The effects of UDP-sugars, UDP and Mg²⁺on uridine diphosphate glucuronosyltransferase activity in human liver microsomes

Abstract

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation.

2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg²⁺ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation.

3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by including Mg²⁺ in the assay. The Mg²⁺ effect can be offset using EDTA, and is dependent on the concentration of UDP-GlcA in the assay.

4. We propose that formation of a Mg²⁺–UDP complex prevents UDP from affecting the enzyme. Our results suggest that 5 mM UDP-GlcA and 10 mM Mg²⁺ be used for UGT assays in fully disrupted HLM.

Keywords:

• Drug metabolism
• glucuronidation
• glycosylation
• in vitro–in vivo extrapolation
• kinetics
• substrate inhibition