Assays On D3


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Assays On D3

  1. 1. ISymposium The use and Interpretation of Assays for Vitamin D and its Metabolites1«2 MICHAEL F. HOLICK Boston University School of Medicine, Vitamin D, Skin, and Bone Research Laboratory, Boston, MA 02118 to form the major circulating form of vitamin D, 25- ABSTRACT Vitamin D is essential for the mainte hydroxyvitamin D (25-OH-D) (1-3) (Fig. 1). The bio nance of calcium and bone metabolism in humans. The logically inert 25-OH-D is then hydroxylated in the recommended daily allowance (RDA) for vitamin D in kidney on carbon 1 to form its biologically active form, the United States of 200 lu (5.0 /ig) ¡seasonable for r commonly known as l«,25-dihydroxyvitamin D adults who receive some exposure to sunlight; however, (1,25(OH)2D) (1-3) (Fig. 1). Circulating 1,25(OH)2D in the absence of any exposure to sunlight, this rec ommendation may be 2 to 3 times lower than that ac is taken up by the small intestine, where it enhances tually required to satisfy the body's needs. Vitamin D the efficiency of intestinal calcium absorption, and by Downloaded from by on June 11, 2008 was first measured by bioassays. However, bioassays the bone, where it triggers differentiation of stem cells became obsolete in light of the revelation that vitamin to osteoclasts, which in turn enhance the mobilization D must be activated first in the liver to 25-hydroxyvi- tamin D (25-OH-D) and then in the kidney to 1,25- of calcium (Fig. 1) (1-3). 1,25 dihydroxycholecalciferol dihydroxyvitamin D [1,25(OH)2D] before becoming also stimulates osteoblasts to make osteocalcin and biologically functional. Current assays measure circu enhances alkaline phosphatase activity in bone. Be lating concentrations of vitamin D, 25-OH-D or sides the intestine and bone, nuclear receptors for 1,25(OH)2D. The serum vitamin D concentration is of 1,25(OH)2D have also been found in a variety of other value for determining the role of sunlight in producing vitamin D in skin and as a provocative test to determine tissues and cells that are not involved in calcium me the absorption of vitamin D in patients with malab- tabolism, including the stomach, brain, pituitary sorption syndromes. The serum concentration of 25- gland, gonads, parathyroid glands, epidermis, dermis, OH-D is most valuable for determining the overall vi monocytes, and activated T and B lymphocytes (1-6). tamin D status of an individual, since it is an average Although the exact physiological action of 1,25(OH)2D of dietary and sunlight-induced vitamin D. The mea surement of the serum 1,25(OH) ,Dconcentrations has in these tissues is unknown, 1,25(OH)2D has been been most useful in evaluating disorders in calcium shown to inhibit the proliferation and to induce the and bone metabolism related to acquired and inborn differentiation of many cell types that possess its re- errors in the conversion of 25-OH-D to 1,25(OH)2D. J. Hutr. 120:1464-1469, 1990. 1 Presented as part of a conference, quot;Nutrition Monitoring and INDEXING KEY WORDS: Nutrition Status Assessment,quot; at the first fall meeting of the Amer ican Institute of Nutrition, Charleston, South Carolina, December • vitamin D • vitamin D metabolites • assays 8-10, 1989. The conference was supported in part by cooperative • sunlight • 25-hydroxyvitamin D agreement with HPU880004-02-1 the DHHS Office of Disease Pre vention and Health Promotion, the USDA Human Nutrition In formation Service, the DHHS National Center for Health Statistics, and the International Life Sciences Institute-Nutrition Foundation. 