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≈KrUGT1A1,VRLM≈VHLM>VrUGT1A1,CLHLM>CLrUGT1A1≈CLrRLM。rUGT1A1(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rUGT1A1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。
Abstract
OBJECTIVE To detect the kenitics of UGT1A1 enzyme in three different systems. METHODS A UPLC-MS/MS method was developed to measure the bilirubin.RESULTS Glucuronidation kinetic constants were analyzed by fitting the Michaelis-Menten equation. As shown in our study, the apparent kinetic parameters of total bilirubin glucuronides were: KRLM>KHLM≈KrUGT1A1,VRLM≈VHLM>VrUGT1A1,CLHLM>CLrUGT1A1≈CLrRLM. It indicated that rUGT1A1 had the strongest binding affinity to bilirubin but could not convert it to bilirubin glucuronidations immediately. On the contrary RLM had a weaker binding affinity to the bilirubin, but could conver it rapidly. CONCLUSION Compared the kinetic in the different systems we could find that HLM almost had the same Km as rUGT1A1 which demonstrated the UGT1A1 enzyme was the primary enzyme mediating the metabolism of bilirubin.The difference parameter between three systems is attributed to the species differences. Summarily our study supplies experimental basis for the further research of the drug interactions.
关键词
UGT1A1酶 /
胆红素 /
肝微粒体 /
代谢
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Key words
UGT1A1 enzyme /
bilirubin /
liver microsome /
metabolism
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中图分类号：
R969.1
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参考文献
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脚注
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基金
国家科技重大专项资助项目(2014ZX09304307-002)
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