C-3 Epimerization of Vitamin D3 Metabolites and Further Metabolism of C-3 Epimers (2004)

Recently, it was revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 of the A-ring. We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 as a major metabolite of 25-hydroxyvitamin D3 (25(OH)D3) and the further metabolism of C-3 epimers of vitamin D3 metabolites. When 25(OH)D3 was incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, and hepatoblastoma cell lines, 3-epi-25(OH)D3 and 24,25 (OH)2D3 were commonly observed as a major and minor metabolite of 25(OH)D3, respectively. 25(OH)D3 was at least as sensitive to C-3 epimerization as 1α, 25(OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction. Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(OH)2D3 rather than 3-epi-25(OH)D3. We also confirmed the existence of 3-epi-25(OH)D3 in the serum of rats intravenously given pharmacological doses of 25(OH)D3. The cultured cells metabolized 3-epi-25(OH)D3 and 3-epi-1α,25(OH)2D3 to 3-epi-24,25(OH)2D3 and 3-epi-1α,24,25(OH)3D3, respectively. In addition, we demonstrated that 3-epi-25(OH)D3 was metabolized to 3-epi-1α,25(OH)2D3 by CYP27B1 and to 3-epi-24,25(OH)2D3 by CYP24 using recombinant Escherichia coli cell systems. 3-Epi-25(OH)D3, 3-epi-1α,25(OH)2D3, and 3-epi-24,25(OH)2D3 were biologically less active than 25(OH)D3, 1α,25(OH)2D3, and 24,25(OH)2D3, but 3-epi-1α,25(OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human myeloid leukemia cells (HL-60). These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of vitamin D3. Recently, it was revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3) were metabolized to their respective epimers of the hydroxyl group at C-3 of the A-ring. We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3 as a major metabolite of 25-hydroxyvitamin D3 (25(OH)D3) and the further metabolism of C-3 epimers of vitamin D3 metabolites. When 25(OH)D3 was incubated with various cultured cells including osteosarcoma, colon adenocarcinoma, and hepatoblastoma cell lines, 3-epi-25(OH)D3 and 24,25 (OH)2D3 were commonly observed as a major and minor metabolite of 25(OH)D3, respectively. 25(OH)D3 was at least as sensitive to C-3 epimerization as 1α, 25(OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction. Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(OH)2D3 rather than 3-epi-25(OH)D3. We also confirmed the existence of 3-epi-25(OH)D3 in the serum of rats intravenously given pharmacological doses of 25(OH)D3. The cultured cells metabolized 3-epi-25(OH)D3 and 3-epi-1α,25(OH)2D3 to 3-epi-24,25(OH)2D3 and 3-epi-1α,24,25(OH)3D3, respectively. In addition, we demonstrated that 3-epi-25(OH)D3 was metabolized to 3-epi-1α,25(OH)2D3 by CYP27B1 and to 3-epi-24,25(OH)2D3 by CYP24 using recombinant Escherichia coli cell systems. 3-Epi-25(OH)D3, 3-epi-1α,25(OH)2D3, and 3-epi-24,25(OH)2D3 were biologically less active than 25(OH)D3, 1α,25(OH)2D3, and 24,25(OH)2D3, but 3-epi-1α,25(OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human myeloid leukemia cells (HL-60). These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of vitamin D3. It is now well established that vitamin D3 is a precursor of the sterol hormone 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). 1The abbreviations used are: 1α,25(OH)2D3,1α,25-dihydroxyvitamin D3; 24,25(OH)2D3, 24R,25-dihydroxyvitamin D3; droxyvitamin 25(OH)D3, 25-hy-D3; 1α,24,25(OH)3D3, 1α,24R,25-trihydroxyvitamin D3; VDR, vitamin D receptor; DBP, vitamin D-binding protein (Gc-globulin); HPLC, high performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; PBS, phosphate-buffered saline; FCS, fetal calf serum; 3-epi-25(OH)D3, 3-epi-25-hydroxyvitamin D3; VDRE, vitamin D-responsive element; R. T., retention time. Vitamin D3 is made from 7-dehydrocholesterol in the skin on exposure to ultraviolet light and metabolized to 25-hydroxyvitamin D3 (25(OH)D3) in the liver. 