HPLC法同时测定大鼠CYP2B、2C、2E酶活性及其应用

Determination of CYP2B,2C,2E activities by high performance liquid chromatography and its application

建立同时测定大鼠肝微粒体中CYP2B、2C、2E酶活性的HPLC法,并用该方法评价氯胺酮对这3种酶的影响。以盐酸安非他酮、甲苯磺丁脲、氯唑沙宗作为CYP2B、CYP2C、CYP2E的特异性底物。以非那西丁为内标,色谱柱为Welchrom C18,流动相为甲醇:0.01 mol/L乙酸铵(pH 5.0)=55∶45,流速为1 mL/min,检测波长为220,247,250,280 nm,柱温为30℃,进样量为40μL。体外孵育反应的蛋白浓度为3.75mg/mL,孵育时间为30min。采用上述方法测定3种底物在对照组和氯胺酮给药组中的代谢速率,结果显示,各底物在线性范围内线性关系良好,相关系数r】0.9991;日内RSD【12.1%,日间RSD【12.5%;方法回收率为84.4%~114.3%,提取回收率为93.65%~100.7%。本方法灵敏、准确、简单,可用于大鼠肝微粒体中CYP2B、2C、2E酶活性的测定。

The HPLC method was established to determine CYP2B,2C,2E activities in rat liver microsome.And the effect of ketamine on CYP2B,2C,2E was evaluated.Bupropion,tolbutamide and chlorzoxazone were employed as the substrates of CYP2B,2C,2E,respectively.The determination was performed on Welchrome C18 column with phenacetin as an internal standard.The mobile phase was methanol:0.01mol/L ammonium acetate(pH 5.0) =55 ∶45.The detection wavelengths were set at 220,247,250,280 nm,the column temperature was 30 ℃ and the injection volume was 40 μL.The substrates' metabolic velocities after incubating with 3.75 mg/mL of liver microsomes for 30 min were determined from the control group and the ketamine group.The results showed that the substrates were linearity within their linearity ranges with the correlation coefficients greater than 0.999 1.The intra-and inter-assay precisions of CYPs substrates were less than 12.1%and 12.5%,the assay recoveries were between 84.4% and 114.3%,and the extract recoveries were93.65%-100.7%.This method is simple,sensitive,accurate and suitable for determining CYP2B,2C,2E activities in rat liver microsome in vitro.