Simultaneous determination of bilirubin and its glucuronides in liver microsomes and recombinant UGT1A1 enzyme incubation systems by HPLC method and its application to bilirubin glucuronidation studies
Simultaneous determination of bilirubin and its glucuronides in liver microsomes and recombinant UGT1A1 enzyme incubation systems by HPLC method and its application to bilirubin glucuronidation studies
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HPLC法同时测定肝微粒体和重组UGT1A1酶孵育体系中胆红素及其葡萄糖醛酸化及其在胆红素葡萄糖醛酸化研究中的应用
DOI：
10.1016/j.jpba.2014.01.025
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发表时间：
2014-04-15
影响因子：
3.4
通讯作者：
Zhang, Peng
中科院分区：
文献类型：
--
作者：
Ma, Guo;Lin, Jiayuan;Zhang, Peng
胆红素是人体重要的内源性物质和肝功能指标，主要通过UGT 1A 1催化的葡萄糖醛酸化作用消除。胆红素及其葡萄糖醛酸苷的不稳定性给体外胆红素葡萄糖醛酸化测定带来了巨大的技术挑战。本研究建立了一种简便、可靠的同时测定非结合胆红素（UCB）及其多种葡萄糖醛酸苷（即胆红素单葡萄糖醛酸苷）的HPLC方法大鼠肝微粒体（RLM）、人肝微粒体（HLM）和重组人UGT 1A 1酶（UGT 1A 1）孵育系统中的BMG（包括BMG 1和BMG 2异构体）和二葡萄糖醛酸（BDG），并将其应用于体外胆红素葡萄糖醛酸化的研究。采用C18色谱柱，以0.1%甲酸水溶液和乙腈为移动的相，在450 nm波长处进行检测，采用线性梯度洗脱，成功分离了UCB、BMG 1、BMG 2、BDG及其异构体。在0.01-2 μ M范围内确认了胆红素的测定线性。低、中、高浓度UCB、BMG 1、BMG 2和BDG（n = 5）的精密度在RSD 0.4- 3.7%范围内，对于日内和日间测定，以平均测定回收率（n = 5）表示的准确度范围为92.8 +/- 1.5%至104.3 +/- 2.2%，平均提取回收率（n = 5）高于91.5 +/-1.0%。胆红素及其葡萄糖醛酸苷的稳定性在反应期间（30 min）的孵育溶液中为37 ℃，在用甲醇处理的孵育样品中为25 ℃持续24 h和-70 ℃持续7 d。此外，我们建立了稳定，可靠的体外孵育系统，并优化了孵育条件，分别通过RLM，HLM和UGT 1A 1表征胆红素葡萄糖醛酸化的动力学。总胆红素葡萄糖醛酸苷生成的动力学参数（TBG，BMG 1、BMG 2和BDG的总和）如下：K-m为0.45 +/- 0.016、0.40 +/- 0.022、0.44 +/- 0.018 μ M，V-max为2.65 +/- 0.057、1.86 +/- 0.029、2.95 +/- 0.036 nmol/mg/min，克林特为5.92 +/- 0.22，RLM、HLM和UGT 1A 1分别为4.70 +/- 0.079、6.72 +/- 0.27 mL/mg/min。胆红素葡萄糖醛酸化反应在选定的底物浓度范围内分别符合Hill方程和Michaelis-Menten方程。此外，BDG和BMG之间的相对比例与酶来源（如RLM、HLM和UGT 1A 1）和胆红素浓度有关。(C)© 2014 Elsevier B. V.保留所有权利。
Bilirubin, an important endogenous substances and liver function index in humans, is primarily eliminated via UGT1A1-catalyzed glucuronidation. Instability of bilirubin and its glucuronides brings substantial technical challenges to conduct in vitro bilirubin glucuronidation assay. In the present study, we developed a simple and robust HPLC method for simultaneous determination of unconjugated bilirubin (UCB) and its multiple glucuronides, i.e. bilirubin monoglucuronides (BMGs, including BMG1 and BMG2 isomers) and diglucuronicie (BDG) in rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human UGT1A1 enzyme (UGT1A1) incubation systems, and applied it to study in vitro bilirubin glucuronidation. UCB, BMG1, BMG2, BDG and their isomers in the incubation mixtures were successfully separated using a C18 column with UV detection at 450 nm and mobile phase consisted of 0.1% formic acid in water and acetonitrile by a linear gradient elution program. Assay linearities of bilirubin were confirmed in the range 0.01-2 mu M. Precision of UCB, BMG1, BMG2 and BDG (n = 5) at low, medium and high concentration was within the range of RSD 0.4-3.7%, accuracy expressed in the mean assay recoveries of them (n = 5) ranged from 92.8 +/- 1.5% to 104.3 +/- 2.2% for intra- and inter-day assays and the mean extraction recoveries of them (n = 5) were above 91.5 +/- 1.0%. Stability of bilirubin and its glucuronides was satisfactory at 37 degrees C in the incubation solutions during the reaction (30 min), 25 degrees C for 24 h and -70 degrees C for 7 d in the processed incubation samples with methanol. Furthermore, we established stable, reliable in vitro incubation systems and optimized the incubation conditions to characterize the kinetics of bilirubin glucuronidation by RLM, HLM and UGT1A1, respectively. The kinetic parameters of formation of total bilirubin glucuronides (TBG, the sum of BMG1, BMG2 and BDG) were as follows: K-m of 0.45 +/- 0.016, 0.40 +/- 0.022, 0.44 +/- 0.018 mu M, V-max of 2.65 +/- 0.057, 1.86 +/- 0.029, 2.95 +/- 0.036 nmol/mg/min, CLint of 5.92 +/- 0.22, 4.70 +/- 0.079, 6.72 +/- 0.27 mL/mg/min by RLM, HLM and UGT1A1, respectively. Bilirubin glucuronidation obeyed the Hill equation by RLM and the Michaelis-Menten equation by HLM and UGT1A1 in the range of substrate concentration selected, respectively. In addition, the relative proportions between BDG and BMGs were in connection with enzyme sources (e.g. RLM, HLM and UGT1A1) and bilirubin concentration. (C) 2014 Elsevier B.V. All rights reserved.