25(OH)D3 is further metabolized to the most active form of vitamin D3, 1α,25(OH)2D3, or the inactive form, 24R,25-dihydroxyvitamin D3 (24,25(OH)2D3), in the kidney (1DeLuca H.F. J. Lab. Clin. Med. 1976; 87: 7-26PubMed Google Scholar, 2Lawson D.E.M. Davie M. Vitam. Horm. 1979; 37: 1-67Crossref PubMed Scopus (22) Google Scholar). 1α,25(OH)2D3 has potent anti-proliferative and cell differentiation-inducing activities in addition to its role in calcium homeostasis. After the expression of various biological activities, 1α,25(OH)2D3 is further metabolized through the C-24 (3Lohnes D. Jones G. J. Biol. Chem. 1987; 262: 14394-14401Abstract Full Text PDF PubMed Google Scholar, 4Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (218) Google Scholar, 5Reddy G.S. Tserng K.-Y. Biochemistry. 1989; 28: 1763-1769Crossref PubMed Scopus (217) Google Scholar, 6Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (1032) Google Scholar)/C-23 (7Ishizuka S. Yamaguchi H. Yamada S. Nakayama K. Takayama H. FEBS Lett. 1981; 134: 207-211Crossref PubMed Scopus (39) Google Scholar, 8Ishizuka S. Ishimoto S. Norman A.W. Biochemistry. 1984; 23: 1473-1478Crossref PubMed Scopus (53) Google Scholar, 9Ishizuka S. Norman A.W. J. Biol. Chem. 1987; 262: 7165-7170Abstract Full Text PDF PubMed Google Scholar, 10Siu-Caldera M.-L. Zou L. Ehrlich M.G. Schwartz E.R. Ishizuka S. Reddy G.S. Endocrinology. 1995; 136: 4195-4203Crossref PubMed Google Scholar) oxidation pathways and the C-3 epimerization pathway (11Bischof M.G. Siu-Caldera M.-L. Weiskopf A. Vouros P. Cross H.S. Peterlok M. Reddy G.S. Exp. Cell Res. 1998; 241: 194-201Crossref PubMed Scopus (80) Google Scholar, 12Brown A.J. Ritter C. Slatopolsky E. Muralidharan K.R. Okamura W.H. Reddy G.S. J. Cell. Biochem. 1999; 73: 106-113Crossref PubMed Scopus (106) Google Scholar, 13Siu-Caldera M.-L. Sekimoto H. Weiskopf A. Vouros P. Muralidharan K.R. Okamura W.H. Bischof M.G. Norman A.W Uskokovic M.R. Schuster I. Reddy G.S. Bone. 1999; 24: 457-463Crossref PubMed Scopus (51) Google Scholar, 14Masuda S. Kamao M. Schroeder N.J. Makin H.L.J. Jones G. Kremer R. Rhim J. Okano T. Biol. & Pharm. Bull. 2000; 23: 133-139Crossref PubMed Scopus (36) Google Scholar). The C-24 oxidation pathway, initiated by C-24 hydroxylation, leads to the conversion of 1α,25(OH)2D3 into a side chain cleavage product, calcitroic acid (4Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (218) Google Scholar, 5Reddy G.S. Tserng K.-Y. Biochemistry. 1989; 28: 1763-1769Crossref PubMed Scopus (217) Google Scholar). The C-23 oxidation pathway, initiated by C-23 hydroxylation, leads to the formation of 1α,25(OH)2D3-26,23-lactone (7Ishizuka S. Yamaguchi H. Yamada S. Nakayama K. Takayama H. FEBS Lett. 1981; 134: 207-211Crossref PubMed Scopus (39) Google Scholar, 8Ishizuka S. Ishimoto S. Norman A.W. Biochemistry. 1984; 23: 1473-1478Crossref PubMed Scopus (53) Google Scholar, 9Ishizuka S. Norman A.W. J. Biol. Chem. 1987; 262: 7165-7170Abstract Full Text PDF PubMed Google Scholar, 10Siu-Caldera M.-L. Zou L. Ehrlich M.G. Schwartz E.R. Ishizuka S. Reddy G.S. Endocrinology. 1995; 136: 4195-4203Crossref PubMed Google Scholar). The newly discovered C-3 epimerization pathway leads to the conversion of the configuration of the hydroxyl group at C-3 of the A-ring and produces 3-epi-1α,25(OH)2D3 from 1α,25(OH)2D3. In view of this modification at the A-ring, the C-3 epimerization pathway is quite different from side chain oxidation pathways. The C-3 epimerization of 1α,25(OH)2D3 was observed in human colon carcinoma-derived Caco-2 cells (11Bischof M.G. Siu-Caldera M.