Differential Metabolic Stability of 4α,25- and 4β,25-Dihydroxyvitamin D and Identification of Their Metabolites.

Vitamin D () is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D () and 4β,25-dihydroxyvitamin D () are both produced from 25-hydroxyvitamin D () by CYP3A4. However, is detectable in serum, whereas is not. We hypothesized that the reason for this is a difference in the susceptibility of and to CYP24A1-mediated metabolism. Here, we synthesized and , and confirmed that has greater metabolic stability than . We also identified 4α,24,25- and 4β,24,25-trihydroxyvitamin D ( and ) as metabolites of and , respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the β-hydroxy group at C4 contributes to the greater metabolic stability of by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1.