Ring hydroxylation of 25-hydroxycholecalciferol by rat renal microsomes

Two metabolites have been isolated from rat renal microsomes incubated with 25-hydroxycholecalciferol. Postmitochondrial supernatant fractions from kidneys of thyroidectomized and parathyroidectomized rats were incubated with magnesium acetate, potassium acetate, an NADPH generating system, and 25-hydroxycholecalciferol at a level of 20 μg/ml postmitochondrial supernatant for 60 min at 30°C. Lipid extracts of the incubation mixtures were purified by silica gel TLC and HPLC. Two peaks were obtained. Metabolite x 2 eluted at 18 min and metabolite x 1 at 23 min when chromatographed on a silica column developed with hexane-isopropanol. Metabolites x 1 and x 2 were found to have maximal absorbance at 265 nm. Both metabolites were periodate sensitive, indicating vicinal hydroxyl groups. Mass spectral analysis of metabolite x 2, which was isolated in greater quantity than metabolite x 1, indicates that metabolite x 2 had resulted from hydroxylation of the A ring. Results indicate that 25-hydroxycholecalciferol is hydroxylated on carbon 2 or carbon 4 by renal microsomes. Metabolites x 1 and x 2, because of similarity in Chromatographic migration and periodate sensitivity, are, perhaps, isomers or 2- and 4-hydroxylated metabolites.