-L. Weiskopf A. Vouros P. Cross H.S. Peterlok M. Reddy G.S. Exp. Cell Res. 1998; 241: 194-201Crossref PubMed Scopus (80) Google Scholar), bovine parathyroid cells (12Brown A.J. Ritter C. Slatopolsky E. Muralidharan K.R. Okamura W.H. Reddy G.S. J. Cell. Biochem. 1999; 73: 106-113Crossref PubMed Scopus (106) Google Scholar), rat osteoblastic UMR 106 and Ros17/2.8 cells (13Siu-Caldera M.-L. Sekimoto H. Weiskopf A. Vouros P. Muralidharan K.R. Okamura W.H. Bischof M.G. Norman A.W Uskokovic M.R. Schuster I. Reddy G.S. Bone. 1999; 24: 457-463Crossref PubMed Scopus (51) Google Scholar), and various cultured cell lines (14Masuda S. Kamao M. Schroeder N.J. Makin H.L.J. Jones G. Kremer R. Rhim J. Okano T. Biol. & Pharm. Bull. 2000; 23: 133-139Crossref PubMed Scopus (36) Google Scholar). From these studies, the C-3 epimerization pathway is assumed to be cell-selective. It was considered that the C-3 epimerization pathway is cell differentiation-related in Caco-2 cells, because 3-epi-1α,25 (OH)2D3 was only observed in confluent, quiescent Caco-2 cells, not proliferating Caco-2 cells (11Bischof M.G. Siu-Caldera M.-L. Weiskopf A. Vouros P. Cross H.S. Peterlok M. Reddy G.S. Exp. Cell Res. 1998; 241: 194-201Crossref PubMed Scopus (80) Google Scholar). 3-Epi-1α,25(OH)2D3 was also isolated as a circulating metabolite of 1α,25(OH)2D3 in rats treated with pharmacological doses of 1α,25(OH)2D3 (15Sekimoto H. Siu-Caldera M.-L. Weiskopf A. Vouros P. Muralidharan K.R. Okamura W.H. Uskokovic M.R. Reddy G.S. FEBS Lett. 1999; 448: 278-282Crossref PubMed Scopus (55) Google Scholar). In addition, synthetic analogs of 1α,25(OH)2D3, e.g. 22-oxacalcitriol (16Kamao M. Tatematsu S. Hatakeyama S. Ozono K. Kubodera N. Reddy G.S. Okano T. J. Biol. Chem. 2003; 278: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), 20-epi-1α,25(OH)2D3 (17Siu-Caldera M.-L. Rao D.S. Astecker N. Weiskopf A. Vouros P. Konno K. Fujishima T. Takayama H. Peleg S. Reddy G.S. J. Cell. Biochem. 2001; 82: 599-609Crossref PubMed Scopus (19) Google Scholar), and 1α,25(OH)2-16-ene-23-yne-D3 (18Reddy G.S. Rao D.S. Siu-Caldera M.-L. Astecker N. Weiskopf A. Vouros P. Sasso G.J. Manchand P.S. Uskokovic M.R. Arch. Biochem. Biophys. 2000; 383: 197-205Crossref PubMed Scopus (25) Google Scholar), have been reported to be metabolized to their respective C-3 epimers. Higashi et al. (19Higashi T. Kikuchi R. Miura K. Shimada K. Hiyamizu H. Ooi H. Iwabuchi Y Hatakeyama S. Kubodera N. Biol. & Pharm. Bull. 1999; 22: 767-769Crossref PubMed Scopus (19) Google Scholar) identified 3-epi-24,25(OH)2D3-24-glucuronide in the bile of rats administered pharmacological doses of 24,25(OH)2D3. 3-Epi-24,25(OH)2D3 was also identified in cell culture media (20Kamao M. Tatematsu S. Reddy G.S. Hatakeyama S. Sugiura M. Ohashi N. Kubodera N. Okano T. J. Nutr. Sci. Vitaminol. 2001; 47: 108-115Crossref PubMed Scopus (22) Google Scholar) and rat plasma (21Higashi T. Ogasawara A. Shimada K. Anal. Sci. 2000; 16: 477-482Crossref Scopus (15) Google Scholar). However, it is unclear whether 25(OH)D3 would be metabolized to its C-3 epimer, 3-epi-25(OH)D3. 25(OH)D3 is the most abundant circulating metabolite of vitamin D3 with a concentration of 20–50 ng/ml under normal conditions (22Napoli J.L. Horst R.L. Vitamin D Metabolism. Martinus Nijhoff, Dordrecht1984: 91-123Google Scholar). 