1 The Planning Committee for the meeting consisted of Drs. He The intensive research activities in the field of vi len A. Guthrie, Roy J. Martin, Linda D. Meyers, James A. Olson, tamin D during the past two decades have made us Catherine E. Woteki, and Richard G. Allison (ex-officio). The sym aware that the quot;sunshine vitaminquot; is more than just posium papers were edited by a committee consisting of Dr. James a fat-soluble vitamin that regulates calcium metabo Allen Olson (coordinator), Dept. of Biochemistry & Biophysics, Iowa State University, Ames, IA; Dr. Cathy C. Campbell, Division of lism. Once vitamin D [the term vitamin D represents vitamin D2 (ergocalciferol) and/or vitamin D3 (chole- Nutritional Sciences, Cornell University, Ithaca, NY; Dr. Roy J. Martin, Dept. of Foods 6k.Nutrition, University of Georgia, Athens, calciferol)] is synthesized in the skin or obtained from GA¡ nd Dr. Catherine E. Woteki, Food & Nutrition Board, National a the diet, it is hydroxylated on carbon 25 in the liver Academy of Sciences, Washington, DC. 0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 11 February 1990. Accepted 11 July 1990. 1464
  2. 2. VITAMIN D ASSAYS 1465 VITAMIN D cutaneous production of previtamin D3 include sea sonal changes, time of day, skin pigmentation, aging, and sunscreen use (10-14). The other major source of vitamin D is from the Liver VITAMIN D-25- diet. In the United States and Canada, the ingestion hydroxylase of fortified milk, which contains 400 IU (10 ¿ig) of either ergocalciferol or cholecalciferol per quart, pro vides twice the United States recommended daily al PO.ond lowance (USRDA) of 200 IU for this essential fat-sol OTHER FACTORS uble substance. The only other major dietary source of vitamin D is from fatty fish and fish liver oils. Vitamin D concentrations were first measured by rat and chick bioassays (15-17). The rat bioassay, 1,25-(OH),-D commonly known as the line test, was widely used to determine the concentration of vitamin D in fortified foods (15). The development of specific assays for vi tamin D and its biologically important metabolites made these bioassays obsolete. The half-life of circulating vitamin D is only 24 h (18). Thus, the serum concentration at any time is de pendent on times of the most recent ingestion of vi Downloaded from by on June 11, 2008 tamin D as well as of the last exposure to sunlight (18, 19). The quot;normalquot; range of serum vitamin D is 0-310 nmol/L (0-120 ng/mL) (18-20). Consequently serum ergocalciferol or cholecalciferol is of little value in de termining the vitamin D status of a patient. Nonethe less, serum vitamin D assays are useful for determining the capacity of human skin to produce vitamin D in response to exposure to solar or simulated solar ultra violet radiation (18). Furthermore, the measurement FIGURE I Schematic representation of the hormonal of circulating concentrations of vitamin D 12-24 h control loop for vitamin D metabolism and function. A re after an oral dose of 50,000 IU (1.25 mg) of ergocal duction in the serum calcium below ~8.8 mg/mL prompts a proportional increase in the secretion of parathyroid hor ciferol provides valuable clinical information for de mone, which enhances the mobilization of calcium stores termining whether a patient with a malabsorption from bone. Parathyroid hormone also promotes the synthesis syndrome can absorb vitamin D (20) (Fig. 3). of 1,25(OH)2D in the kidney, which, in turn, stimulates the Plasma and serum concentrations of vitamin D are mobilization of calcium from the bone and its