Vitamin D

4 Vitamin D 4.1 Background Vitamin D is represented by cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2), which are structurally similar secosteroids derived from the UV irradiation of provitamin D sterols Exposure of the skin to sunlight converts 7-dehydrocholesterol to vitamin D3, which, on reaching the blood capillaries of the dermis, is conveyed to the liver on a specific plasma transport protein Vitamin D2 is produced in plants, fungi, and yeasts by the solar irradiation of ergosterol On irradiation, the provitamins are initially converted to previtamin D, which undergoes thermal transformation to vitamin D In parts of the world where there is ample sunlight throughout the year, the human vitamin D requirement is met entirely by biogenesis in the skin; otherwise, populations depend on dietary sources of the vitamin Vitamin D itself is biologically inactive and must be metabolized to 1a,25-dihydroxyvitamin D [1a,25(OH)2D], which acts as a hormone in controlling calcium homeostasis and regulating the growth of various cell types In the liver, the vitamin D of both cutaneous and dietary origin is hydroxylated to 25-hydroxyvitamin D [25(OH)D], which is released without delay into the bloodstream On reaching the kidneys, this circulating metabolite undergoes the second hydroxylation to 1a,25(OH)2D This hormone stimulates the intestine to absorb calcium and phosphate, and acts with parathyroid hormone to mobilize calcium, accompanied by phosphate, from the bone fluid compartment into the bloodstream 1a,25-Dihydroxyvitamin D is also involved in the formation of osteoclasts – giant cells that are solely responsible for the resorption of bone matrix Resorption is an essential process for the development, growth, maintenance, and repair of bone Another metabolite, 24R,25-dihydroxyvitamin D [24R,25(OH)2D], is the initial product in the catabolism of 1a,25(OH)2D and plays a crucial role in bone formation and repair of bone fractures The hormonal effects of 1a,25(OH)2D are mediated via a specific intracellular receptor, which belongs to the superfamily of steroid receptors After binding to 1a,25(OH)2D, the receptor– ligand complex binds to vitamin D responsive elements on the DNA and, depending on the © 2006 by Taylor & Francis Group, LLC 107 Vitamin D 108 target cell, it regulates the expression of a number of genes involved in calcium homeostasis or in the control of cell proliferation and differentiation Vitamin D deficiency in children causes rickets, in which the bones not develop properly and become deformed through inadequate deposition of calcium and phosphorus The equivalent disease in adults is called osteomalacia, in which the newly deposited bone matrix fails to mineralize adequately, causing the bones to become brittle Toxicity can result from chronic oversupplementation of vitamin D, but not from unlimited exposure to sunshine or the consumption of usual diets The physiological basis for toxicity is bone demineralization, causing increased serum calcium levels The excess calcium is deposited in the kidneys, causing hypertension, cardiac insufficiency, and renal failure 4.2 4.2.1 Chemical Structure, Biopotency, and Physicochemical Properties Structure and Biopotency Irradiation of the parent steroid causes breakage of the B ring at the 9,10-carbon bond, resulting in the conjugated triene system of double bonds The numbering system of the carbon atoms of the vitamin D molecule is identical to that of the parent steroid (Figure 4.1) Vitamin D2 (C28H44O, MW ¼ 396.6) and vitamin D3 (C27H44O, MW ¼ 384.2) differ structurally only in the C-17 side chain, which in vitamin D2 has a double bond and an additional methyl group (Figure 4.1) Both compounds occur naturally with 5,6 double bond in the cis configuration The biological potencies of vitamins D2 and D3 in humans are essentially equal The circulating metabolite 25(OH)D3, which is found in significant quantities in