Investigation of C-3 Epimerization Mechanism of 24,25-Dihydroxyvitamin D₃ in Rat Using Liquid Chromatography/Mass Spectrometry

Abstract

Studies on C-3 epimerization of 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] were performed. 3-Epi-24,25(OH)₂D₃ was found to circulate in the blood of rats administered 24,25(OH)₂D₃, which was confirmed in comparison with a standard sample based on its chromatographic behavior during inclusion high-performance liquid chromatography using γ-cyclodextrinas a mobile phase additive and data obtained from liquid chromatography (LC)/mass spectrometry (MS) and gas chromatography/MS. The C-3 epimerization mechanism was investigated in in vitro experiments using [3α-²H]-24,25(OH)₂D₃. The disappearance of deuterium was monitored by LC/MS. As a result, it was clarified that the 3-epimer was formed via the 3-oxo-form as an intermediate. This epimerization was also found to be controlled by the enzyme existing in the liver cytosol fraction and to require NAD and NADPH as coenzymes.