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目的:测定大鼠肝微粒体中细胞色素P4503A(CYP3A)的活性,并对其体外孵育条件进行优化。方法:采用1’-羟基咪哒唑仑的生成量表示肝中CYP3A的活性,高效液相色谱法测定肝微粒体中1’-羟基咪哒唑仑的浓度,用单因素试验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究肝CYP3A酶促动力学。结果:1’-羟基咪哒唑仑在18.20~1 820.00μg.L-1范围内线性关系良好;体外优化的孵育条件为5μmol.L-1咪哒唑仑、0.102mg肝微粒体、孵育15min;肝CYP3A酶促动力学参数Km为1.67μmol.L-1,Vmax为95.15pmol.min-1.mg-1。结论:HPLC法操作简便、灵敏、快速,适用于肝微粒体中1’-羟基咪哒唑仑的浓度测定,肝CYP3A孵育条件及其酶促动力学研究为研究经CYP3A代谢的药物相互作用及其他物质对CYP3A酶的影响提供理论依据。
OBJECTIVE: To determine the activity of cytochrome P4503A (CYP3A) in rat liver microsomes and to optimize its in vitro incubation conditions. Methods: The CYP3A activity in the liver was expressed by the amount of 1’-hydroxymidazolam. The concentration of 1’-hydroxymidazolam in the liver microsome was determined by high performance liquid chromatography (HPLC) The incubation conditions were optimized and the liver CYP3A enzymatic kinetics was studied by linear Lineweaver-Burk double reciprocal mapping. Results: The linearity of 1’-hydroxymidazolam was good in the range of 18.20-1 820.00μg.L-1. The optimal incubation conditions in vitro were 5μmol.L-1 midazolam and 0.102mg liver microsomes, incubated for 15min ; Liver CYP3A enzymatic kinetic parameters Km 1.67μmol.L-1, Vmax 95.15pmol.min-1.mg-1. Conclusion: The HPLC method is simple, sensitive and rapid for the determination of 1’-hydroxymidazolam in liver microsomes. The incubation conditions and enzymatic kinetics of CYP3A in liver microsomes were studied. In order to study the drug interactions via CYP3A metabolism and The effect of other substances on CYP3A enzyme provides a theoretical basis.