant extravasation of red cells, pre- dized to Fe3+, which is catalytically inactive; activity is sumably as a result of the increased fragility of blood restored only by ascorbate, which reduces it back to capillaries. Fe2+. The oxidation of Fe2+ is the consequence of a side-reaction rather than the main reaction of the At a later stage there is also hemorrhage of the enzyme, which explains how 15–30 cycles of enzyme gums, beginning in the interdental papillae and pro- activity can occur before there is significant loss of gressing to generalized sponginess and bleeding. This activity in the absence of ascorbate, and why the con- is frequently accompanied by secondary bacterial sumption of ascorbate is not stoichiometric. infection and considerable withdrawal of the gum from the necks of the teeth. As the condition pro- Pro-oxidant and antioxidant roles of ascorbate gresses, there is loss of dental cement, and the teeth Ascorbate can act as a radical-trapping antioxidant, become loose in the alveolar bone and may be lost. reacting with superoxide and a proton to yield hydro- gen peroxide, or with the hydroxy radical to yield Wounds show only superficial healing in scurvy, water. In each instance the product is the monodehy- with little or no formation of (collagen-rich) scar droascorbate radical. Thus, as well as reducing the tissue, so that healing is delayed and wounds can tocopheroxyl radical formed by interaction of readily be reopened. The scorbutic scar tissue has only α-tocopherol in membranes with lipid peroxides, about half the tensile strength of that normally ascorbate acts to trap the oxygen radicals that would formed. otherwise react to form lipid peroxides. Advanced scurvy is accompanied by intense pain in At high concentrations, ascorbate can reduce the bones, which can be attributed to changes in bone molecular oxygen to superoxide, being oxidized to mineralization as a result of abnormal collagen syn- thesis. Bone formation ceases and the existing bone

The Vitamins 183 becomes rarefied, so that the bones fracture with The requirement for vitamin C to prevent clinical minimal trauma. scurvy is less than 10 mg/day. However, at this level of intake wounds do not heal properly because of the The name scurvy is derived from the Italian scor- requirement for vitamin C for the synthesis of colla- butico, meaning an irritable, neurotic, discontented, gen in connective tissue. An intake of 20 mg/day is whining, and cranky person. The disease is associated required for optimum wound healing. Allowing for with listlessness and general malaise, and sometimes individual variation in requirements, this gives a ref- changes in personality and psychomotor performance erence intake for adults of 30 mg/day, which was the and a lowering of the general level of arousal. These British recommended daily allowance (RDA) until behavioral effects can be attributed to impaired syn- 1991. thesis of catecholamine neurotransmitters, as a result of low activity of dopamine β-hydroxylase. The 1991 British reference nutrient intake (RNI) for vitamin C is based on the level of intake at which Most of the other clinical signs of scurvy can be the plasma concentration rises sharply, showing that accounted for by the effects of ascorbate deficiency on requirements have now been met, tissues are satu- collagen synthesis, as a result of impaired proline and rated and there is spare vitamin C being transported lysine hydroxylase activity. Depletion of muscle car- between tissues, available for excretion. This criterion nitine, due to impaired activity of trimethyllysine and of adequacy gives an RNI of 40 mg/day for adults. γ-butyrobetaine hydroxylases, may account for the lassitude and fatigue that precede clinical signs of The alternative approach to determining require- scurvy. ments is to estimate the total body content of vitamin C, then measure the rate at which it is metabolized, Anemia in scurvy by giving a test dose of radioactive vitamin. This is Anemia is frequently associated with scurvy, and may the basis of both the former US RDA of 60 mg/day be either macrocytic, indicative of folate deficiency, or for adults and the Netherlands RDA of 80 mg/day. hypochromic, indicative of iron deficiency. Indeed, it also provides an alternative basis for the RNI of 40 mg/day. Folate deficiency may be epiphenomenal, since the major dietary sources of folate are the same as those The problem lies in deciding what is an appropriate of ascorbate. However, some patients with clear meg- body content of vitamin C. The studies were per- aloblastic anemia respond to the administration of formed on subjects whose total body vitamin C was vitamin C alone, suggesting that there may be a role estimated to be 1500 mg at the beginning of a deple- of ascorbate in the maintenance of normal pools of tion study. However, there is no evidence that this is reduced folates, although there is no evidence that any a necessary, or even a desirable, body content of the of the reactions of folate is ascorbate dependent. vitamin. It is simply the body content of the vitamin of a small group of people eating a self-selected diet Iron deficiency in scurvy may well be secondary to rich in fruit. There is good evidence that a total body reduced absorption of inorganic iron and impaired content of 900 mg is more than adequate. It is three mobilization of tissue iron reserves (see below). At the times larger than the body content at which the first same time, the hemorrhages of advanced scurvy will signs of deficiency are observed, and will protect cause a significant loss of blood. against the development of any signs of deficiency for several months on a completely vitamin C-free diet. There is also evidence that erythrocytes have a shorter half-life than normal in scurvy, possibly as a There is a further problem in interpreting the results. result of peroxidative damage to membrane lipids The rate at which vitamin C is metabolized varies with owing to impairment of the reduction of tocopher- the amount consumed. This means that as the experi- oxyl radical by ascorbate. mental subjects become depleted, so the rate at which they metabolize the vitamin decreases. Thus, calcula- Vitamin C requirements tion of the amount that is required to maintain the body content depends on both the way in which the Vitamin C illustrates extremely well how different results obtained during depletion studies are extrapo- criteria of adequacy, and different interpretations of lated to the rate in subjects consuming a normal diet experimental data, can lead to different estimates and the amount of vitamin C in that diet. of requirements, and to reference intakes ranging between 30 and 90 mg/day for adults.