24R ,25(OH) 2 D 3 ] is one of the major metabolites of vitamin D 3 , which is being examined as an anti-osteoporosis medicine 1 , and much interest is focused on the metabolism of 24R,25(OH) 2 D 3 . In a previous paper of this series, we reported the in vitro glucuronidation of 24R,25(OH) 2 D 3 using rat liver microsome as an enzyme source, in which its 3-and 24-glucuronides (G) were formed as the main products together with a small amount of the corresponding 25G. 2 On the other hand, we also reported that substrate specificity was observed between the in vitro glucuronidation of 25-hydroxyvitamin D 3 [25(OH)D 3 ] and that of its pro-form. 3 The former and the latter gave 3G and 25G as the main product, respectively, which means that glucuronidation at the sterically hindered tert-hydroxy group occurred mainly on the pro-form.In the present work, we examined whether this specificity was also observed on 24R,25(OH) 2 D 3 and its proform [24R,25(OH) 2 proD 3 ]. Furthermore, the in vitro glucuronidation of the stereoisomer, 24S,25(OH) 2 D 3 , was compared with that of 24R,25(OH) 2 D 3 ( Fig. 1).
Experimental
Materials and reagents24R,25(OH) 2 D 3 was obtained from Duphar (Amsterdam, The Netherlands) and 24R,25(OH) 2 proD 3 was synthesized in our laboratories by a known method.4 24S,25(OH) 2 D 3 was supplied by Kureha Chemical Co. (Tokyo, Japan). 24R,25(OH) 2 D 3 G 2 and 24R,25(OH) 2 proD 3 G (-3G, -24G and -25G) were synthesized in our laboratories; the syntheses of the latter are described below. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was synthesized from 4-phenylurazole (Nacalai Tesque, Kyoto, Japan) and purified by sublimation prior to use.5 Isolute C18 (EC) cartridges (500 mg: International Sorbent Tech., Hengoed, UK) were obtained from Uniflex (Tokyo), and piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20) 6 was prepared in our laboratories. The other reagents and solvents were commercially available and of analytical grade.
InstrumentsHigh-performance liquid chromatography (HPLC) was performed on a Shimadzu LC-10AT chromatograph (Kyoto) equipped with a Shimadzu SPD-10A UV (265 nm) or a Shimadzu SPD-M6A photodiode array UV (200 -340 nm) detector. J'sphere ODS-H80 (4 µm, 150×4.6 mm i.d.) and J'sphere ODS-M80 (4 µm, 150×4.6 mm i.d.) (YMC, Kyoto) columns were used at a flow rate of 1 ml/min at 30˚C. The pH of the mobile phase containing AcONH 4 or NaClO 4 was adjusted to the desired values with AcOH or HClO 4 , respectively.Liquid chromatography/tandem mass spectrometry (LC/MS n ) was performed using a Finnigan MAT LCQ (San Jose, CA, USA) connected to a JASCO PU-980 (Tokyo) chromatograph and electrospray ionization (ESI) was used. The following conditions were used for the analysis. The spray needle voltage was 5 kV; the heated capillary temperature, the sheath gas flow rate and the auxiliary gas were set at 200˚C, 70 units and 20 units, respectively. The capillary voltage was 1 or -1 V and the tube lens offset was 10 or -10 V in the positive-or the negative-ion mode, respectively. A YMC-Pack Pro C18 (5 µ...