Identification of 25,26-dihydroxyvitamin D3 as a rat renal 25-hydroxyvitamin D3 metabolite

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Abstract
25,26-Dihydroxyvitamin D3 [25,26-(OH)2D3] was unequivocally identified as a major renal microsomal metabolite of 25-hydroxyvitamin D3 in rats fed a vitamin D sufficient diet. The structural assignment was based on a comparison of the high-performance liquid chromatograms of synthetic and in vitro generated 25,26-(OH)2D3 through 4 different systems, the UV absorbance and mass spectral characteristics of biological 25,26-(OH)2D3, and the chromatographic and mass spectral characteristics of the sodium metaperiodate cleavage product of the metabolite. The enzymic synthesis of 25,26-(OH)2D3 was inhibited 60-80% by a semipurified goat anti-rat NADPH-cytochrome P-450 reductase. This implicates cytochrome P-450 as the probable terminal oxidase of the 25-hydroxyvitamin D3-26-hydroxylase system. The methodology used to assay rat renal 25-OH-D3 hydroxylases is discussed.

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