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.

人UDP-葡糖醛酸糖基转移酶1A1（UGT1A1）是最重要的结合酶之一，负责胆红素和其他内源性物质以及许多不同的外源性化合物的代谢和解毒。解密与人类疾病相关的UGT1A1并表征小分子对UGT1A1活性的影响需要可靠的工具来探测这种关键酶在复杂生物基质中的功能。在此，已经开发并验证了一种易于使用的测定方法，该方法使用带有荧光检测的液相色谱法（LC-FD）对各种生物基质中的UGT1A1活性进行高度选择性和灵敏的监测。新开发的基于LC-FD的测定法在灵敏度，特异性，精密度，定量线性范围和稳定性方面得到了证实。它的主要优点之一是，与使用相同探针底物的以前的检测方法相比，检测和定量的下限降低了约100倍，从而可实现定量定量的活性酶，比其他任何方法都可靠。精密度测试表明，该测定的日内和日间变化均小于5.5％。此外，新开发的测定法也已成功用于筛选和表征小分子对活细胞中UGT1A1表达水平的调节作用。总体而言，已开发出一种易于使用的基于LC-FD的测定方法，用于各种生物系统中超灵敏的UGT1A1活性测量，为探索UGT1A1在人类疾病，与异种生物的相互作用，