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

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,5 μm),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V_max与K_m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0.1~10 μmol·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V_max为0.21 nmol·min^-1·mg protein^-1,K_m 为 20.39 μmol·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 Shimadzu Shim-Pack VP-ODS(150 mm 4.6 mm,5 μm) and mobile phase of 100 mmol·L^-1 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^-1 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 CYP1A2 and the incubation time was 60 min.The kinetic parameters was shown that V_max was 0.21 nmol·min^-1·mg protein^-1 and K_m was 20.39 μmol·L^-1. Conclusions:: The method is steady,accurate and suitable for assaying CYP1A2 activity which can be used to evaluate the pharmacokinetics of CYP1A2 in rat liver microsomes.

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