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目的基于二相代谢酶UGT1A1酶介导胆红素代谢过程,测定比较不同种属肝微粒体体系中UGT1A1酶动力学参数。方法采用高分辨质谱对胆红素及其葡萄糖醛酸代谢物进行定性鉴别,采用UPLC测定法,分别以胆红素一级、二级和总代谢产物生成量对胆红素底物浓度作图,求得动力学参数。结果实验结果发现,以总代谢产物计,KRLM>KHLM≈Kr UGT1A1,VRLM≈VHLM>Vr UGT1A1,CLHLM>CLr UGT1A1≈CLrRLM。r UGT1A1(人重组UGT1A1酶)酶体系中UGT1A1酶Km值最小,表明其与胆红素亲和能力最强,Vmax值最小,不能及时将其代谢转化;与此相反,大鼠肝微粒体(RLM)体系中UGT1A1酶与胆红素的亲和能力最弱,但转化效果最佳;人肝微粒体(HLM)体系中UGT1A1酶对胆红素亲和能力较强,转化能力适中。结论通过3个体系比较发现,人肝微粒体与r UGT1A1体系Km值接近,确证了胆红素在肝微粒体中的葡萄糖醛酸化反应主要由UGT1A1介导,人肝微粒体和大鼠肝微粒体体系差别主要由酶及其环境的种属差异导致,本实验的相关数据为研究因诱导或抑制UGT1A1酶引起的药物相互作用及毒性提供了实验依据。
OBJECTIVE: To determine the kinetic parameters of UGT1A1 in different species of liver microsomes based on the bilirubin metabolism mediated by the UGT1A1 enzyme. Methods The bilirubin and its glucuronic acid metabolites were qualitatively identified by high resolution mass spectrometry. UPLC assay was used to determine the concentrations of primary and secondary bilirubin and total metabolites, respectively, on the concentrations of bilirubin substrate , Obtained kinetic parameters. Results The experimental results showed that KRLM> KHLM≈Kr UGT1A1, VRLM≈VHLM> Vr UGT1A1, CLHLM> CLr UGT1A1≈CLrRLM based on the total metabolites. UGT1A1 enzyme (UGT1A1 enzyme) enzyme system UGT1A1 enzyme Km value is the smallest, indicating that the strongest affinity with bilirubin, Vmax value of the smallest, can not be metabolized in time; In contrast, rat liver microsomal ( UGT1A1 enzyme has the weakest affinity with bilirubin, but the transformation effect is the best. In the human liver microsome (HLM) system, the UGT1A1 enzyme has strong affinity to bilirubin and moderate transformation ability. Conclusion The comparison of three systems shows that the Km values of human liver microsomes and r UGT1A1 system are similar, confirming that the glucuronidation reaction of bilirubin in liver microsomes is mainly mediated by UGT1A1, and that human liver microsomes and rat liver microsomes The difference of the body system is mainly caused by the difference of the species of the enzyme and its environment. The data of this experiment provide the experimental basis for studying the drug interaction and toxicity caused by inducing or inhibiting the UGT1A1 enzyme.