微粒体体系中胆红素及其代谢产物测定方法的建立

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(rhUGT1A1)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、rhUGT1A1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200 μmol·L^-1)600 μL终止反应;本实验最适宜反应时间为10 min(HLM、rhUGT1A1 温孵 体系)及15 min(RLM 温孵 体系),酶蛋白浓度均为0.5 mg·mL^-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rhUGT1A1酶动力学特征提供了实验基础。

Objective: To establish an evaluation method for the determination of bilirubin and bilirubin glucuronidations in human liver microsomes (HLM), rat liver microsomes (RLM), and recombined human UDP-glucuronosyltransferases1A1 enzyme (rhUGT1A1), so as to lay a foundation for further study of the bilirubin metabolism. Methods: A UPLC-MS/MS method was established to verify and quantify the bilirubin and bilirubin glucuronidations in three incubation systems. Furthermore, a robust assay was performed to optimize the terminal condition, incubation time, and protein concentration. Results: The bilirubin and bilirubin glucuronidations(two isomers of monoglucuronides and diglucuronide) were identified by UPLC-MS/MS. The terminal condition was 600 μL ice methanol and acetonitrile(2:1)(containing ascorbic acid 200 μmol·L^-1). The proper incubation time was 10 min(HLM and rhUGT1A1 incubation systems)and 15 min(RLM incubation systems). The suitable protein concentration was 0.5 mg·mL^-1. Conclusion: The established method is stable and sensitive, and can be applied to the determination of bilirubin and bilirubin glucuronidations in HLM, RLM, and rhUGT1A1.Our results also provide an experimental foundation for the further study of UGT1A1 enzyme kinetics.

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