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

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.