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目的:建立人肝微粒体(HLM),大鼠肝微粒体(RLM)及重组人UDP-葡萄糖醛酸转移酶1A1(rh UGT1A1)体系中底物胆红素及其葡萄糖醛酸代谢产物的测定方法,为进一步研究药物影响胆红素肝代谢奠定基础。方法:采用UPLC及MS/MS联用技术对胆红素及其代谢产物进行定性及定量研究,确定检测方法。优化HLM、RLM、rh UGT1A1体系终止反应条件、反应时间及反应蛋白浓度,确定反应体系条件。结果:初步鉴定了底物胆红素及其代谢物单葡萄糖醛酸胆红素(BMG1及BMG2)及双葡萄糖醛酸胆红素(BDG)。确定了体系终止反应条件为反应结束时,加入冰乙腈-甲醇(2∶1,含抗坏血酸浓度为200μmol·L-1)600μL终止反应;本实验最适宜反应时间为10 min(HLM、rh UGT1A1温孵体系)及15 min(RLM温孵体系),酶蛋白浓度均为0.5 mg·m L-1。结论:本实验所建立的肝微粒体体系中底物胆红素及其代谢产物的测定方法简便、可行,为进一步研究rh UGT1A1酶动力学特征提供了实验基础。
OBJECTIVE: To establish a method for determination of substrate bilirubin and its glucuronic acid metabolites in human liver microsomes (HLM), rat liver microsomes (RLM) and recombinant human UG-glucuronosyltransferase 1A1 (rh UGT1A1) system Method to lay the foundation for the further study of drugs affecting bilirubin liver metabolism. Methods: The methods of UPLC and MS / MS were used to qualitatively and quantitatively determine bilirubin and its metabolites. Optimization of the terminating reaction conditions, reaction time and reaction protein concentration of HLM, RLM, rh UGT1A1 system, to determine the reaction system conditions. Results: The substrate bilirubin and its metabolites monoglucuronidase (BMG1 and BMG2) and bis-glucuronil bilirubin (BDG) were preliminarily identified. At the end of the reaction, the reaction was terminated by adding 600 μL of ice-acetonitrile-methanol (2:1, containing ascorbic acid at 200μmol·L-1). The optimal reaction time was 10 min (HLM, rh UGT1A1 temperature Incubation system) and 15 min (RLM incubation system), enzyme protein concentrations were 0.5 mg · m L-1. Conclusion: The method of determination of substrate bilirubin and its metabolites in liver microsomes established in this experiment is simple and feasible, which provides the experimental basis for further study of enzyme kinetics characteristics of rh UGT1A1.