Objective

Uridine diphospho-glucuronosyltransferases (UGTs) are membrane-bound enzymes that catalyze the conjugation of glucuronic acid onto a diverse set of xenobiotics. Horses efficiently and extensively glucuronidate a number of xenobiotics, including opioids, making UGTs an important group of drug-metabolizing enzymes for the clearance of drugs. Recombinant enzymes have allowed researchers to characterize the metabolism of a variety of drugs. The primary objective was to clone, express and characterize equine UGTs using drugs characterized as UGT substrates in other species. A secondary objective was to characterize the in vitro metabolism of morphine in horses.

Study design

In vitro drug metabolism study using liver microsomes and recombinant enzyme systems.

Animals

Liver microsomes and RNA from tissue collected from two Thoroughbred mares euthanized for other reasons.

Methods

Based on homology to the human UGT2B7, four equine UGT variants were expressed: UGT1A1, UGT2A1, UGT2B31 and UGT2B4. cDNA sequences were cloned and resulting protein expressed in a baculovirus expression system. Functionality of the enzymes was assessed using 4-methylumbelliferone, testosterone, diclofenac and ketoprofen. Recombinant enzyme, control cells, equine liver microsomes and human UGT2B7 supersomes were then incubated with morphine. Concentrations of metabolites were measured using liquid chromatography–tandem mass spectrometry and enzyme kinetics determined.

Results

4-Methylumbelliferone was glucuronidated by all expressed equine UGTs. Testosterone glucuronide was not produced by any of the expressed enzymes, and diclofenac glucuronide and ketoprofen glucuronide were produced by UG2A1 and UGT1A1, respectively. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide.

Conclusions and clinical relevance

This is the first successful expression of functional recombinant equine UGTs. UGT2B31 contributes to the glucuronidation of morphine; however, it is probably not the main metabolizing enzyme. These results warrant further investigation of equine UGTs, including expression of additional enzymes and further characterization of UGT2B31 as a contributor to morphine metabolism.

中文翻译：

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马尿苷二磷酸-葡萄糖醛酸糖基转移酶1A1、2A1、2B4、2B31：吗啡代谢的cDNA克隆，表达和初步表征。

目的

尿苷二磷酸-葡萄糖醛酸糖基转移酶（UGT）是膜结合酶，可催化葡萄糖醛酸与多种异源生物的结合。马能有效，广泛地葡萄糖醛酸酯化多种异生素，包括阿片类药物，使UGT成为清除药物的重要药物代谢酶类。重组酶使研究人员能够表征多种药物的代谢。主要目的是使用其他物种中以UGT为底物的药物克隆，表达和鉴定马UGT。第二个目的是表征马体内吗啡的体外代谢。

学习规划

使用肝微粒体和重组酶系统进行体外药物代谢研究。

动物

出于其他原因，对从两只纯种母马处以安乐死的肝脏微粒体和组织中的RNA。

方法

基于与人UGT2B7的同源性，表达了四种马UGT变体：UGT1A1，UGT2A1，UGT2B31和UGT2B4。克隆cDNA序列，并在杆状病毒表达系统中表达所得蛋白质。使用4-甲基伞形酮，睾丸激素，双氯芬酸和酮洛芬评估酶的功能。然后将重组酶，对照细胞，马肝微粒体和人UGT2B7超体与吗啡一起孵育。使用液相色谱-串联质谱法测量代谢物的浓度，并确定酶动力学。

结果

所有表达的马UGT均对4-甲基伞形酮进行了葡萄糖醛酸化。睾丸激素葡糖醛酸苷不是由任何表达的酶产生的，双氯芬酸葡糖醛酸苷和酮洛芬葡糖醛酸苷分别是由UG2A1和UGT1A1产生的。UGT2B31将吗啡代谢为吗啡-3-葡糖醛酸和低浓度的吗啡-6-葡糖醛酸。

结论与临床意义

这是功能性重组马UGT的首次成功表达。UGT2B31有助于吗啡的葡萄糖醛酸化；但是，它可能不是主要的代谢酶。这些结果需要进一步研究马的UGT，包括表达其他酶并进一步表征UGT2B31作为吗啡代谢的贡献者。