absorption determined by: 1) extracting the lipid-soluble vitamin from the intestine. Reproduced with permission (38). D from 1 to 2 mL of serum or plasma, 2) separating vitamin D from its metabolites and lipid contaminants in the extract by rapid reverse-phase cartridge chro- ceptor (1-3, 7). These insights have been of great value matography, 3) further resolving the vitamin D frac for the pharmacologie use of 1,25(OH)2D3 in the hy- tion by straight phase high-performance liquid chro- perproliferative skin disorder psoriasis (7-9). matography (HPLC), and 4) quantitating the amount of vitamin D by its ultraviolet absorbance in the HPLC eluate or by a competitive protein binding assay (18, 21-23). The intra-assay and interassay variations are PHOTOSYNTHESIS, ABSORPTION, ~10 and 12%, respectively. AND ASSAYS FOR VITAMIN D Sunlight, which contains wavelengths between 290 and 315 nm, photolyzes provitamin D3 (7-dehydro- DETERMINATION AND CLINICAL UTILITY cholesterol) in the skin to previtamin D3 (Fig. 2). Pre OF THE CIRCULATING CONCENTRATION vitamin D3, a thermally labile compound, undergoes OF 25-HYDROXYVITAMIN D an internal isomerization of its double bonds to form the thermodynamically stable vitamin D3 (2), which The half-life of circulating 25-OH-D is ~3 wk (2). is then translocated from the epidermis into the dermal Therefore, its steady-state concentration summates the capillary bed (Fig. 2). Factors that can influence the concentrations of vitamin D derived both from the
  3. 3. 1466 HOLICK SUN SUN 7- DEHYDROCHOLESTEROL Downloaded from by on June 11, 2008 BLOOD DBP-D3 DBP FIGURE 2 Schematic representation of the formation of previtamin D3 in the skin during exposure to the sun, the thermal isomerization of previtamin D3 to vitamin D3/ and the specific translocation of vitamin D3 by the vitamin D-binding protein (DBP) into the circulation. During continual exposure to the sun, previtamin D3 also photoisomerizes reversibly to Iumisterol3 and tachysterol3, which are biologically inert photoproducts (i.e., they do not stimulate intestinal calcium absorption). Because the DBP has no affinity for Iumisterol3 and has minimal affinity for tachysterol3/ the translocation of these photoisomers into the circulation is negligible. Subsequently, these photoproducts are sloughed off during the natural turnover of the skin. When previtamin D3 stores are depleted (due to its thermal isomerization to D3), however, lumisterol and tachysterol, upon exposure to UV radiation, will photoisomerize to preD3. Reproduced with permission of the American Association for the Advancement of Science (39). diet and from photo-formation over a several weeks is considered to be normal. Vitamin D intoxication is to several months. The separate measurement of 25- usually associated with 25-OH-D concentrations OH-cholecalciferol and 25-OH-ergocalciferol was above 375 nmol/L (150 ng/mL), with attendant hy- originally thought to provide information about sun percalcemia and hyperphosphatemia (2). light-induced vs. dietary sources of vitamin D (24). Serum concentrations of 25-OH-D are measured in However, since milk and multivitamin preparations several ways (25-30). A competitive protein-binding are now fortified with both forms of the vitamin, the assay is often used. The vitamin D-binding protein, separate measurement of these metabolites is of little which has a very high affinity for 25-OH-D (26-28), value. binds the ligand in a lipid extract of serum or plasma The 25-OH-D assay is most valuable for determin (0.1 mL). However, other vitamin D metabolites can ing