Assay of mephenytoin metabolism in human liver microsomes by high-performance liquid chromatography

Abstract

The metabolism of mephenytoin to its two major metabolites, 4-OH-mephenytoin (4-OH-M) and 5-phenyl-5-ethylhydantoin (nirvanol) was studied in human liver microsomes by a reversed phase HPLC assay. Because of preferential hydroxylation of S-mephenytoin in vivo, microsomes (5–300 μg protein) were incubated separately with S- and R-mephenytoin. After addition of phenobarbital as internal standard, the incubation mixture was extracted with dichloromethane. The residue remaining after evaporation was dissolved in water and injected on a 60 × 4.6-mm reversed-phase column (5 μ-C-18). Elution with acetonitrile/methanol/sodium perchlorate (20 m m, pH 2.5) led to almost baseline separation of mephenytoin, metabolites, and phenobarbital. Quantitation was performed by uv-absorption at 204 nm by the internal standard method. Propylene glycol was found to be the best solvent for mephenytoin, but inhibited the reaction noncompetitively. 4-OH-M and nirvanol could be detected at concentrations in the incubation mixture as low as 40 and 80 n m, respectively. The rates of metabolite formation were linear with time and protein concentration. The reaction was found to be substrate stereoselective. At substrate concentrations below 0.5 mm S-mephenytoin was preferentially hydroxylated to 4-OH-M, while R-mephenytoin was preferentially demethylated to nirvanol at all substrate concentrations tested (25–1600 μ m). These data provide a mechanistic explanation for the stereospecific pharmacokinetics in vivo. The dependence of both metabolic reations on NADPH and the inhibition by CO suggest that they are mediated by cytochrome P-450-type monooxygenases. These data, together with the recent discovery of a genetic polymorphism causing an exclusive deficiency of the 4-hydroxylation of S-mephenytoin, suggest that hydroxylation and demethylation of S-mephenytoin are catalyzed by different cytochrome P-450 isozymes.