A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

Anal Biochem. 1985 May 15;147(1):174-9. doi: 10.1016/0003-2697(85)90024-7.


A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • Catalysis
  • Chromatography, Thin Layer
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 Enzyme System / metabolism*
  • Humans
  • Kinetics
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / metabolism*
  • Oxidoreductases / metabolism


  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Oxidoreductases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C19 protein, human
  • Cytochrome P-450 CYP1A2
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 CYP2D6