1D Proton NMR Spectroscopic Determination of Ethanol and Ethyl Glucuronide in Human Urine

Abstract

Forensic and legal medicine require reliable data to indicate excessive alcohol consumption. Ethanol is oxidatively metabolized to acetate by alcohol dehydrogenase and non-oxidatively metabolized to ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanol, or fatty acid ethyl esters (FAEE). Oxidative metabolism is too rapid to provide biomarkers for the detection of ethanol ingestion. However, the nonoxidative metabolite EtG is a useful biomarker because it is stable, non-volatile, water soluble, highly sensitive, and is detected in body fluid, hair, and tissues. EtG analysis methods such as mass spectroscopy, chromatography, or enzyme-linked immunosorbent assay techniques are currently in use. We suggest that nuclear magnetic resonance (NMR) spectroscopy could be used to monitor ethanol intake. As with current conventional methods, NMR spectroscopy doesn't require complicated pretreatments or sample separation. This method has the advantages of short acquisition time, simple sample preparation, reproducibility, and accuracy. In addition, all proton-containing compounds can be detected. In this study, we performed 1 H NMR analyses of urine to monitor the ethanol and EtG. Urinary samples were collected over time from 5 male volunteers. We confirmed that ethanol and EtG signals could be detected with NMR spectroscopy. Ethanol signals increased immediately upon alcohol intake, but decreased sharply over time. In contrast, EtG signal increased and reached a maximum about 9 h later, after which the EtG signal decreased gradually and remained detectable after 20-25 h. Based on these results, we suggest that 1 H NMR spectroscopy may be used to identify ethanol non-oxidative metabolites without the need for sample pretreatment.

Automated analysis of large-scale NMR data generates metabolomic signatures and links them to candidate metabolites

“…To better understand why module #240 correlates more strongly to the alcohol consumption biomarker while being a worse match to ethanol than module #57 (Figure 3), we studied whether any of its features point to other compounds related to ethanol metabolism. Indeed, we found that this module contains three (Nicholas et al 2006) and more recently in human urine of alcohol drinkers (Kim et al 2013) . To confirm EtG as a possible match for ISA module #240, we added its features as extracted from (Nicholas et al 2006) to the metabomatching library manually, since EtG had no entry in UMDB.…”

Automated Analysis of Large-Scale NMR Data Generates Metabolomic Signatures and Links Them to Candidate Metabolites

“…Indeed, we found that this module contains three features, at 1.26, 3.52, and 4.47 ppm, that individually correlate more strongly to CDT (0.40 [0.34, 0.45], 0.29 [0.23, 0.35], 0.33 [0.27, 0.39], respectively) than the features mapping to ethanol. Interestingly, these features appeared to be close to those of ethyl glucuronide (EtG), a direct product of ethanol nonoxidative metabolism by conjugation with uridine diphosphate (UDP)-glucuronic acid, which had previously been detected in 1 H NMR spectra of liver extracts and more recently in human urine of alcohol drinkers . To confirm EtG as a possible match for ISA module #240, we added its features as extracted from Nicholas et al to the metabomatching library manually, since EtG had no entry in UMDB.…”

Meeting report: The biology of genomes and proteomes

“…Inhaled toluene is metabolized to hippuric acid in the liver and excreted in the urine. In another example, Dr. Kim found that the biomarker ethyl glucuronide could be used instead of ethanol as an indicator of alcohol consumption (Kim et al 2013a). Dr. Kim applied NMR-based metabolomics to tests of environmental toxicity and successfully found many biomarkers in zebrafish for each chemical treatment tested, including organic solvents, pesticides, and medicines (Yoon et al 2013).…”