the vitamin D status of an individual. For its assay, interfere with this assay, even though they usually commercial diagnostic laboratory services and assay represent <10% of the total binding activity (29). To kits are available. The normal circulating concentra increase the specificity, 25-OH-D can first be separated tion of 25-OH-D is usually reported to be between 20 from vitamin D and its metabolites by a rapid straight- nmol/L (8 ng/mL) and 150 nmol/L (60 ng/mL). Serum phase silica cartridge chromatography (30), followed values below 25 nmol/L (10 ng/mL) are generally con by the competitive protein-binding assay. sidered to indicate impending or frank vitamin D de Circulating concentrations of 25-OH-ergocalciferol ficiency. Although most diagnostic laboratories report and 25-OH-cholecalciferol can be accurately measured the upper limit of the normal range for 25-OH-D to by first chromatographing a lipid extract from 1 mL be 150 nmol/L (60 ng/mL), circulating concentrations of serum or plasma on a rapid straight-phase or reverse- of 250 nmol/L (100 ng/mL) in lifeguards after a full phase cartridge followed by straight-phase HPLC. The summer of exposure to sunlight is not uncommon and amounts of 25-OH-ergocalciferol and 25-OH-chole-
  4. 4. VITAMIN D ASSAYS 1467 80 n 1,25(OH)2D receptor recognizes l,25(OH)2cholecal- ciferol better than l,25(OH)2ergocalciferol (2, 29). As a result, this assay underestimated the total circulating concentration of 1,25(OH)2D. This assay has been im proved by using the bovine thymus 1,25(OH)2D re ceptor (29, 33), which reacts equally well with l,25(OH)2ergocalciferol and l,25(OH)2cholecalciferol. In addition, the HPLC step has now been replaced by a rapid silica cartridge Chromatographie step (34, 35). A bioassay using cultured rat calvarÃ-a an detect pi- c cogram quantities of 1,25(OH)2D in the circulation (36). However, this assay is very time consuming and requires a tissue culture facility. It is most useful in verifying the results from a competitive receptor binding assay. The half-life of circulating 1,25(OH)2D has been es 24 48 72 timated to be between 4 and 6 h (37). The normal range HOURS of serum values is between 38 and 144 pmol/L (16- FIGURE 3 Serum vitamin D concentrations in seven pa 60 pg/mL). As vitamin D deficiency develops, the body tients with diarrheal syndromes after a single oral dose of responds by increasing the production and secretion Downloaded from by on June 11, 2008 50,000 IU (1.25 mg) of ergocalciferol. For comparison, the of parathyroid hormone (Fig. 1). Parathyroid hormone means and standard errors of vitamin D concentrations in turn enhances the 1-hydroxylation of 25-OH-D (1- measured in seven normal control subjects after a similar 3) (Fig. 1). Thus, secondary hyperparathyroidism ac dose are indicated by the closed circles and dotted lines celerates the conversion of 25-OH-D to 1,25(OH)2D (— •€”). Note that two patients, one with Crohn's ileo- â colitis (patient F) and one with ulcerative colitis (patient G), (1-3). Since the circulating concentration of 25-OH- had essentially normal absorption curves. Five patients, D is about three orders of magnitude higher than however, showed a dramatic lack of response, with no values 1,25(OH)2D, even very low levels of 25-OH-D can above 10 ng/mL. Reproduced with permission (20). provide enough substrate for the formation of some 1,25(OH)2D. In vitamin D deficiency, vitamin D is also efficiently converted to 25-OH-D (18). As a result, calciferol in the eluate are quantitatively determined a hospital patient with vitamin D