25(OH)D3 is the immediate precursor of the active and hormonal form, 1α,25(OH)2D3, and the inactive form, 24,25(OH)2D3. Metabolism of 25(OH)D3 is important for regulation of the biological activity of vitamin D3. In the present study, we investigated the metabolism of 25(OH)D3 in rat osteosarcoma cells (UMR 106) and identified 3-epi-25(OH)D3 using 1H NMR spectroscopy and LC-MS techniques. 3-Epi-25(OH)D3 was detected in various cell models cultured with 25(OH)D3in vitro and in the serum of rats administered pharmacological doses of 25(OH)D3in vivo. We also demonstrated that 3-epi-25(OH)D3 was further metabolized by 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24). The results indicated that 25(OH)D3 was metabolized through the C-3 epimerization pathway like 1α,25(OH)2D3 and 24,25(OH)2D3. C-3 epimerization is a common metabolic pathway for the major metabolites of vitamin D3. Materials—3-Epi-25(OH)D3, 3-epi-1α,25(OH)2D3, and 3-epi-24,25(OH)2D3 were synthesized in our laboratory. 25(OH)D3, 1α, 25(OH)2D3, and 24,25(OH)2D3 were obtained from Solvay-Duphar Co. (Weesp, The Netherlands). 1α,24R,25-Trihydroxyvitamin D3 (1α, 24,25(OH)3D3) was kindly provided by Kureha Chemical Industry Co., Ltd. [26,27-methyl-3H]1α,25(OH)2D3 (179 Ci/mmol) and [23,24-3H]25(OH)D3 (82 Ci/mmol) were purchased from Amersham Biosciences. Deuterized chloroform (CDCl3, 99.8%, NMR analytical grade) was purchased from Euriso-top (Gif-Sur-Yvette, France). Organic solvents of HPLC grade were obtained from Wako Pure Chemical Industries, Ltd. Cell Culture—A rat osteosarcoma cell line (UMR 106), human osteosarcoma cell line (MG-63), human colon adenocarcinoma cell line (Caco-2), porcine kidney cell line (LLC-PK1), and human hepatoblastoma cell line (Hep G2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). UMR 106 and MG-63 cells were maintained in Dulbecco's modified Eagle's medium containing 10% FCS. Caco-2 cells were maintained in minimum essential medium containing 10% FCS and 1% non-essential amino acids. LLC-PK1 cells were maintained in Medium 199 containing 5% FCS. Hep G2 cells were maintained in minimum essential medium containing 10% FCS, 1% non-essential amino acids, and 1% sodium pyruvate. All culture media contained penicillin (100 IU/ml) and streptomycin (100 μg/ml). Cells were cultured at 37 °C in a humidified atmosphere of CO2 in air with a change of medium every 3 days. In the experiments described below, vitamin D3 compounds were added to the culture medium in an ethanolic solution, the final concentration in the medium never exceeding 0.1% (v/v). Generation of 25(OH)D3, 1α,25(OH)2D3, 24,25(OH)2D3, 3-Epi-25(OH)D3, and 3-Epi-1a,25(OH)2D3 Metabolites in Cultured Cells— UMR 106, MG-63, and LLC-PK1 cells (2 × 106) and Caco-2 and Hep G2 cells (4 × 106) were seeded in 150-mm culture dishes and cultured for 4 days to late log phase. The medium was removed, and cells were washed with phosphate-buffered saline without calcium, magnesium (PBS(–)) and then in medium containing 1% bovine serum albumin in the presence of 10 μm of 25(OH)D3, 1α,25(OH)2D3, 24,25(OH)2D3, 3-epi-25(OH)D3, or 3-epi-1α,25(OH)2D3 for 48 h at 37 °C. For measurements of vitamin D3 metabolites, three 150-mm culture dishes were used for each culture. For time course experiments, the cells were incubated with 5 μm of 25(OH)D3, 3-epi-25(OH)D3, or 1 μm of 1α,25(OH)2D3, 3-epi-1α,25(OH)2D3 for a period ranging from 1 to 48 h. Purification of Metabolites—Lipid extraction was performed according to the method of Bligh and Dyer (23Bligh E.G. Dyer W.J. Can. J. Biochem. 