Discovery and Structural Analysis of Metabolites of Vitamin D₃ Lactones

25-Hydroxyvitamin D₃-23,26-lactone (1) and 1α,25-dihydroxyvitamin D₃-23,26-lactone (2) have long been considered as among the end metabolites of vitamin D₃. Recently, however, we found that these lactones exhibit biological activity related to the β-oxidation of fatty acids. We hypothesized that a metabolic pathway might exist to inactivate their physiological activity. Here, by means of metabolic experiments with a variety of cytochrome P450 (CYP) enzymes, we show that CYP3A4 metabolizes the lactones. The metabolites were presumed to be hydroxylated at C4 based on the previous reports showing that metabolism of 25-hydroxyvitamin D₃ by CYP3A4 along with the current LC-MS analysis. To confirm this, we chemically synthesized 4α,25(OH)₂D₃-23,26-lactone (3), 4β,25(OH)₂D₃-23,26-lactone (4), 1α,4α,25(OH)₃D₃-23,26-lactone (5), and 1α,4β,25(OH)₃D₃-23,26-lactone (6). HPLC analysis using these authentic compounds as standards revealed that 1 was metabolized to 3 and 4, while 2 was metabolized exclusively to 6 by CYP3A4. Docking studies suggest that the hydroxyl group at C1 in 2 forms hydrogen bonds with Ser119 and Arg212 of CYP3A4, contributing to the fixation of C4β on heme iron in the CYP, thereby resulting in stereoselective hydroxylation at C4.