deficiency who has by their UV absorption at either 254 or 265 nm (21, previously obtained a very small quantity of vitamin 25, 29, 30). D from food or exposure to sun can have low or un- The assay for serum 25-OH-D has clinical utility detectable circulating concentrations of 25-OH-D in determining the vitamin D status of patients with while having low, normal, or even high circulating intestinal malabsorption syndromes or with severe concentrations of 1,25(OH)2D (1, 2, 18, 38). Thus hepatic failure, as well as of the very young and the serum 1,25(OH)2D concentrations are of little value elderly (2), who may be at risk of vitamin D deficiency. in the evaluation of vitamin D deficiency. Needless to say, in an absolute vitamin D deficiency state, circu lating concentrations of 1,25(OH)2D are undetectable. DETERMINATION AND CLINICAL UTILITY The measurement of circulating concentrations of OF THE CIRCULATING CONCENTRATION 1,25(OH)2D have been of great value to clinicians for OF 1,25-DIHYDROXYVITAMIN D the evaluation of patients with acquired and inherited disorders of 1,25(OH)2D metabolism (1-3, 29, 38). Specific assays for 1,25(OH)2D (29, 31-33) in serum Serum 1,25(OH)2D levels are routinely assayed by and plasma are based on the separation of minute commercial diagnostic laboratories and can be mea quantities of 1,25(OH)2D from lipid contaminants and sured as well by assay kits. Patients with chronic other vitamin D metabolites. The concentration of renal failure, hyperphosphatemia, hypoparathyroid- l,25(OH)2cholecalciferol is then determined by a ism, pseudohypoparathyroidism, tumor-induced os competitive receptor binding assay using a nuclear/ teomalacia, hypercalcemia of malignancy (in most cytosolic receptor for 1,25(OH)2D (31-33). Initially cases), or vitamin D-dependent rickets type I [an in 1,25(OH)2D in a lipid extract of 2 to 3 mL of serum born error that markedly reduces the conversion of or plasma was separated from other vitamin D metab 25-OH-D to 1,25(OH)2D] often have low circulating olites by straight phase HPLC. The 1,25(OH)2D frac concentrations of 1,25(OH)2D (2, 29, 38). Serum con tion was then subjected to a competitive protein-bind centrations of 1,25(OH)2D are elevated above the nor ing assay that used the chick cytosolic 1,25(OH)2D mal range in patients with primary hyperparathyroid receptor (31, 32). However, the chick intestinal ism, vitamin D-dependent rickets type II; [an inborn
  5. 5. 1468 HOLICK error in which the recognition of 1,25(OH)2D by target 11. WEBB, A. R., KLINE, L. & HOLICK, M. F. (1988) Influence of tissue receptors is defective]; chronic granulomatous season and latitude on the cutaneous synthesis of vitamin D3 : exposure to winter sunlight in Boston and Edmonton will not disorders such as sarcoidosis, tuberculosis, and sili promote vitamin D3 synthesis in human skin. /. Clin. Endocrino!. cosis; and lymphoma (in some patients) (2, 29, 38). Metab. 67: 373-378. 12. CLEMENS, . L., HENDERSON, . L., ADAMS,J. S. &.HOLICK,M. F. T S (1982) Increased skin pigment reduces the capacity of skin to synthesize vitamin D3. Lancet 74-76. CONCLUSION 13. HOLICK,M. F., MATSUOKA,L. Y. & WORTSMAN,J. (1989) Age, vitamin D, and solar ultraviolet radiation. Lancet ii: 1104-1105. 14. MATSUOKA,L. Y., IDE, L., WORTSMAN,J., MACL.AUGHLIN, . & J Vitamin D is essential for the maintenance of cal HOLICK, M. F. (1987) Sunscreens suppress cutaneous vitamin cium and bone metabolism throughout our lives. The D3 synthesis. /. Clin. Endocrino!. Metab. 