Proton nuclear magnetic resonance spectroscopic determination of ethanol-induced formation of ethyl glucuronide in liver.

Ethyl glucuronide (ethyl-beta-D-6-glucosiduronic acid, EtG), a unique metabolite of ethanol, has received much recent attention as a sensitive and specific biological marker of ethanol consumption. Formed in the liver via conjugation of ethanol with activated glucuronate, EtG remains detectable in serum, plasma, and hair for days after ethanol abuse. Thus far, gas chromatography-mass spectrometry and enzyme-linked immunosorbent assays have been developed to detect trace quantities of EtG for forensic purposes, but reports of the nuclear magnetic resonance (NMR) properties of EtG have been scarce. Herein we present the first report of EtG determination using proton NMR spectroscopy. We collected 700-MHz proton spectra of liver extracts from rats treated with a 4-day binge ethanol protocol (average ethanol dose: 8.6g/kg/day). An unexpected signal (triplet, 1.24 ppm) appeared in ethanol-treated liver extracts but not in control samples; based on chemical shift and multiplicity, we suspected EtG. We observed quantitative hydrolysis of the unknown species to ethanol while incubating our samples with beta-glucuronidase, confirming that the methyl protons of EtG were responsible for the triplet at 1.24 ppm. This study demonstrates that proton NMR spectroscopy is capable of detecting EtG and that future NMR-based metabolomic studies may encounter this metabolite of ethanol.