Analysis of C-3 epimerization in (24R)-24,25-dihydroxyvitamin D3 catalyzed by hydroxysteroid dehydrogenase

Studies on the C-3 epimerization in (24R)-24,25-dihydroxyvitamin D3 [24R,25(OH)2D3] were performed using hydroxysteroid dehydrogenases (HSDs). 3-Epi-24R,25(OH)2D3 was formed from 24R,25(OH)2D3 by the catalysis of 3α- or β-HSD. These HSDs also catalyzed the C-3 epimerization in 3-epi-24R,25(OH)2D3 to form 24R,25(OH)2D3. 24R,25(OH)2D3 and its C-3 epimer were separated by inclusion high-performance liquid chromatography using γ-cyclodextrin (γ-CD) as the mobile phase additive or a γ-CD bonded chiral column. The product derived from the intermediate during the C-3 epimerization was isolated from the incubation specimens and identified as (7Z)-(24R)-24,25-dihydroxy-9,10-secocholesta-4,7,10(19)-trien-3-one by several instrumental analyses including 1H-nuclear magnetic resonance spectrometry. The occurrence of this compound strongly proves that the formation of the C-3 epimer by HSD involves a dehydrogenation process. The present study suggests that HSDs may catalyze the C-3 epimerization of vitamin D compounds and modulate their concentrations and biological activities in animals and humans.