animal-derived foods, induces all the responses of vitamin D3, but its biological activity is five-times higher based on its ability to enhance intestinal calcium absorption [1] Animal tissues contain a small proportion of vitamin D esterified with both saturated and unsaturated fatty acids All the esters have biological activity equivalent on a molar basis to that shown by cholecalciferol [2] 4.2.2 Physicochemical Properties 4.2.2.1 Appearance and Solubility Vitamins D2 and D3 are white to yellowish crystalline powders, which are insoluble in water; soluble in 95% ethanol, acetone, fats, and oils; and readily soluble in chloroform and ether © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability (a) 12 13 11 10 A B D C 14 (b) 17 109 18 R H3C 16 12 C 11 15 H 10 A Vitamin D3 R = 21 H3C 20 22 21 H3C 20 22 H 17 26 CH3 24 H 23 17 Vitamin D2 R = 15 14 19 CH2 16 HO H 17 13 D 25 CH 27 28 H CH3 26 CH3 24 23 25 CH 27 FIGURE 4.1 Structural relationship of (a) the parent steroid nucleus to (b) vitamin D 4.2.2.2 Stability in Nonaqueous Solution In solution, but not in the solid state, vitamins D2 and D3 exhibit reversible thermal isomerization to their corresponding previtamins, forming an equilibrium mixture Equations and calculations have been published to determine the ratio of previtamin D to vitamin D as a function of temperature and reaction time [3] The previtamin D/vitamin D ratios and the equilibration times attained at different temperatures are given in Table 4.1 [4] When equilibrated at 208C, the ratio of previtamin D to vitamin D is 7:93 The isomerization rates of vitamins D2 and D3 are virtually equal [5] and are not affected by solvent, light, or catalysis [4] Like vitamin A, the stability of vitamin D in fats and oils corresponds to the stability of the fat itself Vitamin D, however, is more stable than vitamin A under comparable conditions Once freed from the protection of the food matrix, vitamin D is susceptible to decomposition by oxygen and light Conditions which promote destruction of vitamin D include exposure of thin films to air (especially with heat), and dispersion of an alcoholic solution of the vitamin into an aqueous phase in the presence of dissolved oxygen [6] The vitamin is stable toward alkali, but © 2006 by Taylor & Francis Group, LLC Vitamin D 110 TABLE 4.1 The Previtamin D and vitamin D Equilibrium Temperature (8C) %Previtamin D %Vitamin D Equilibrium Timea 11 13 16 22 28 35 98 96 93 91 89 87 84 78 72 65 16 yr 350 days 30 days 10 days 3.5 days 1.3 days 0.5 days 0.1 days 30 min 220 10 20 30 40 50 60 80 100 120 a The time necessary to reach equilibrium, starting with pure vitamin D or pure previtamin D Source: From Mulder, F.J., de Vries, E.J., and Borsje, B., J Assoc Off Anal Chem., 54, 1168, 1971 With permission under conditions of even mild acidity the molecule isomerizes to form the 5.6-trans and isotachysterol isomers (Figure 4.2), neither of which possesses any significant antirachitic activity [7] 4.3 4.3.1 Vitamin D in Foods Occurrence As with vitamin A, fish-liver oils are exceedingly rich in vitamin D However, natural foods contain only very small amounts and significant sources are confined to a limited number of animal products Fatty fish, such as herring, sardines, pilchards, and tuna, are rich natural food sources; smaller amounts are found in mammalian liver, eggs, and dairy products (Table 4.2) [8] The concentration of vitamin D3 in milk shows a seasonal variation, which is related to the amount of sunlight available for vitamin D biogenesis in the cow Milk also contains vitamin D2, but in smaller concentrations than vitamin D3 The D2 vitamer is derived by UV irradiation of ergosterol in sun-dried green forage (hay); ergosterol cannot be converted by the animal into vitamin D2 The vitamin D activity in animal products is contributed by both vitamin D and its immediate metabolite, 25(OH)D Typical values of this metabolite (micrograms per 