HPLC法测定大鼠UGT1A6的酶活性及体外动力学分析

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,5 μm),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0.5~100 μmol·L^-1,回归方程为Y=5973.12X+571.58 (r=0.9999),最低检测限(LOD)为0.1 μmol·L^-1(S/N≥3),最低定量限(LLOQ)为0.5 μmol·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24.63 μmol·L^-1,V_max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 V_max 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.