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

Biochem J. 1988 Mar 1;250(2):527-32. doi: 10.1042/bj2500527.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcitriol / pharmacology*
  • Carbon Monoxide / pharmacology
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / pharmacology
  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 Enzyme System*
  • Dihydroxycholecalciferols / metabolism*
  • Fasting
  • Gas Chromatography-Mass Spectrometry
  • Guinea Pigs
  • Hydroxylation
  • Kidney Cortex / drug effects
  • Kidney Cortex / enzymology*
  • Male
  • Mitochondria / drug effects
  • Mitochondria / enzymology
  • Oxidation-Reduction
  • Steroid Hydroxylases / metabolism*
  • Vitamin D3 24-Hydroxylase

Substances

  • Dihydroxycholecalciferols
  • Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone
  • 23,25-dihydroxyvitamin D3
  • Carbon Monoxide
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Vitamin D3 24-Hydroxylase
  • Calcitriol