100 g) are bovine muscle, 0.2 –0.3; bovine liver, 0.3– 0.5; bovine kidney, 0.5 – 1.0 [9]; and egg yolk, 1.0 [10] Supplementation of chicken feed with vitamin D increases the content © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability (a) 111 (b) R CH2 R C 3H OH OH FIGURE 4.2 Structures of (a) 5,6-trans vitamin D and (b) isotachysterol of both vitamin D3 and 25(OH)D3 in egg yolk [11] 25-Hydroxyvitamin D is of nutritional significance: in milk, for example, it accounts for 75% of the total vitamin D activity as estimated by the calcium transport assay [12] In the United States and Canada, fluid milk is supplemented by law with vitamin D to a level of 400 international units (IU) per quart (10 mg/0.95 l) to meet the recommended dietary allowance (RDA) of 10 mg In the United Kingdom, the Margarine Regulations [13] state that “every ounce of margarine shall contain not less than 80 IU and not more than 100 IU of vitamin D,” equating to limits of between and mg/100 g Other foodstuffs commonly enriched with vitamin D include skimmed milk powder, evaporated milk, milk-based beverages, breakfast cereals, dietetic products of all kinds, infant formulas, and soup powders Vitamin D is either added as an oily solution or in combination with a vitamin A formulation [14] Reports of water-soluble vitamin D sulfate present in large quantities in milk [15,16] have not been confirmed in later studies [17] The water-soluble vitamin D activity in milk can now be explained by the presence of protein-bound 25(OH)D [18] In any event, synthetic vitamin D sulfate has been shown to possess negligible biological activity [19] The RDA of vitamin D for humans exposed to inadequate sunlight is 10 mg It has been estimated that to maintain adequate plasma 25-hydroxyvitamin D levels without any input from skin irradiation © 2006 by Taylor & Francis Group, LLC Vitamin D 112 TABLE 4.2 Vitamin D Content of Various Foods Micrograms of Vitamin D per 100 g Edible Portion Food Cow milk, whole, pasteurized Butter Cheese, cheddar Egg, chicken, whole, raw Beef, trimmed lean, raw, average Lamb, trimmed lean, raw, average Pork, trimmed lean, raw, average Chicken meat, raw, average Liver, lamb, fried Cod, raw, fillets Haddock, raw, fillets Herring, raw grilled Pilchards, canned in tomato sauce Sardines, canned in brine, drained Sardines, canned in oil, drained Sardines, canned in tomato sauce Tuna, canned in brine, drained Tuna canned in oil, drained Tr 0.9 0.3 1.8 0.5a 0.4a 0.5a 0.1a 0.9a Tr Tr 19.0 (range 7–31) 16.1 14.0 4.6 5.0 8.0 3.6 3.0 Note: Tr, trace a The total vitamin D activity for meat, meat products and poultry is taken as the sum of vitamin D3 and five times 25-hydroxyvitamin D3 Source: From Food Standards Agency, McCane and Widdowon’s The Composition of Foods, 6th summary ed., Royal Society of Chemistry, Cambridge, 2002 With permission would require ingestion of at least 12.5 mg of vitamin D per day in the form of dietary supplements [20] Except for eggs and fatty fish, a serving of food containing only natural sources of the vitamin D would probably supply less than mg [21] 4.3.2 Stability Vitamin D is destroyed in oxidizing fats; otherwise, food processing, cooking, and storage of foods not generally affect the vitamin’s activity The vitamin will withstand smoking of fish, pasteurization and sterilization of milk, and spray drying of eggs [22] Indyk et al [23] evaluated the stability of supplemental vitamin D3 in spray-dried milk Measured losses through the pasteurization, high-pressure evaporation, and drying processes were demonstrated to be statistically insignificant (P 0.05) Milk should not be exposed to © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 113 light during processing and storage because the vitamin D present can be oxidized to an inactive 5,6-epoxide by riboflavin-photosensitized singlet oxygen [24] 4.3.3 Expression of Dietary Values One IU of vitamin D is the activity obtained