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An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems.
J Pharm Anal. 2020 Jun; 10(3):263-270.JP

Abstract

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

Authors+Show Affiliations

Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.School of Life Science and Medicine, Dalian University of Technology, Panjin, 124221, China.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, 00014, Finland.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.Department of Pharmacy, Huashan Hospital, Fudan University, Shanghai, China.Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32612873

Citation

Zhu, Ya-Di, et al. "An Ultra-sensitive and Easy-to-use Assay for Sensing Human UGT1A1 Activities in Biological Systems." Journal of Pharmaceutical Analysis, vol. 10, no. 3, 2020, pp. 263-270.
Zhu YD, Pang HL, Zhou QH, et al. An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems. J Pharm Anal. 2020;10(3):263-270.
Zhu, Y. D., Pang, H. L., Zhou, Q. H., Qin, Z. F., Jin, Q., Finel, M., Wang, Y. N., Qin, W. W., Lu, Y., Wang, D. D., & Ge, G. B. (2020). An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems. Journal of Pharmaceutical Analysis, 10(3), 263-270. https://doi.org/10.1016/j.jpha.2020.05.005
Zhu YD, et al. An Ultra-sensitive and Easy-to-use Assay for Sensing Human UGT1A1 Activities in Biological Systems. J Pharm Anal. 2020;10(3):263-270. PubMed PMID: 32612873.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems. AU - Zhu,Ya-Di, AU - Pang,Hui-Lin, AU - Zhou,Qi-Hang, AU - Qin,Zi-Fei, AU - Jin,Qiang, AU - Finel,Moshe, AU - Wang,Yi-Nan, AU - Qin,Wei-Wei, AU - Lu,Yin, AU - Wang,Dan-Dan, AU - Ge,Guang-Bo, Y1 - 2020/05/23/ PY - 2020/02/14/received PY - 2020/05/14/revised PY - 2020/05/14/accepted PY - 2020/7/3/entrez PY - 2020/7/3/pubmed PY - 2020/7/3/medline KW - LC-FD KW - Modulators KW - N-butyl-4-(4-hydroxyphenyl)-1,8-naphthalimide KW - UGT1A1 SP - 263 EP - 270 JF - Journal of pharmaceutical analysis JO - J Pharm Anal VL - 10 IS - 3 N2 - The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme. SN - 2214-0883 UR - https://www.unboundmedicine.com/medline/citation/32612873/An_ultra-sensitive_and_easy-to-use_assay_for_sensing_human_UGT1A1_activities_in_biological_systems L2 - https://linkinghub.elsevier.com/retrieve/pii/S2095-1779(20)30165-9 DB - PRIME DP - Unbound Medicine ER -
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