Development and preliminary application of a simple assay of S-mephenytoin 4-hydroxylase activity in human liver microsomes

Eur J Clin Pharmacol. 1993;44(6):559-62. doi: 10.1007/BF02440859.


We have developed a simple HPLC assay to measure the activity of S-mephenytoin 4-hydroxylase in human liver microsomes, and have assessed its practical applicability by determining the kinetic parameters of the enzyme in 10 different human liver samples. The recovery of 4-hydroxymephenytoin and phenobarbital (the internal standard) after the precipitation of microsomal protein was almost complete, and the coefficients of variation for the intra- and interassay measurement of S-mephenytoin 4-hydroxylase activity were < 6.4 and 8.0%, respectively. Eadie-Hofstee plots for the formation of 4-hydroxymephenytoin gave a straight line for all of the 10 samples studied. There was large interindividual variability in the kinetic parameters estimated: 4.6- (36 to 166 microM), 11.8- (0.9 to 10.6 nmole/mg protein/h) and 30.1- times (0.10 to 3.01 microliters/mg protein/min) for Km, Vmax and Vmax/Km, respectively. The mean (+/- SD) Km, Vmax and Vmax/Km were 72.4 +/- 40.4 microM, 4.23 +/- 2.88 nmole/mg protein/h and 1.33 +/- 1.02 microliters/mg protein/min, respectively. Thus, the assay was sufficiently accurate and reproducible to permit estimation of the kinetic parameters of S-mephenytoin 4-hydroxylase in human liver microsomes, and it appears to be applicable to an in vitro study of the possible involvement of S-mephenytoin-type oxidation polymorphism in drug metabolism.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aryl Hydrocarbon Hydroxylases*
  • Chromatography, High Pressure Liquid / methods*
  • Cytochrome P-450 CYP2C19
  • Cytochrome P-450 Enzyme System / analysis*
  • Humans
  • Mephenytoin / analogs & derivatives
  • Mephenytoin / analysis
  • Mephenytoin / pharmacokinetics
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / analysis*
  • Oxidation-Reduction
  • Polymorphism, Genetic


  • 4-hydroxymephenytoin
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C19 protein, human
  • Cytochrome P-450 CYP2C19
  • Mephenytoin