25-Hydroxylase activity in subcellular fractions from human liver. Evidence for different rates of mitochondrial hydroxylation of vitamin D2 and D3

25-Hydroxylation of vitamin D2 and D3 was studied in subcellular fractions from human liver, using a technique based on isotope dilution-mass spectrometry. The mitochondrial fraction fortified with isocitrate catalysed 25-hydroxylation of vitamin D3 at a rate of about 10 pmol/mg protein xmin. Under the same conditions, the rate of 25-hydroxylation of vitamin D2 was less than 2 pmol/mg protein × min. Crude microsomes fortified with NADPH catalysed 25-hydroxylation of vitamin D3 to a very low extent, and this activity was not linear with the amount of microsomal protein. A higher rate of conversion was obtained with a partially purified cytochrome P-450 fraction in the presence of NADPH-cytochrome P-450 reductase and NADPH. This fraction also catalysed 25-hydroxylation of 1α-hydroxyvitamin D3 and 5β-cholestane-3α, 7α, 12α-triol. 25-Hydroxylation of vitamin D2 could not be detected, neither with crude microsomes, nor with the microsomal cytochrome P-450 fraction. Since the assay for 25-hydroxyvitamin D2 was ...