HPLC-RF测定小鼠肝微粒体中CYP2A5的酶活性

Determination the activity of CYP2A5 in mouse liver microsomes by HPLC-RF

目的以香豆素为探针底物建立测定小鼠肝微粒体中CYP2A5酶活性的方法。方法用Waters Symmetry C18柱;流动相为0.1%醋酸-乙腈=70∶30;流速为1.0 mL·min-1,柱温30℃,E x=340 nm,E m=458 nm。香豆素与小鼠肝微粒体在37℃孵育20 min,加入含内标的冰乙腈终止,4℃、12000 r·min-1离心15 min,取上清液,用HPLC测定其代谢产物7-羟基香豆素的含量,以GraphPad Prism 5.0作图计算酶动力学参数。结果 7-羟基香豆素与内标两者分离良好。7-羟基香豆素的检测限为2 nmol·L-1(S/N≥3),线性范围为8～800nmol·L-1。日内日间精密度RSD小于5%,回收率为91.3%～101.4%。香豆素羟化反应的K m为2.15μmol·L-1;V max为9.74 pmol·(min·mg protein)-1。结论该方法稳定可靠,灵敏度高,适于体外CYP2A5酶活性的测定。

Objective To develop a HPLC method for the determination of the activity of cytochrome P450 2A5 （CYP2A5）in mouse microsomes with coumarin as the probe drug. Methods An waters symmetry Cts col-umn was used. The mobile phase was water（0. 1% acetic acid）and aceto-nitrile（70： 30）. The flow rate was 1.0 mL · min^-1. The column tempera- ture was 30 ℃ and the RF wave length was Ex =340 nm, Em =458 nm. Coumarin was incubated with mouse liver microsomes in vitro at 37 ℃ for 20 min and stopped by addition ice acetonitrile which contained inter- nal standard substances and centrifuged （12000 r · min^-1） for 15 min, then the production 7 - hydroxyeoumarin was analyzed by HPLC - RF. Results The lowest detection limit of 7 -hydroxyeoumarin was 2 nmol · L^-1 （S/N≥3）, and the calibration curve was linear from 8 nmol · L^-1 to 800 nmol · L^-1. The intra-day and inter-day relative standard devi-ations were less than 5% respectively. The method recoveries ranged from 91.3 % - 101.4%. The kinetic parameters,Km was 2. 15 μmol · L^-1 and Vmax was 9. 74 pmol · （min · mg protein）^-1. Conclusion This method is reliable and the results can accurately indicate CYP2A5 enzyme activity, which can be used for the kinetics study of CYP2A5 in liver microsomes.