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目的测定大鼠肠微粒体中细胞色素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肠中CYP3A的活性,反相高效液相色谱法测定肠微粒体中1′-羟基咪哒唑仑的浓度,用单因素实验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究肠CYP3A酶促动力学。结果1′-羟基咪哒唑仑在9.10～910.00ng.mL-1内线性关系良好;体外优化的孵育条件为10μmol.L-1咪哒唑仑、4μg肠微粒体、孵育20min;肠CYP3A酶促动力学参数Km为7.61μmol.L-1,Vmax为1.26nmol.min-1.mg-1。结论HPLC操作简便、灵敏、快速,适用于肠微粒体中1′-羟基咪哒唑仑的浓度测定,肠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 intestinal microsomes and to optimize its in vitro incubation conditions. Methods The activity of CYP3A in intestine was expressed by the amount of 1’-hydroxyl midazolam, and the concentration of 1’-hydroxymidazolam in intestinal microsome was determined by reverse-phase high performance liquid chromatography In vitro incubation conditions were optimized, and linearized Lineweaver-Burk double reciprocal mapping was used to study intestinal CYP3A enzymatic kinetics. Results The linearity of 1’-hydroxymidazolam in the range of 9.10-910.00 ng.mL-1 was good. The optimal incubation conditions were 10 μmol.L-1 midazolam, 4 μg intestinal microsomes and incubated for 20 min. The intestinal CYP3A enzyme The kinetic parameters Km was 7.61μmol.L-1, Vmax was 1.26nmol.min-1.mg-1. Conclusion HPLC is simple, sensitive and rapid and is suitable for the determination of 1’-hydroxymidazolam in enteric microsome and the incubation conditions and enzymatic kinetics of enteric CYP3A. To study the interaction of CYP3A metabolites and other substances The effect of CYP3A enzyme provides a theoretical basis.