from 0.025 mg of crystalline cholecalciferol in a bioassay Despite a recommendation by an expert committee in 1970 that the IU be abandoned, the vitamin D content of foods is often expressed in such units There is as yet no consensus on which conversion factor should be used for expressing the content of 25(OH)D in IU For calculation of the “total” vitamin D activity present in foods, a factor of [1 IU ¼ 0.005 mg 25(OH)D] has been used [18] 4.3.4 Applicability of Analytical Techniques The estimation of the very low concentrations of indigenous vitamin D in foodstuffs is difficult owing to the need to remove interfering subtances, such as cholesterol, vitamin A, and vitamin E, which are invariably present in gross excess (Table 4.3) [25] Most of the published HPLC methods for determining vitamin D in foods are concerned with estimating the vitamin D content in supplemented products, such as milk in various forms, infant formulas, and margarine In supplemented foods, the amount of naturally occurring vitamin D (if any) is usually considered to be negligible, and it is deemed necessary to determine only the vitamin D that is added Even so, supplementation levels are very low (e.g., 7.5– 12.5 mg/100 g in milk powder) [26] and the determination of vitamin D is by no means a simple task A bioassay will account for the activity of previtamin D as well as vitamin D and its various active metabolites A valid estimate of the TABLE 4.3 Ratios of Vitamins A and E and Cholesterol to Vitamin D in Some Foodstuffs Ratio Relative to Vitamin D on a Weight Basis Foodstuff Whole milk Ox liver Whole egg D A E Cholesterol 1 1,500 6,000 75 5,000 1,400 500 600,000 300,000 80,000 Source: From Osborne, D.R and Voogt, P., The Analysis of Nutrients in Foods, Academic Press, London, 1978 With permission © 2006 by Taylor & Francis Group, LLC Vitamin D 114 vitamin D value of a food should therefore represent “potential vitamin D,” that is, the sum of the vitamin D and previtamin D contents When determining naturally occurring vitamin D in animal products for nutritional evaluation purposes, 25(OH)D3 should be included, because this metabolite contributes significantly to the total biological activity, particularly in milk 25-Hydroxyvitamin D3 is present in dairy products, eggs, and meat tissues in sufficient concentration to permit its determination by HPLC, using an absorbance detector In bovine milk, the concentration of this metabolite is less than ng/ml [27]; hence it is usually determined by a competitive protein-binding assay after fractionation of the extracted sample by HPLC [28] 4.4 Intestinal Absorption, Transport, and Metabolism The following discussion of absorption, transport, and metabolism is taken from more a detailed account in a book by Ball [29] published in 2004 The vitamin D activity in the human diet is contributed mainly by free vitamin D, with 25(OH)D making a quantitatively small but nutritionally significant contribution Ingested vitamin D is solubilized within mixed micelles in the duodenum and passively absorbed in the jejunum along with other lipids Esters of vitamin D, if present, are hydrolyzed during solubilization [18] A cytosolic vitamin D-binding protein (cDBP), with a preference for binding 25(OH)D, has been isolated from rat enterocytes [30,31] and may play a role in the absorption of 25(OH)D Vitamin D is incorporated into chylomicrons within the enterocytes and the released chylomicrons convey the vitamin in the mesenteric lymph to the systemic circulation During the journey in the lymph, an appreciable amount of the vitamin D in the chylomicrons is transferred to the serum vitamin D-binding protein (sDBP) [32] This protein has a higher affinity for 25(OH)D than for vitamin D [33] and is present in large excess of the normal blood contents of its ligands [34] After lipolysis of the chylomicrons, the vitamin D remaining on the chylomicron remnants, and also the vitamin D bound to