Side chain metabolism of vitamin D3 in osteosarcoma cell line UMR-106. Characterization of products.

Abstract

Previous work has shown that 25-hydroxyvitamin D3 (25-OH-D3) and 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) may be metabolized in the mammalian kidney through a side chain oxidation pathway resulting in C23-C24 cleavage, yielding 24,25,26,27-tetranor-23-OH-D3. In the present study, we have used UMR-106 clonal osteoblast cells to demonstrate that products of the side chain oxidation pathway are produced by an osteoblast-like cell. Cells cultured on microcarrier beads and incubated in the presence of pharmacological levels of substrate (1.4 microM, either 25-OH-D3 or 1,25-(OH)2D3) produced sufficient quantities of metabolite to allow identification through mass spectrometry. In addition, putative metabolites were identified through comigration with authentic standards on three high pressure liquid chromatography systems, chemical modification by NaBH4 and periodate, and UV spectral characterization. The pathway was undetectable unless the cells had been exposed to 1,25-(OH)2D3 prior to incubation with substrate. We have shown that 1,25-(OH)2D3 induces the 24-hydroxylase and perhaps also the other enzymes of this pathway in the bone cell. Although we used pharmacological concentrations of substrate to demonstrate the existence of the side chain oxidation pathway in bone cells, physiological levels of 25-OH-D3 or 1,25-(OH)2D3 were also metabolized through the pathway, at least as far as the penultimate product. We speculate that the side chain oxidation pathway may be ubiquitous among vitamin D target tissues.