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.
Chiba
K
K
Division of Clinical Pharmacology, National Medical Center, Tokyo, Japan.
Manabe
K
K
Kobayashi
K
K
Takayama
Y
Y
Tani
M
M
Ishizaki
T
T
D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D006801:Humans; D008617:Mephenytoin / Q000031:analogs & derivatives / Q000032:analysis / Q000493:pharmacokinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis*; D010084:Oxidation-Reduction; D011110:Polymorphism, Genetic
C025687:4-hydroxymephenytoin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D008617:Mephenytoin