sDBP, is initially taken up by the liver [35] Absorption of 25-hydroxyvitamin D is faster than that of vitamin D in normal humans and in patients with fat malabsorption syndromes [36] Dueland et al [37] compared the distribution of [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 in mesenteric lymph after simultaneous instillation of these compounds in the duodenum of rats They showed that vitamin D3 is mainly carried with chylomicrons, whereas 25(OH)D3 is carried mainly by proteins The protein responsible for carrying the bulk of 25(OH)D and part of the vitamin D is very likely sDBP The results suggested that the portal circulation plays only a minor role in © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 115 the total absorption of vitamin D and 25(OH)D The association of 25(OH)D3 with sDBP rather than with chylomicrons implies that absorption of 25(OH)D3 is far less dependent on bile salts Thus 25(OH)D3 is an important pharmacological agent in cases of bile salt deficiency Three key enzymes are involved in the conversion of vitamin D to active hormonal forms: vitamin D-25-hydroxylase in the liver converts vitamin D to 25(OH)D; a multicatalytic 1a-hydroxylase in the kidney converts 25(OH)D to 1a,25(OH)2D; and 24R-hydroxylase, another renal multicatalytic enzyme, converts 25(OH)D to 24R,25(OH)2D The mammalian liver does not store vitamin D to any significant effect, and any vitamin D which is not metabolized in the liver is stored in the adipose tissue and skeletal muscle The liver metabolite, 25(OH)D, is the major circulating form of vitamin D In contrast to the rapid hepatic uptake of dietary vitamin D and inactivation of surplus amounts, the supply of cutaneous vitamin D to the liver is gradual, allowing continuous prolonged production of 25(OH)D Thus, the plasma concentration of this metabolite is maintained, even when exposure of the skin to sunlight occurs only intermittently The hydroxylating enzyme, vitamin D-25-hydroxylase, is only loosely regulated, and circulating concentrations of 25(OH)D continue to rise in response to ingestion of pharmacological doses of vitamin D Levels can rise to more than 400 ng/ml, leading to vitamin D toxicity In contrast, extensive UV irradiation of the skin does not cause hypervitaminosis D, and raises 25(OH)D levels in plasma to no more than 80 ng/ml [20] Almost all the 25(OH)D produced in the liver is released without delay into the bloodstream, where it becomes tightly bound to sDBP The normal concentration range of plasma 25(OH)D is 10– 40 ng/ml The blood constitutes the largest single body pool of 25(OH)D, since extrahepatic tissues take up only small amounts During vitamin D deprivation, the 25(OH)D blood pool is maintained through the prolonged release of vitamin D from its skin reservoir and from its muscle and adipose tissue storage sites Plasma concentrations of the highly potent 1a,25(OH)2D are kept within the range of 25 to 70 pg/ml by reciprocal changes in the rates of synthesis and degradation at the cellular level 4.5 Bioavailability In the only known study of vitamin D bioavailability from natural sources [18], the average relative bioavailability of vitamin D2 from meat sources was estimated to be ca 60% as compared with a vitamin supplement Vitamin D given with milk has been reported to be –10 times more potent than that given with oil, the stimulatory factor being attributable © 2006 by Taylor & Francis Group, LLC 116 Vitamin D to the lactalbumin fraction [38] It is not known whether enhanced absorption or some other factor is responsible for the greater apparent biological activity of vitamin D given in milk The serum 25(OH)D concentration reflects vitamin D stores in humans, and its measurement by protein-binding assay is an index of nutritional status [39] In the rat, chronic ethanol ingestion promotes the biliary loss of 25(OH)D and this loss may be a contributing factor in the impaired vitamin D status in