Isolation, Identification and Biological Activity of 24R, 25-Dihydroxy-3-epi vitamin D₃: A Novel Metabolite of 24R, 25-Dihydroxyvitamin D₃ Produced in Rat Osteosarcoma Cells (UMR 106)

Abstract

We recently identified 1α, 25-dihydroxy-3-epi-vitamin D₃ [1 α, 25(OH)₂-3-epi-D₃] as a metabolite of 1 α, 25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] produced in rat osteosarcoma cells (UMR 106). We now report the isolation of 24R, 25-dihydroxy-3-epi-vitamin D₃ [24R, 25 (OH)₂-3-epi-D₃] as a metabolite of 24R, 25-dihydroxyvitamin D₃ [24R, 25(OH)₂D₃] by high-performance liquid chromatography (HPLC) with chiral column and its structure assignment by proton nuclear magnetic resonance (¹H-NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. We also demonstrated the production of 24R, 25(OH)₂-3-epi-D₃ in two other cell lines [human colon carcinoma cells (Caco-2) and porcine kidney cells (LLC-PK₁)] which were previously shown to convert 1 α, 25(OH)₂D₃ into 1 α, 25 (OH)₂-3-epi-D₃. It can be seen that the production of 24R, 25 (OH)₂-3-epi-D₃ from 24R, 25(OH)₂D₃ is lower than that of 1 α, 25 (OH)₂-3-epi-D₃ from 1α, 25(OH)₂D₃ in all the cells studied. 24R, 25(OH)₂-3-epi-D₃ was found to be inactive in terms of its ability to bind to the vitamin D receptor (UDR), in inhibiting proliferation and in inducing differentiation of human promyelocytic leukemia cells (HL-60). Thus, our study indicates that the C-3 epimerization pathway is common to both lα, 25(OH)₂D₃ and 24R, 25(OH)₂D₃ and may play an important role in modulating the concentration and the biological activity of these two major vitamin D₃ metabolites in target tissues.