title: 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.
authors.affiliation: Laboratory for Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Jos
authors.initials: J
authors.lastname: Hoogmartens
authors.forename: Ann
authors.initials: A
authors.lastname: Van Schepdael
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009543:Nifedipine / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D013739:Testosterone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D009543:Nifedipine