64: 1165-1168. major source of vitamin D is casual exposure to sun 15. STEENBOCK, & BLACK,A. (1924) The reduction of growth- H. light. In the absence of exposure to sunlight, the av promoting and calcifying properties in a ration by exposure to erage daily requirement for vitamin D is probably 2 ultraviolet light. /. Bio!. Chem. 61: 408-422. 16. STEENBOCK, . & KLETZIEN,S. W. F. (1932) The reaction of H to 3 times higher than the USRDA of 200 IU (5 fig) chickens to irradiated ergosterol and irradiated yeast as con (Paris, P., Bondi, K., Luria, S. and Holick, M. F., un trasted with the natural vitamin D in fish liver oil. /. Bio!. Chem. published results). Among assays for vitamin D and 97: 249-264. its metabolites, the assay of serum 25-OH-D levels 17. WADDELL,J. (1934) The provitamin D of cholesterol. I. The has the most utility for determining the vitamin D antirachitic efficacy of irradiated cholesterol. /. Bio!. Chem. 105: 711-739. status of an individual. Circulating concentrations of 18. CLEMENS, . L., ADAMS,J. S. & HOLICK,M. F. (1982) Measure T vitamin D and of 1,25(OH)2D, however, can be of Downloaded from by on June 11, 2008 ment of circulating vitamin D in man. Clin. Chim. Acta 121: value for clinicians who are evaluating malabsorption 301-308. syndromes and acquired and inherited disorders of 25- 19. ADAMS,J. A., CLEMENS,T. L., PARRISH,J. A. & HOLICK, M. F. OH-D metabolism, respectively (1-3, 29, 38). (1981) Vitamin D synthesis and metabolism after ultraviolet radiation of normal and vitamin D deficient subjects. N. Eng!. /. Med. 306: 722-725. 20. Lo, C. W., PARIS,P. W., CLEMENS, . L., NOLAN, J. &. HOLICK, T M. F. (1985) Vitamin D absorption in healthy subjects and in LITERATURE CITED patients with intestinal malabsorption syndromes. Am. J. Clin. Nutr. 42: 644-649. 1. DELuCA, H. (1988) The vitamin D story: a collaborative effort 21. JONES,G. (1978) Assay of vitamin D2 and D3 in human plasma of basic science and clinical medicine. FASEB /. 2: 224-236. by high performance liquid chromatography. Clin. Chem. 24: 2. HOLICK,M. F. (1989) Vitamin D: biosynthesis, metabolism, and 287-298. mode of action. In: Endocrinology, pp. 902-926, vol. 2, 22. CHEN, T. C., TURNER, A. K. & HOLICK,M. F. (1990) A method (DeGroot, L. }., CahUl, G. F. Jr., Martini, L., Nelson, D. H., for the determination of the circulating concentration of vitamin Odell, W. D., Potts, J. T. Jr., Steinberger, E., and Winegrad, D. /. Nutr. Biochem. (in press). A. I., eds.) Gruñeand Stratton, New York. 23. HOLLIS,B. W., Roos, B. A. & LAMBERT, . W. (1981) Vitamin P 3. REICHEL,H., KOEFFLER, P. & NORMAN,A. W. (1989) The role H. D in plasma: quantitation by a nonequilibrium ligand binding of the vitamin D endocrine system in health and disease. N. assay. Steroids 37: 609-619. Engl./. Med. 320:981-991. 24. HADDAD, H. G. & HAHN, T. J. (1973) Natural and synthetic 4. STUMPF,W. E., SAR, M., REID, F. A., et al. (1979) Target cells sources of circulating 25-hydroxyvitamin D in man. Nature 244: for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kid 515-517. ney, skin, pituitary, and parathyroid. Science 206: 1188-1190. 25. CHEN, T., TURNER, A. & HOLICK,M. F. (1990) Method for de 5. TSOUKAS, . D., PROVVEDINE, M. &.MANOLAGAS, C. (1984) C D. S. termination of the circulating concentration of 25-hydroxyvi 1,25-Dihydroxyvitamin D3, a novel immuno-regulatory hor mone. Science 221: 1438-1440. tamin D. /. Nutr. Biochem. (in press). 6. BHALLA,A. K., CLEMENS,T., AMENTO, E., HOLICK, M. F. &. 26. HADDAD,J. G. & CHUY,K. J. (1971 ) Competitive protein binding KRANE, S. M. (1983) Specific high-affinity receptors for 1,25- radioassay for 25-hydroxycholecalciferol. /. Clin. Endocrinol. Metab. 