以右美沙芬为探针测定大鼠肝微粒体中CYP2D6酶活性及动力学分析

Determination of CYP2D6 activity in rat liver microsomes and study on its kinetics with dextromethorphan probe in vitro

目的建立大鼠肝微粒体中细胞色素P450(CYP)2D6酶活性的检测方法,以右美沙芬为探针底物进行体外酶动力学分析,为相关药物代谢研究提供参考依据。方法采用高效液相色谱-荧光检测(HPLC-FLD)方法提高检测灵敏度。通过优化大鼠肝微粒体孵育体系的反应条件,建立稳定的动力学评价方法。以Graphpad prism 5.01软件绘制米曼动力学曲线并计算V_(max)与K_m值。结果右美沙芬与其代谢产物右啡烷分离良好且无其他内源性物质干扰。右啡烷检测限为5 nmol·L^(-1)(S/N>3),定量下限为0.015μmol·L^(-1),线性范围为0.015~7.5μmol·L^(-1)。测定方法重现性好且稳定。经优化条件后测定,不同浓度的右美沙芬在0.2 mg·mL^(-1)蛋白浓度下,孵育10 min,测得动力学参数V_(max)为(1.075±0.060)nmol·min^(-1)·mg^(-1)pro,K_m为(7.470±0.983)μmol·L^(-1)。结论高效液相色谱结合荧光检测法可以有效提高灵敏度,此大鼠微粒体孵育体系的建立可应用于体外CYP2D6酶活性的测定及酶动力学研究。

AIM To establish a method for measuring CYP2D6 activity in rat liver microsomes, and investigate the enzyme kenetics of CYP2D6 using dextromethorphan as a probe- substrate in order to better understand of the relevant drug metabolism. METHODS Analytes were examined by high- performance liquid chromatography with fluorescence detector （HPLC- FLD） in order to improve the detection sensitivity. By optimizing the reaction conditions of incubation system for rat liver microsomes, a method for evaluating CYP2D6 activity and enzyme kinetics was established. The kinetic parameters, V/max and Km were calculated byGraphpad prism 5.01. RESULTS Dextromethorphan and its metabolites （dextrorphan） were separated well without endogenous substances interference. The detectable limit of dextrorphan was 5 μmol· L-1 （S/N 〉 3） and the quantitative lower limit was 0.015 μmol· L-1. The linear range of calibration curve was from 0.015 μmol·L-1 to 7.5 μmol·L-1. The reproducibility and stability of this method were reliable. The kinetic parameters, V/max and Km were （ 1.075 ± 0.060） nmol·min-1 mg-l pro and （7.470 ± 0.983） μmol·L-i, respectively. CONCLUSION The high-performance liquid chromatography with fluorescence detection can effectively improve the sensitivity. Moreover, the rat microsomal incubation system can be applied for CYP2D6 activity measurement and enzyme kinetics studies in vitro.