Kinetic study of cytochrome P450 3A4 activity on warfarin by capillary electrophoresis with fluorescence detection.
The use of capillary electrophoresis (CE) for the determination of cytochrome P450 3A4 (CYP3A4) activity with R-warfarin as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, 10-hydroxywarfarin, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 microm i.d., 60 cm effective length); 50 mM sodium phosphate buffer (pH 6.5); 23 kV (90 microA) applied voltage; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 37 degrees C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of 10-hydroxywarfarin from R-warfarin in the presence of CYP3A4 were calculated to be 166 +/- 12 microM and 713 +/- 14 pmol/min/nmol (or 91.4 pmol/min/mg) CYP3A4, respectively.
Zhang
Jie
J
Laboratory of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmaceutical Sciences, K. U. Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium.
Ha
Pham Thi Thanh
PT
Lou
Yijia
Y
Hoogmartens
Jos a
Ja
Van Schepdael
Ann
A
D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007700:Kinetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014859:Warfarin / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*
D014859:Warfarin; C065719:10-hydroxywarfarin; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human