高效液相色谱法测定鼠肝微粒体中CYP1A2酶的活性及动力学考察

HPLC determination of activity of CYP1A2 and its pharmacokinetics in rat liver microsomes

目的:建立以非那西丁为探针的高效液相色谱-紫外检测的实验方法,测定大鼠肝微粒体中CYP1A2酶活性并对其进行动力学考察。方法:采用Shimadzu Shim-Pack VP-ODS柱(150 mm×4.6 mm,5μm),流动相为:100 mmol·L-1磷酸二氢钠缓冲液(pH 4.3)和乙腈,梯度洗脱,流速为1.0 mL·min-1,柱温为室温,检测波长245 nm。非那西丁与大鼠肝微粒体在37℃温孵60 min,加入冰甲醇终止,12000 r.min-1离心10 min,取上清进行HPLC分析,以Lineweaver-Burk作图计算Vmax与Km值。结果:非那西丁、对乙酰氨基酚及其内标间乙酰氨基酚三者分离良好且无内源性干扰。对乙酰氨基酚最低检测限50 nmol·L-1,线性范围0.1～10μmol·L-1。日内日间精密度均小于10%。回收率大于75%。动力学考察表明选择甲醇作为终止试剂效果较好,非那西丁在0.2 mg·mL-1大鼠肝微粒体体系中孵育60 min,测得动力学参数Vmax为0.21 nmol·min-1.mgprotein-1,Km为20.39μmol·L-1。结论:该方法稳定,结果能准确地反映CYP1A2酶的活性,可以用于相关动力学研究。

Objective：To establish a method evaluates cytochrome p450 1A2 （CYP1A2） activity using phenacetin as a probe by high - performance liquid chromatography （ HPLC ） - UV detection. Methods： Column for the Shi- madzu Shim- Pack VPODS （150 mm 4. 6 ram, 5 μm） and mobile phase of 100 mmol·L-L phosphate buffer （pH 4. 3 ） -acetonitrile were used. Detection wavelength was 245 nm. Phenacetin was incubated with rat liver microsomes at 37℃ for 60 min and the reaction was stopped by cold methanol. The reactive liquid was centrifuged at 12000 r min - 1 for 10 min. Finally, the supernatant was analyzed by HPLC. Results： Phenacetin, acetamidophenol and 3 - acetamidophenol were perfectly separated. The detection limit of phenacetin was 50 nmol L- and the linear range of method was 0. 1 μmol L-1 to 10 μmol L-1 The intraday and interday relative standard deviations were less than 10% respectively. The method recoveries were more than 75%. The methanol was selected as reagent to terminate the reaction catalyzed by CYP1 A2 and the incubation time was 60 rain. The kinetic parameters was shown that Vmax was 0. 21 nmol min-1 mg protein-1 and Km was 20. 39 μmol L-1. Conclusions：The method is stead- y,accurate and suitable for assaying CYP1A2 activity which can be used to evaluate the pharmacokinetics of CYP1 A2 in rat liver microsomes.