114991-26-3Relevant articles and documents
Validated assay for the evaluation of multiple glucuronidation activities in human liver microsomes via liquid chromatography-tandem mass spectrometry
Shi, Rong,Yang, Yuanyuan,Zhong, Jie,Wang, Tianming,Ma, Yueming
, p. 56132 - 56138 (2014)
A sensitive and high-throughput liquid chromatography-tandem mass spectrometry system was developed and validated for the simultaneous determination of major human hepatic UDP-glucuronyltransferase forms in human liver microsomes. The analytes were detected using a triple-quadrupole mass spectrometer equipped with an electrospray ionization source in the negative ion and selected reaction monitoring modes. The method provided satisfactory linear concentration range, accuracy, precision, and stability. The developed method was successfully applied to the enzyme kinetic study of estradiol-3-O-glucuronidation, 4-methylumbelliferone-O-glucuronidation, propofol-O-glucuronidation, and 3-azido-3-deoxythymidine glucuronidation in human liver microsomes.
Accurate prediction of glucuronidation of structurally diverse phenolics by human UGT1A9 using combined experimental and in silico approaches
Wu, Baojian,Wang, Xiaoqiang,Zhang, Shuxing,Hu, Ming
experimental part, p. 1544 - 1561 (2012/07/27)
Purpose: Catalytic selectivity of human UGT1A9, an important membrane-bound enzyme catalyzing glucuronidation of xenobiotics, was determined experimentally using 145 phenolics and analyzed by 3D-QSAR methods. Methods: Catalytic efficiency of UGT1A9 was determined by kinetic profiling. Quantitative structure activity relationships were analyzed using CoMFA and CoMSIA techniques. Molecular alignment of substrate structures was made by superimposing the glucuronidation site and its adjacent aromatic ring to achieve maximal steric overlap. For a substrate with multiple active glucuronidation sites, each site was considered a separate substrate. Results: 3D-QSAR analyses produced statistically reliable models with good predictive power (CoMFA: q 2=0.548, r2=0.949, r pred 2 =0.775; CoMSIA: q2=0.579, r2=0.876, rpred2 =0.700). Contour coefficient maps were applied to elucidate structural features among substrates that are responsible for selectivity differences. Contour coefficient maps were overlaid in the catalytic pocket of a homology model of UGT1A9, enabling identification of the UGT1A9 catalytic pocket with a high degree of confidence. Conclusion: CoMFA/CoMSIA models can predict substrate selectivity and in vitro clearance of UGT1A9. Our findings also provide a possible molecular basis for understanding UGT1A9 functions and substrate selectivity.
Identification of human UGT2B7 as the major isoform involved in the O-glucuronidation of chloramphenicol
Chen, Mei,LeDuc, Barbara,Kerr, Stephen,Howe, David,Williams, David A.
experimental part, p. 368 - 375 (2011/04/16)
Chloramphenicol (CP), a broad spectrum antibiotic, is eliminated in humans by glucuronidation. The primary UGT enzymes responsible for CP O-glucuronidation remain unidentified. We have previously identified the 3-O-CP (major) and 1-O-CP (minor) glucuronides by β-glucuronidase hydrolysis, liquid chromatography-tandem mass spectrometry, and 1D/2D H NMR. Reaction phenotyping for the glucuronidation of CP with 12 expressed human liver UGT isoforms has identified UGT2B7 as having the highest activity for 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. The kinetics of CP 3-O-glucuronidation by pooled human liver microsomes (HLMs) exhibited biphasic Michaelis-Menten kinetics with the apparent high-affinity K m1 and low-affinity Km2 values of 46.0 and 1027 μM, whereas expressed UGT2B7 exhibited Michaelis-Menten kinetics with the apparent Km value of 109.1 μM. The formation of 1-O-CP glucuronide by pooled HLM and expressed UGT2B7 exhibited substrate inhibition kinetics with apparent Km values of 408.2 and 115.0 μM, respectively. Azidothymidine (AZT) and hyodeoxycholic acid (substrates of UGT2B7) inhibited 3-O- and 1-O-CP glucuronidation in pooled HLMs. In 10 donor HLM preparations, both CP 3-O- and CP 1-O-glucuronidation showed a significant correlation with AZT glucuronidation (UGT2B7) (rs = 0.85 and rs = 0.83, respectively) at 30 μMCP, whereas no significant correlation was observed between CP 3-O-glucuronidation and serotonin glucuronidation (UGT1A6) or propofol glucuronidation (UGT1A9) at this CP concentration. These results suggest that UGT2B7 is the primary human hepatic UDP-glucuronosyltransferase isoform catalyzing 3-O- and 1-O-CP glucuronidation with minor contributions from UGT1A6 and UGT1A9. Copyright
