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

Abstract

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 less sensitive than that for 25-hydroxyvitamin D₃, these experiments do not rule out the presence of some 25-hydroxylase activity towards vitamin D₂ in the microsomes. The results are discussed in relation to previous work in which a lower toxicity has been reported for vitamin D₂ than for vitamin D₃ in some mammalian species.

Key Words:

• human liver
• metabolism
• microsomal cytochrome P-450
• mitochondria
• vitamin D₂
• vitamin D₃