Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada.
Comp Biochem Physiol C Toxicol Pharmacol. 2021 Oct;248:109114. doi: 10.1016/j.cbpc.2021.109114. Epub 2021 Jun 18.
Hepatic glucuronidation represents an important phase II biotransformation reaction in both mammals and fish. The kinetic characteristics of uridine 5'-diphosphate (UDP) glucuronosyltransferases (UDPGTs) in rainbow trout liver microsomes were examined using p-nitrophenol (p-NP) as an aglycone and UDP-glucuronic acid (UDPGA) as a glucuronyl donor according to an existing protocol. The kinetic data obtained with varying concentrations of p-NP best fit the Hill equation and UDPGT activity was successfully induced following an i.p. injection of β-naphthoflavone (β-NF). The assay was subsequently adapted to a microplate method for determination of UDPGT activity in microsomal samples obtained from rainbow trout as well as Nile tilapia. In contrast to rainbow trout, UDPGT activity of Nile tilapia was best described by Michaelis-Menten kinetics. Based on the linearity of p-NP glucuronide formation, a p-NP concentration of 0.60 mM and a UDPGA concentration of 6.89 mM were determined to be suitable for assaying UDPGT activity in samples from rainbow trout and Nile tilapia. The microplate method offers several advantages over the historical assay; most notably it enables the observation of successive kinetics which ensures that enzyme activity is calculated in the most linear (initial) rate of the reaction. It also provides practical advantages in terms of ease-of-use and efficiency. This may be relevant to researchers investigating exposure of wild or farmed fish to environmental or feed-borne contaminants which are substrates of UDPGTs.
肝葡萄糖醛酸化是哺乳动物和鱼类中重要的 II 相生物转化反应。根据现有的方案，使用对硝基苯酚（p-NP）作为糖苷配基和 UDP-葡萄糖醛酸（UDPGA）作为葡萄糖醛酸供体，研究了虹鳟鱼肝微粒体中尿苷 5′-二磷酸（UDP）葡萄糖醛酰基转移酶（UDPGTs）的动力学特征。用不同浓度的 p-NP 获得的动力学数据最符合 Hill 方程，并且通过腹腔注射β-萘黄酮（β-NF）成功诱导了 UDPGT 活性。该测定法随后被改编为微板法，用于测定从虹鳟鱼和尼罗罗非鱼获得的微粒体样品中的 UDPGT 活性。与虹鳟鱼不同，尼罗罗非鱼的 UDPGT 活性最适合米氏动力学描述。基于对硝基苯酚葡萄糖醛酸形成的线性，确定 0.60 mM 的 p-NP 浓度和 6.89 mM 的 UDPGA 浓度适合测定来自虹鳟鱼和尼罗罗非鱼的样品中的 UDPGT 活性。与历史测定法相比，微孔板法具有几个优势；最值得注意的是，它能够观察连续的动力学，从而确保在反应的最线性（初始）速率下计算酶活性。它在易用性和效率方面也具有实际优势。这可能与研究环境或饲料源性污染物对野生或养殖鱼类暴露的研究人员有关，这些污染物是 UDPGT 的底物。