alcoholics [40] Human subjects receiving a high-fiber diet exhibited a reduced plasma half-life of 25(OH)D3, indicating a more rapid elimination of the metabolite from the body [41] Smoking seems to depress the serum levels of 25(OH)D, 1a,25(OH)2D, and parathyroid hormone [42] This may partly account for the decreased bone mass and increased fracture risk seen among smokers later in life [43] References Ovesen, L., Brot, C., and Jakobsen, J., Food contents and biological activity of 25-hydroxyvitamin D: a vitamin D metabolite to be reckoned with? Ann Nutr Metab., 47, 107, 2003 Lawson, E., Vitamin D, in Fat-Soluble Vitamins, Their Biochemistry and Applications, Diplock, A.T., Ed., Heinemann, London, 1985, p 76 Keverling Buisman, J.A., Hanewald, K.H., Mulder, F.J., Roborgh, J.R., and Keuning, K.J., Evaluation of the effect of isomerization on the chemical and biological assay of vitamin D, J Pharm Sci., 57, 1326, 1968 Mulder, F.J., de Vries, E.J., and Borsje, B., Chemical analysis of vitamin D in concentrates and its problems 12 Analysis of fat-soluble vitamins, J Assoc Off Anal Chem., 54, 1168, 1971 Hanewald, K.H., Mulder, F.J., and Keuning, K.J., Thin-layer chromatography assay of vitamin D in high-potency preparations, J Pharm Sci., 57, 1308, 1968 Chen, P.S., Jr., Terepka, R., Lane, K., and Marsh, A., Studies of the stability and extractability of vitamin D, Anal Biochem., 10, 421, 1965 DeLuca, H.F., Vitamin D, in Vitamin D Handbook of Lipid Research, DeLuca, H.F., Ed., Vol 2, Plenum Press, New York, 1978, p 69 Food Standards Agency, McCance and Widdowson’s The Composition of Foods, 6th summary ed., Royal Society of Chemistry, Cambridge, 2002 Koshy, K.T and VanDerSlik, A.L., High-performance liquid chromatographic method for the determination of 25-hydroxycholecalciferol in the bovine liver, kidney, and muscle, J Agric Food Chem., 25, 1246, 1977 10 Mattila, P., Piironen, V., Uusi-Rauva, E., and Koivistoinen, P., Determination of 25-hydroxycholecalciferol content in egg yolk by HPLC, J Food Comp Anal., 6, 250, 1993 11 Mattila, P., Lehikoinen, K., Kiiskinen, T., and Piironen, V., Cholecalciferol and 25-hydroxycholecalciferol content of chicken egg yolk as affected by the cholecalciferol content of feed, J Agric Food Chem., 47, 4089, 1999 © 2006 by Taylor & Francis Group, LLC Vitamins in Foods: Analysis, Bioavailability, and Stability 117 12 Reeve, L.E., Jorgensen, N.A., and DeLuca, H.F., Vitamin D compounds in cow’s milk, J Nutr., 112, 667, 1982 13 Margarine Regulations, Statutory Instrument, No 1867, as amended, Her Majesty’s Stationery Office, London, 1967 14 Kla¨ui, H.M., Hausheer, W., and Huschke, G., Technological aspects of the use of fat-soluble vitamins and carotenoids and of the development of stabilized marketable forms, in Fat-Soluble Vitamins, Morton, R.A., Ed., Pergamon Press, New York, 1970, p 113 15 Asano, T., Hasegawa, T., Suzuki, K., Masushige, S., Nose, T., and Suzuki, T., Determination of vitamin D3-sulfate in milk by high-performance liquid chromatography, Nutr Rep Int., 24, 451, 1981 16 Le Boulch, N., Cancela, L., and Miravet, L., Cholecalciferol sulfate identification in human milk by HPLC, Steroids, 39, 391, 1982 17 Okano, T., Kuroda, E., Nakao, H., Kodama, S., Matsuo, T., Nakamichi, Y., Nakajima, K., Hirao, N., and Kobayashi, T., Lack of evidence for existence of vitamin D and 25-hydroxyvitamin D sulfates in human breast and cow’s milk, J Nutr Sci Vitaminol., 32, 449, 1986 18 van den Berg, H., Bioavailability of vitamin D, Eur J Clin Nutr., 51 (Suppl 1), S76, 1997 19 Reeve, L.E., DeLuca, H.F., and Schnoes, H.K., Synthesis and biological activity of vitamin D3-sulfate, J Biol Chem., 256 (2), 823, 1981 20 Fraser, D.R., The physiological economy of vitamin D, Lancet, I (8331), 969, 1983 21 Parrish, D.B., Determination of vitamin D in foods: a review, CRC Crit Rev Food Sci Nutr., 12, 29, 1979 22 Bender, A.E., Food Processing and Nutrition, Academic Press, London, 