免疫性肝损伤过程中CYP2C19体内外活力测定的方法学建立  

Methodological establishment of CYP2C19 activity in vivo and in vitro with immune liver injury in rodents

作　　者：李晓霞 刘凤婷 王涛 林琴 薛永志[1] LI Xiao-xia;LIU Feng-ting;WANG Tao;LIN Qin;XUE Yong-zhi(Institute of Pharmacolinetics and Liver Molecular Pharmacology,Dept of Pharmacology,Baotou Medical College,Baotou,Inner Mongolia 014060,China)

机构地区：[1]包头医学院药物代谢与肝脏分子药理研究所,药理学教研室,内蒙古包头014060

出　　处：《中国药理学通报》2020年第11期1622-1626,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81460567);内蒙古自然科学基金资助项目(No 2009MS1104,2014MS0813,2019MS08198);内蒙古教育厅资助项目(No NJ03148,NJZY13244)。

摘　　要：目的在免疫性肝损伤过程中建立细胞色素P4502C19(CYP2C19)代谢活力的体内体外高效液相色谱(HPLC)检测方法。方法采用单次静脉注射卡介苗(BCG)125 mg·kg^-1,建立免疫性肝损伤大鼠或小鼠模型。HPLC法检测奥美拉唑钠(omeprazole,OME)与其羟基化代谢产物5-羟基OME(5-OHOME)的血浆药物浓度随时间的变化规律,建立大鼠体内实验的HPLC检测方法。采用差速离心法或钙沉淀法制备肝微粒体样本,与OME在37℃孵育,用其代谢率(5'-OHOME/OME)来反映体外CYP2C19酶的活性。结果OME从0.05~50 mg·L^-1浓度范围内峰面积与浓度线性关系良好,R^2=0.993。OME日内差异(RSD)分和日间差异(RSD)均小于10%;免疫性肝损伤导致CYP2C19的代谢活力下调。结论体内静脉给药法和体外钙沉淀法实验方法均有稳定、快速、灵敏度高等优点,可用于OME与其代谢产物5'-OHOME的测定,用于检测CYP2C19的代谢活力;免疫性肝损伤导致CYP2C19的代谢活力下调。Aim To establish a high-performance liquid chromatography(HPLC)to evaluate the metabolic activity of cytochrome P4502C19(CYP2C19)with immune liver injury in vivo and in vitro.Methods A rat or mouse model of immune liver injury was replicated by a single intravenous injection of BCG(125 mg·kg^-1).The plasma drug concentration of omeprazole(OME)and its hydroxylated metabolite 5-hydroxyOME(5-OHOME)with time was detected by HPLC method to establish the experimental method in vivo in rats.Liver microsome samples were prepared by differential centrifugation or calcium precipitation,incubated with OME at 37℃,and their metabolic rate(5'-OHOME/OME)was used to reflect the activity of CYP2C19 enzyme in vitro.Results OME had a good linear relationship from 0.05 to 50 mg·L^-1 concentration range,R^2=0.993.The OME intraday RSD was 1.72% and 1.16%,respectively,and each was less than 5%;the daytime RSD was 3.99% and 5.39%,respectively,and each was less than 10%.The metabolic activity of CYP2C19 was down-regulated in immune liver injury.Conclusions Both the in vivo intravenous administration method and the in vitro calcium precipitation method have the advantages of stability,rapidity and high sensitivity.They can be used for the determination of OME and its metabolite 5'-OHOME,and for the detection of the metabolic activity of CYP2C19.Immune liver injury is responsible for the reduction of the metabolic activity of CYP2C19.

关 键 词：免疫性肝损伤 CYP2C19 细胞色素P450 肝微粒体 高效液相色谱 奥美拉唑 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R345.99[医药卫生—基础医学]