Assay and properties of 25-hydroxyvitamin D3 23-hydroxylase. Evidence that 23,25-dihydroxyvitamin D3 is a major metabolite in 1,25-dihydroxyvitamin D3-treated or fasted guinea pigs.

Incubation of 25-hydroxyvitamin D3 with kidney cortex mitochondria from 1,25-dihydroxyvitamin D3-treated guinea pigs resulted in the formation of 23,25-dihydroxyvitamin D3 as the major product. The identity of the product was verified by g.c.-m.s. and quantification was performed by h.p.l.c. The rates of the reaction were in the range 1.0-1.8 pmol/min per mg of mitochondrial protein (at 37 degrees C), which were 5-10 times the rates of formation of 24,25-dihydroxyvitamin D3. In mitochondrial preparations from untreated guinea pigs, the rate of 23-hydroxylation was below detection limit (0.02 pmol/min per mg of mitochondrial protein). Fasting the animals for 24 h induced the 23-hydroxylase almost as efficiently as treatment with 1,25-dihydroxyvitamin D3, with a concomitant depression of the 1 alpha-hydroxylase. The 23-hydroxylase reaction required oxidizable substrate, was decreased by low O2 partial pressures and inhibited by CO or the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It was stimulated by the respiratory-chain inhibitors rotenone, antimycin A and KCN. These results indicate that the guinea-pig renal mitochondrial 23-hydroxylase is a cytochrome P-450 and that the reducing equivalents are primarily supplied by NADPH via the energy-dependent transhydrogenase.