1957; 37: 911-917Crossref Scopus (42855) Google Scholar) as modified by Makin et al. (4Makin G. Lohnes D. Byford V. Ray R. Jones G. Biochem. J. 1989; 262: 173-180Crossref PubMed Scopus (218) Google Scholar). Lipid was extracted from cells and medium with methanol and dichloromethane. The organic layer, containing unchanged substrate and lipid-soluble metabolites, was evaporated under nitrogen gas to dryness, and the residue was redissolved in hexane/2-propanol/methanol (HIM) (88:10:2, v/v/v). HPLC was carried out using a model 600 pump and a model 996 photodiode array detector (Waters Associates, Milford, MA). Elution was performed on a Zorbax SIL column (4.6 × 250 mm, Dupont Instruments), using HIM (88:10:2), at a flow rate of 1.0 ml/min (first HPLC system). Metabolites were identified based on UV characteristics of the vitamin D cis-triene system (λmax = 265 nm, λmin = 228 nm). The same metabolites were also separated on a Sumichiral OA-2000 column (4.6 × 250 mm, Sumika Chemical Analysis Service, Ltd., Osaka, Japan) using 2-propanol/hexane (96.5:3.5 or 94.5:5.5, v/v) or a Zorbax CN column (4.6 × 250 mm, Dupont Instruments) using HIM (88:10:2) at a flow rate of 1.0 ml/min (second HPLC system). Concentrations of stock solutions of vitamin D3-related compounds were determined spectrophotometrically using a molar extinction coefficient, ϵ265 = 18,200. 1H NMR and LC-MS Analyses—The 500-MHz 1H NMR spectra of the isolated metabolites were measured on a Varian VXR-500 (1H, 499.9 MHz). Purified metabolites (3 μg) were dissolved in 40 μl of CDCl3 with a very small portion of CHCl3 (7.24 ppm, used as an internal standard for 1H NMR spectroscopy) and transferred into a nano probe. Two-dimensional COSY and two-dimensional NOESY spectra were obtained as described previously (16Kamao M. Tatematsu S. Hatakeyama S. Ozono K. Kubodera N. Reddy G.S. Okano T. J. Biol. Chem. 2003; 278: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 20Kamao M. Tatematsu S. Reddy G.S. Hatakeyama S. Sugiura M. Ohashi N. Kubodera N. Okano T. J. Nutr. Sci. Vitaminol. 2001; 47: 108-115Crossref PubMed Scopus (22) Google Scholar). LC-MS analysis was carried out with a QuattroII (Micromass, Manchester, UK) equipped with an electrospray ionization source in the positive ion mode. An HPLC system consisting of a MAGIC2002 Micro-LC full system (Michrom Bio Resources) and a Develosil ODS-HG-5 column (2.0 × 150 mm, Nomura Chemical, Tokyo, Japan) was used. As a mobile phase, methanol, 10 mm ammonium acetate (50:50, v/v, A) and methanol, 10 mm ammonium acetate (98:2, v/v, B) were used, and a linear gradient elution was run from an A/B ratio of 65:35 to 20:80 at a flow rate of 0.2 ml/min. The column temperature was maintained at 40 °C. Mass spectra were obtained by averaging each peak and subtracting the background. In Vivo Metabolism of 25(OH)D3 in Rats—Male Wistar rats (Japan SLC, Hamamatsu, Japan), weighing ∼300 g, were used in experiments, following adaptation to laboratory conditions for at least 5 days. Three rats were given a bolus dose of 500 μg of 25(OH)D3 intravenously. At 6 h after dosing, each rat was killed, and the blood was collected, heparinized, and immediately centrifuged. About 5 ml of serum was obtained from each rat. Lipid extraction of serum and purification of 25(OH)D3 metabolites were performed as described under "Purification of Metabolites." Metabolism of 3-Epi-25(OH)D3 in Cultures of Recombinant E. coli Cells Expressing CYP27B1 and CYP24 —Co-expression plasmids for human CYP27B1, bovine adrenodoxin, and NADPH-adrenodoxin reductase were constructed as described (pKSNdl-CYP27B1) (24Sawada N. Sakaki T. Kitanaka S. Takeyama K. Kato S. Inouye K. Eur. J. Biochem. 