1978, p 27 23 Indyk, H., Littlejohn, V., and Woollard, D.C., Stability of vitamin D3 during spray-drying of milk, Food Chem., 57, 283, 1996 24 King, J.M and Min, D.B., Riboflavin-photosensitized singlet oxygen oxidation product of vitamin D2, J Am Oil Chem Soc., 79, 983, 2002 25 Osborne, D.R and Voogt, P., The Analysis of Nutrients in Foods, Academic Press, London, 1978, p 183 26 Woollard, D.C., Quality control of the fat-soluble vitamins in the New Zealand dairy industry, Food Technol Australia, 39, 250, 1987 27 Koshy, K.T and VanDerSlik, A.L., 25-Hydroxycholecalciferol in cow milk as determined by high-performance liquid chromatography, J Agric Food Chem., 27, 650, 1979 28 Hollis, B.W and Frank, N.E., Quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human milk, Methods Enzymol., 123H, 167, 1986 29 Ball, G.F.M., Vitamins: Their Role in the Human Body, Blackwell Publishing Ltd., Oxford, 2004, p 188 30 Teegarden, D., Meredith, S.C., and Sitrin, M.D., Isolation and characterization of a 25-hydroxyvitamin D binding protein from rat enterocyte cytosol, J Nutr Biochem., 8, 195, 1997 31 Teegarden, D., Nickel, K.P., and Shi, L., Characterization of 25-hydroxyvitamin D binding protein from intestinal cells, Biochem Biophys Res Commun., 275, 845, 2000 © 2006 by Taylor & Francis Group, LLC 118 Vitamin D 32 Dueland, S., Pedersen, J.I., Helgerud, P., and Drevon, C.A., Transport of vitamin D3 from rat intestine, J Biol Chem., 257 (1), 146, 1982 33 Brown, A., Dusso, A., and Slatopolsky E., Vitamin D, in The Kidney, Seldin, D.W and Giebisch, G., Eds., 3rd ed., Vol 1, Lippincott, Williams and Wilkins, Philadelphia, 2000, p 1047 34 Haddad, J.G and Walgate, J., 25-Hydroxyvitamin D transport in human plasma, J Biol Chem., 251 (16), 4803, 1976 35 Dueland, S., Helgerud, P., Pedersen, J.I., Berg, T., and Drevon, C.A., Plasma clearance, transfer, and distribution of vitamin D3 from intestinal lymph, Am J Physiol., 245, E326, 1983 36 Davies, M., Mawer, E.B., and Krawitt, E.L., Comparative absorption of vitamin D3 and 25-hydroxyvitamin D3 in intestinal disease, Gut, 21, 287, 1980 37 Dueland, S., Pedersen, J.I., Helgerud, P., and Drevon, C.A., Absorption, distribution, and transport of vitamin D3 and 25-hydroxyvitamin D3 in the rat, Am J Physiol., 245, E463, 1983 38 Holmes, R.P and Kummerow, F.A., The relationship of adequate and excessive intake of vitamin D to health and disease, J Am Coll Nutr., 2, 173, 1983 39 Burnand, B., Sloustkis, D., Gianoli, F., Cornuz, J., Rickenbach, M., Paccaud, F., and Burckhardt, P., Serum 25-hydroxyvitamin D: distribution and determinants in the Swiss population, Am J Clin Nutr., 56, 537, 1992 40 Gascon-Barre´, M and Joly, J.-G., The biliary excretion of [3H]-25-hydroxyvitamin D3 following chronic ethanol administration in the rat, Life Sci., 28, 279, 1981 41 Batchelor, A.J and Compston, J.E., Reduced plasma half-life of radio-labelled 25-hydroxyvitamin D3 in subjects receiving a high-fibre diet, Br J Nutr., 49, 243, 1983 42 Brot, C., Jørgensen, N.R., and Sørensen, O.H., The influence of smoking on vitamin D status and calcium metabolism, Eur J Clin Nutr., 53, 920, 1999 43 Law, M.R and Hackshaw, A.K., A meta-analysis of cigarette smoking, bone mineral density and risk of hip fracture: recognition of a major effect, Br Med J., 315, 841, 1997 © 2006 by Taylor & Francis Group, LLC ... Pilchards, canned in tomato sauce Sardines, canned in brine, drained Sardines, canned in oil, drained Sardines, canned in tomato sauce Tuna, canned in brine, drained Tuna canned in oil, drained Tr... Vitamin D2 (C28H44O, MW ¼ 396.6) and vitamin D3 (C27H44O, MW ¼ 3 84.2 ) differ structurally only in the C-17 side chain, which in vitamin D2 has a double bond and an additional methyl group (Figure 4.1 )... supplemented foods, the amount of naturally occurring vitamin D (if any) is usually considered to be negligible, and it is deemed necessary to determine only the vitamin D that is added Even so,