Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis

Abstract

An electrophoretically mediated microanalysis method for the determination of CYP3A4 activity using testosterone and nifedipine as substrates was developed. Initially, the enzymatic reaction was performed off-line and the samples were subsequently injected into the capillary by pressure. The CYP3A4 activity was determined by quantitation of the reactant cofactor, NADPH. To further optimize, speed-up and miniaturize the enzyme assay, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product cofactor, NADP, employing the plug-plug mode of electrophoretically mediated microanalysis. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 260 nm. This setup resulted in a fully automated assay, which can be carried out in less than 35 min. Using the Lineweaver-Burk equation, the Michaelis constants (K(m)) for the oxidation of testosterone and nifedipine by CYP3A4 were calculated to be 58.6+/-8.3 and 19.1+/-2.4 microM, respectively, which are consistent with off-line assay and previously reported values.