1999; 265: 950-956Crossref PubMed Scopus (51) Google Scholar). Coexpression plasmids for CYP24, adrenodoxin, and adrenodoxin reductase were reported previously (pKSNdl-CYP24) (25Sakaki T. Sawada N. Komai K. Shiozawa S. Yamada S. Yamamoto K. Ohyama Y. Inouye K. Eur. J. Biochem. 2000; 267: 6158-6165Crossref PubMed Scopus (161) Google Scholar). Recombinant Escherichia coli cells transfected with the above plasmids (JM109/pKSNdl-CYP27B1 or JM109/pKSNdl-CYP24) were grown in TB medium (26Akiyoshi-Shibata M. Sakaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (146) Google Scholar) containing 50 μg/ml ampicillin at 26 °C. The induction of transcription was initiated by addition of isopropylthio-β-d-galactoside at a final concentration of 1 μm when the cell density (OD660) reached 0.5. Then 50 μm of 25(OH)D3 or 3-epi-25(OH)D3 was added to the culture, and the cells were incubated for 48 h. Lipid extraction and purification of metabolites were performed as described under the "Purification of Metabolites." Recombinant E. coli cells transfected with pKSNdl derived from pKK233-3 (JM109/pKSNdl) were used for control experiments. VDR and DBP Binding Assay—The binding affinity of 1α,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, and their C-3 epimers for the VDR was tested using a calf thymus 1α,25(OH)2D3 receptor assay (Yamasa Co., Chiba, Japan). The receptor was incubated at 20 °C for 1 h with increasing concentrations of a vitamin D3 compound, and then 15,000 dpm of [3H]1α,25(OH)2D3 was added and incubated for 1 h at 20 °C as described previously (16Kamao M. Tatematsu S. Hatakeyama S. Ozono K. Kubodera N. Reddy G.S. Okano T. J. Biol. Chem. 2003; 278: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 20Kamao M. Tatematsu S. Reddy G.S. Hatakeyama S. Sugiura M. Ohashi N. Kubodera N. Okano T. J. Nutr. Sci. Vitaminol. 2001; 47: 108-115Crossref PubMed Scopus (22) Google Scholar, 27Masuda S. Byford V. Kremer R. Makin H.L.J. Kubodera N. Nishii Y. Okazaki A. Okano T. Kobayashi T. Jones G. J. Biol. Chem. 1996; 271: 8700-8708Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The binding affinity of 1α,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, and their C-3 epimers for the vitamin D-binding protein (DBP) was tested using plasma from vitamin D-deficient rats (28Suda T. DeLuca H.F. Tanaka Y. J. Nutr. 1970; 100: 1049-1052Crossref PubMed Scopus (297) Google Scholar). The displacement of [3H]25(OH)D3 from vitamin D-deficient rat plasma diluted (1:70,000) in 3.5 mm barbiturate buffer (pH 8.6) containing 0.13 m NaCl and 0.1% ovalbumin was measured following the addition of the compounds as described previously (16Kamao M. Tatematsu S. Hatakeyama S. Ozono K. Kubodera N. Reddy G.S. Okano T. J. Biol. Chem. 2003; 278: 1463-1471Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 20Kamao M. Tatematsu S. Reddy G.S. Hatakeyama S. Sugiura M. Ohashi N. Kubodera N. Okano T. J. Nutr. Sci. Vitaminol. 2001; 47: 108-115Crossref PubMed Scopus (22) Google Scholar, 27Masuda S. Byford V. Kremer R. Makin H.L.J. Kubodera N. Nishii Y. Okazaki A. Okano T. Kobayashi T. Jones G. J. Biol. Chem. 1996; 271: 8700-8708Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Transfection and Luciferase Activity Assay—MG-63 was maintained in Dulbecco's modified Eagle's medium supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml), and 10% dextran-coated charcoal-treated FCS. Cells (2 × 105) were suspended in 2 ml of the medium and transfected with 0.5 μg of luciferase reporter plasmid (pGVB2 vector, Toyo Ink Co., Ltd., Tokyo, Japan) containing a human osteocalcin gene promoter (–848/+10) including a VDRE (29Ozono K. Liao J. Kerner S.A. Scoot R.A. Pike J.W. J. Biol. Chem. 1990; 265: 