In vitro formation of ethyl glucuronide and ethyl sulfate

Aims: Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are used as markers of alcohol consumption in various clinical and forensic settings. In controlled studies, their concentration considerably varies between subjects. Knowledge on glucuronosyltransferases (UGT) and sulfotransferases (SULT) catalyzing formation of EtG and EtS formation is as moderate as diverging. A possible influence of nutritional components such as plant-derived phenols on the formation rates has not been addressed. Methods: Formation rates of EtG and EtS from ethanol via recombinant human UTGs (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) and recombinant SULTs (SULT1A1, 1A3, 1B1, 1E1, 2A1), respective kinetics and the inhibitory potential of quercetin, kaempferol and resveratrol were determined. Analysis was performed following either solid phase extraction due to severe ion suppression of EtG or direct injection in the case of EtS by LC/MS/MS. Results: All enzymes under investigation formed EtG and EtS with UGT1A9 showing the highest glucuronidation rate and SULT1A1 exhibiting the highest sulfonation activity. Data for all enzymes could best be described by Michaelis-Menten kinetics. Formation of EtG was significantly reduced following co-incubation with quercetin and kaempferol, except UGT2B15. Resveratrol inhibited conjugation of ethanol via UGT1A1 and UGT1A9. All phenolic compounds decreased activity of SULTs towards ethanol. Inhibition was reversible and competitive for most enzymes; mechanism-based inhibition was evident for UGT2B7 and SULT2A1 with regard to quercetin and SULT1E1 with regard to kaempferol. Conclusions: Conjugation of ethanol occurs via multiple UGTs and SULTs. Beside known polymorphisms of UGT and SULT family members, common nutritional components influence formation of EtG and EtS. The results warrant further studies but may partly serve as an explanation for the variable formation of both biomarkers in man.