HPLC法测定大鼠UGT1A6的酶活性及体外动力学分析  

HPLC determination of activity of UGT1A6 in rat liver microsomes and study on pharmacokinetics

作　　者：郭延垒[1] 于超[1] 杨竹[1] 王应雄[1] 张艳辉[1] 张有金[1] 

机构地区：[1]重庆医科大学生命科学研究院,重庆400016

出　　处：《药物分析杂志》2012年第4期717-720,696,共5页Chinese Journal of Pharmaceutical Analysis

基　　金：重庆市自然科学基金(CSTC;2009BA5083)

摘　　要：目的:建立灵敏、稳定的高效液相色谱法,以对硝基酚为探针底物,评价体外大鼠肝微粒体中UGT1A6酶活性。方法:色谱柱为Agilent Zorbax SB-C18柱(250 mm×4.6 mm,5μm),流动相为20 mmol.L-1磷酸二氢钾缓冲液-甲醇(50∶50,V/V),流速0.8 mL.min-1,柱温30℃,检测波长280 nm。对硝基酚与大鼠肝微粒体在37℃共同孵育一段时间后,加入冰乙腈终止反应,于4℃下12000 r.min-1离心15 min后,吸取上清进行HPLC分析,以双倒数作图法计算酶动力学参数。结果:对硝基酚保留时间为8.95 min,线性范围为0.5～100μmol.L-1,回归方程为Y=5973.12X+571.58(r=0.9999),最低检测限(LOD)为0.1μmol.L-1(S/N≥3),最低定量限(LLOQ)为0.5μmol.L-1,回收率为104.5%～105.5%,日内、日间RSD均小于10%,温孵体系中其他内源性物质不干扰测定;计算酶动力学参数Km为24.63μmol.L-1,Vmax为18.87 nmol.min-1.mg.protein-1。结论:该方法灵敏度高、稳定性好,适合体外对硝基酚的测定,可用于大鼠体外UGT1A6酶活性的评价及酶动力学研究。Objective:To develop a stable and sensitive method with p-nitrophenol as probe drug in order to evaluate the activity of UDP-glucuronosyltransferases(UGT1A6) in rat liver microsomes by the high-performance liquid chromatograph(HPLC).Methods:An Agilent Zorbax SB-C18(250 mm×4.6 mm,5 μm) column was used.The mobile phase was methanol and 20 mmol·L-1 KH2PO4 solution(50:50,V/V).The flow rate was 0.8 mL·min-1.The UV wave length was set at 254 nm and the column temperature at 30 ℃.p-Nitrophenol was incubated with rat liver microsomes in vitro at 30 ℃ and stopped by addition ice acetonitrile and centrifuged(12000 r·min-1) for 3 min and further analyzed by HPLC.Results:The retention time of P-Nitrophenol is 8.95 min.The concentration range was 0.5～100 μmol·L-1 and the regression equation was Y=5973.12X+571.58(r=0.9999).The lowest detectable limit(LOD) was 0.1 μmol·L-1(S/N≥3) and the lower limit of quantification(LLOQ) was 0.5 μmol·L-1.The Abstraction recoveries were 104.5%-105.5%.The intra-day and inter-day relative standard deviations were all less than 15%.There were no endogenous substances existing in the incubation system which interfered with the determination of the analyses of interest.For the rat liver microsomes Km was 24.63 μmol·L-1 and Vmax was 18.87 nmol·min-1·mg·protein-1.Conclusion:The method is stable and highly sensitive for determination of P-Nitrophenol in vitro and suitable for the evaluation of UGT1A6 activity in vitro.

关 键 词：高效液相色谱法 对硝基酚探针底物 大鼠肝微粒体 尿苷二磷酸葡萄糖醛酸转移酶(UGT) 酶活性评价 酶动力学研究中 

分 类 号：R917[医药卫生—药物分析学]