Substantial amounts of endogenous and xenobiotic compounds are transformed to ß-D-Glucuronides in the human body. The conjugation reaction, which requires uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid as a cosubstrate is catalyzed by UDP-glucuronosyltransferases (UGTs). Beta-glucuronidase (GUSB), which is widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants, opposes this reaction: ß-D-glucuronides are hydrolyzed to ß-D-glucuronic acid and the corresponding aglycon in a configuration retaining manner. This study investigated GUSB activity in the presence of ethanol.

Phenolphthalein-ß-D-glucuronide (PHPH-G), 4-nitrophenol-ß-D-glucuronide (4NP-G), morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide were used as substrates. Enzymatic reactions were carried out at 37°C in buffer solution fortified with different amounts of ethanol (0–70% by volume) using GUSB from different sources (E. coli, H. pomatia, bovine and human recombinant). The enzymatic reactions were stopped after 60minutes and all samples were tested for ß-D-ethyl glucuronide (EtG) by a validated LC/MS/MS assay. In incubations of PHPH-G and 4NP-G, the release of aglyca was additionally measured by colorimetrical methods.

It was found that EtG, which is a minor metabolite of ethanol but one of the most important direct biomarkers of alcohol consumption in man today, builts up from all investigated ß-D-glucuronides by GUSB activity in the presence of ethanol. The glucuronyl transfer reaction, which was neither observed in absence of ethanol nor in absence of GUSB, is detectable but minor at ethanol concentrations which are commonly observed in blood and tissues after consumption of alcoholic beverages. At higher ethanol concentrations (>5% by volume), the transfer reaction is predominant and the majority of glucuronyl from ß-D-glucuronides is transferred to ethanol. Different types of donor substrates yielded different amounts of EtG in the presence of the same GUSB-activity and different types of GUSB yielded different amounts of EtG from the same donor substrate.

The detection of EtG in several settings independent from human UGT-metabolism (e.g., EtG post collection synthesis in E.coli contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the observed mechanism. A contribution of GUSB activity to EtG kinetics in man is suspected and should be further investigated since high concentrations of ethanol (oral intake of alcoholic beverages) and high concentrations of ß-D-glucuronides (hepatobiliary secretion) are present in the gut, where the main GUSB activity is localized. Finally, the findings could be of interest in context of EtG hair analysis, since the prerequisites for in vitro formation of EtG in this study may also be found on head. There is an additional biochemical path and another source of EtG which deserves further attention in context of alcohol biomarker applications.