大鼠肝细胞色素P4503A活性测定及其孵育条件的优化  

Determination of the activity of cytochrome P450 3A in rat liver microsomes by HPLC and optimization of the incubated conditions in vitro

作　　者：任秀华[1,2] 曹磊[2] 斯陆勤[2] 薛柯雯[2] 裘军[2] 李高[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院药学部,湖北武汉430030 [2]华中科技大学同济医学院药学院,湖北武汉430030

出　　处：《中国医院药学杂志》2010年第10期809-812,共4页Chinese Journal of Hospital Pharmacy

基　　金：国家自然科学基金资助项目(编号:30572265)

摘　　要：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18.20～1820．00ug·L-1范围内线性关系良好；体外优化的孵育条件为5umol·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1．67umol·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 CYP3A and optimize the incubated conditions in the liver microsomes of rats. METHODS The formation of 1 ＇-hydroxymidazolam determined by HPLC was used to indicate the activity of CYP3A. Single effect experimental design was used to optimize the incubated conditions and the enzyme kinetics was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots （1/velocity vs. 1/ [substrate]）. RESULTS Calibration curves of 1 ＇-hydroxymidazolam were linear in the range of 18. 20-1 820. 00ug.L-1 （ R2 = 0. 999 9）. The optimal incubated conditions were 5 umol. L- 1,0. 102 mg and 15 min for midazolam concentration, liver microsomes and incubation time, respectively. The parameters of CYP3A in the liver mierosomes for Km and Vmax were 1. 67 umol.L-1 and 95. 15 pmol.min - 1.mg -1 , respectively. CONCLUSION The HPLC method is simple, accurate and rapid, suitable for the determination of 1 ＇-hydroxymidazolam in the liver microsomes. The incubated conditions of CYP3A and its kinetics can be applied for studying on the interaction between drugs metabolized by CYP3A.

关 键 词：CYP3A活性 肝微粒体 高效液相色谱法 1’-羟基咪哒唑仑 优化 大鼠 

分 类 号：R969[医药卫生—药理学]