21881-21888Abstract Full Text PDF PubMed Google Scholar) or a rat cyp24 gene promoter (–291/+9) including two VDREs (30Ohyama Y. Ozono K. Uchida M. M. T. T. Yamamoto J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) and μg of (pGVB2 vector, Toyo Ink Co., as an internal control using The cells were incubated with 1α,25(OH)2D3 or the vitamin D3 compounds for 48 h. The luciferase activities of the cell were measured with a luciferase assay system Ink Co., according to the measured as luciferase activity was with the luciferase activity of the same cells determined with the luciferase assay system as a control Ink Co., M. Sci. S. A. PubMed Scopus Google Scholar). Activity Assay—The human leukemia cells were kindly provided by M. Cells were cultured at 37 °C in medium Co., supplemented with 10% FCS and For flow cells were in culture and cultured for 3 days with 1α,25(OH)2D3 or the vitamin D3 compounds group of cells was washed with and in containing and and incubated at 37 °C for 1 h. Cells were washed with and incubated with 0.5 ml of containing at 4 °C for 20 The cells were with a flow equipped with an nm, and the cell was using Analysis of Cell the analysis of cell expression of cells were cultured for 3 days with 1α,25(OH)2D3 or the vitamin D3 compounds under the same conditions as for flow group of cells was washed with and the cells (2 × were in μl of containing 1% bovine serum albumin and 1% sodium Then the cells were incubated with 10 μl of human for at The cells were washed with and then in μl of containing was detected on a at an of and of were as the which is the of the and the with cells Metabolism of 25(OH)D3 in UMR 106 of 10 μm of 25(OH)D3 with UMR 106 cells for 48 from the media with the cells were to a HPLC using a Zorbax SIL column The major peak 2 to the of 25(OH)D3 time and 3-epi-25(OH)D3 was in a 5 and The was then to a HPLC using a Sumichiral OA-2000 column for the of 3-epi-25(OH)D3 from 25(OH)D3 The peak which the UV of vitamin D3 228 nm, 265 the same retention time as 3-epi-25(OH)D3 but a different retention time to 25(OH)D3. The metabolite to peak was not when 25(OH)D3 was incubated with the medium It is from the in 1 that peak is the major metabolite of 25(OH)D3 produced in UMR 106 was in to by 1H NMR and LC-MS which the UV of D3 228 nm, was also observed in a control with cells, only 4 in the HPLC system to of 24,25(OH)2D3 and 3-epi-24,25(OH)2D3 was and as described previously (20Kamao M. Tatematsu S. Reddy G.S. Hatakeyama S. Sugiura M. Ohashi N. Kubodera N. Okano T. J. Nutr. Sci. Vitaminol. 2001; 47: 108-115Crossref PubMed Scopus (22) Google Scholar). 24,25(OH)2D3 was contained in this 3-epi-24,25(OH)2D3 was not detected not of 3-Epi-25(OH)D3 by 1H NMR and LC-MS Analyses—The 1H based on and two-dimensional NOESY spectra of synthetic and the 25(OH)D3 metabolite in I. In the 1H NMR the most 25(OH)D3 and 3-epi-25(OH)D3 was the of 3-epi-25(OH)D3, The of of peak was observed at the same as 3-epi-25(OH)D3 in with 25(OH)D3. an to be for the C-3 epimerization in the A-ring of 25(OH)D3. The from the were of an unchanged side chain as well as the cis-triene In the LC-MS spectra of 25(OH)D3 and 3-epi-25(OH)D3, and were observed at and In the of peak and were also observed at and peak was considered to be a or a of 25(OH)D3. From the results of HPLC, 1H and LC-MS peak be as 3-epi-25(OH)D3, a of a hydroxyl group at C-3 of the A-ring of NMR and LC-MS of 25(OH)D3 NMR 1H and in in (1H, = (1H, (1H, (1H, = (1H, = (1H, = (1H, = (1H, (1H, (1H, = (1H, (1H, (1H, = (1H, = (1H, = (1H, = (1H, (1H, metabolite (1H, = (1H, (1H, (1H, = (1H, = (1H, = (1H, = (1H, (1H, Chemical in in