CYP2Cl g-Mediated (S)-Mephenytoin 4-Hydroxylation Assayed by High-Performance Liquid Chromatography with Radiometric Detection

CYP2C 19 is one of at least three CYP2C enzymes expressed in human hver (
1
,
2
), although the abundance of this P450 is generally less than that of etther CYP2C8 or CYP2C9 (
3
). Considerable interindividual differences occur in hepatic CYP2C19 content (
1
,
2
), primarily owmg to a genetic polymorphism in this enzyme (4). Approximately 15-20% of Orientals and 3% of Caucasians exhibit the CYP2C19 poor-metabolizer phenotype, which is associated with at least two mutant alleles, designated CYP2C 19ml and CYP2C 19m2 (
4
). Studies with c
DNA
-expressed CYP2C 19 have identified several drugs as substrates for this polymorphically expressed P450, including omeprazole (
5
,
6
), diazepam (
7
), cyclophosphamlde (
3
), and lfosfamide (
3
). However, the precise role of human hepatic CYP2C19 in drug biotransformation is poorly understood because mhibitory monospecific antiCYP2C19 antibodies are not available and a CYP2C19-specific chemical inhibitor has yet to be identified. Recent correlational analyses have led to the conclusion that CYP2C19 is a major P450 responsible for (S)-mephenytoin 4’-hydroxylation activity in human liver microsomes (
1
,
2
). This is supported by the finding that
cDNA
-expressed CYP2C19 has a 50-to lOO-fold greater turnover number than CYP2C8, CYP2C9, or CYP2C 18 for (S)-mephenytoin 4−hydroxylatlon (
2
). As a result, (S)-mephenytom 4’-hydroxylase activity is frequently used as a catalytic monitor for human hepatic CYP2C19. In addmon, (S)-mephenytom 4’-hydroxylation is a useful indicator of the catalytic activity of cDNA-expressed CYP2C 19 (
2
). Varrous analytical methods have been developed to measure the enzymatic formation of 4’-hydroxymephenytoin, including isocratrc, reversed-phase hrgh performance hqutd chromatography (
8
). This chapter describes a method for the determination of (S)-mephenytoin 4’-hydroxylation by gradient, reversedphase high performance llqurd chromatography using
14
C-labeled substrate and radiometrrc detection.