33: 992-995. dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes 27. BELSEY, ., CLARK,M. B. BERNAT,M., GLOWACKI,J., HOLICK, R following activation. /. Clin. Endocrino!. Metab. 57: 13008- M. F., DELUCA, H. F. & POTTS, J. T. (1974) The physiologie 13010. significance of plasma transport of vitamin D and metabolites. 7. HOLICK,M. F. (1989) 1,25-Dihydroxyvitamin D3 and the skin: Am. /. Med. 57: 50-56. a unique application for the treatment of psoriasis. Proc. Soc. 28. HOLLIS,B. W., BURTON,J. H. & DRAPER,H. H. (1977) Abinding Exper.Med. 19:246-257. assay for 25-hydroxycalciferol and 24R,25-dihydroxycalciferols 8. MORIMOTO,S. & KUMAHARA, . (1985) A patient with psoriasis Y using bovine plasma globulin. Steroids 30: 285-293. cured by la-hydroxyvitamin D3. Med. /. Osaka L7niv. 35: 51. 29. HORST, R. (1984) Recent advances in the quantitation of vitamin 9. SMITH, E. L. & HOLICK,M. F. (1987) The skin: the site of vitamin D and vitamin D metabolite. In: Vitamin D: Basic and Clinical D3 synthesis and a target tissue for its metabolite 1,25-dihy Aspects, pp. 423-478 (R. Kumar, ed.), Martinus Nijhoff, Boston. droxyvitamin D3. Steroids 49: 103-131. 30. ADAMS,J. S., CLEMENS, . L. & HOLICK,M. F. (1981) Silica Sep- T 10. HOLICK, M. F. (1990) Vitamin D and the skin: photobiology, Pak preparative chromatography for vitamin D and its metab physiology, and therapeutic efficacy for psoriasis. In: Bone and olites. /. Chromatogr. 226: 198-201. Mineral Research, |N. M. Heersche and J. A. Kanis, eds.), Annual 31. BRUMBAUGH, F., HAUSSLER, H., BURSAC, . M. & HAUSSLER, P. D. K Series, Vol. 7, Elsevier, Amsterdam, in press. M. R. (1974) Filter assay for 1,25-dihydroxyvitamin D3. Utili-
  6. 6. VITAMIN D ASSAYS 1469 zation of the hormone's target tissue chromatin receptor. Bio (1978) A bioassay capable of measuring 1 picogram of 1,25- chemistry 13: 4091-4097. dihydroxyvitamin D3. /. Clin. Endocrinol. Metab. 46: 891-896. 32. EISMAN,J. A., HAMSTRA,A. J., KREAM,B. E. & DELucA, H. F. 37. GRAY,R. W., CALDAS,A. E., WILZ, D. R., LEMANN,J. a SMITH, (1976) A sensitive, precise and convenient method for the de G. A. (1978) Execretion of 3H-1,25-dihydroxyvitamin D3 in termination of 1,25-dihydroxyvitamin D in human plasma. healthy adults /. Clin. Endocrinol. Metab. 46: 756-765. Arch. Biochem. Biophys. 176: 235-243. 38. HOLICK,M. F., KRANE,S. M., & POTTS,J.T., JR. (1986) Calcium, 33. REINHARDT,T. A., HORST, R. L., ORF, J. W. & HOLLIS,B. W. phosphorus, and bone metabolism: calcium regulating hor (1984) A microassay for 1,25-dihydroxyvitamin D not requiring mones. In: Harrison's Principles of Internal Medicine, pp. 1889- high performance liquid chromatography: application to clinical 1900, (E. Braunwald, K. J. Isselbacher, R. G. Petersdorf, J. D. studies. /. Clin. Endocrino!. Metab. 58: 91-98. Wilson, J. B. Martin, A. S. Fauci, eds.) 11th ed, McGraw-Hill, 34. HOLLIS,B. W. (1986) Assay of circulating 1,25-dihydroxyvitamin New York. D involving a novel single-cartridge extraction and purification 39. HOLICK, M. F. (1987) Vitamin D and the kidney. Kidney Int. procedure. Clin. Chem. 32: 2060-2063. 32:912-929. 35. CHEN, T. C., TURNER,A. K. fin.HOLICK,M. F. (1990) A method 40. HOLICK, M. F., MACLAUGHLIN,J. A. & DOPPELT,S. H. (1981) for the determination of the circulating concentration of 1,25- Regulation of cutaneous previtamin D3 photosynthesis in man: dihydroxyvitamin D. /. Nutr. Biochem. (in press). skin pigment is not an essential regulator. Science 211: 590- 36. STERN, P. H., HAMSTRA,A. J., DELUCA, H. F. & BELL,N. H. 593. Downloaded from by on June 11, 2008