Postcollection Synthesis of Ethyl Glucuronide by Bacteria in Urine May Cause False Identification of Alcohol Consumption

Department of Clinical Neuroscience, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden.
Clinical Chemistry (Impact Factor: 7.91). 11/2007; 53(10):1855-7. DOI: 10.1373/clinchem.2007.089482
Source: PubMed


Ethyl glucuronide (EtG) is a minor ethanol metabolite used as a specific marker to document recent alcohol consumption; confirm abstinence in treatment programs, workplaces, and schools; and provide legal proof of drinking. This study examined if bacterial pathogens in urine may enable postsampling synthesis of EtG and ethyl sulfate (EtS) from ethanol, leading to clinical false-positive results.
Urine specimens with confirmed growth of Escherichia coli, Klebsiella pneumoniae, or Enterobacter cloacae were stored at room temperature in the presence of ethanol. Ethanol was either added to the samples or generated by inoculation with the fermenting yeast species Candida albicans and glucose as substrate. EtG and EtS were measured by LC-MS.
High concentrations of EtG (24-h range 0.5-17.6 mg/L) were produced during storage in 35% of E. coli-infected urines containing ethanol. In some specimens that were initially EtG positive because of recent alcohol consumption, EtG was also sensitive to degradation by bacterial hydrolysis. In contrast, EtS was completely stable under these conditions.
The presence of EtG in urine is not a unique indicator of recent drinking, but might originate from postcollection synthesis if specimens are infected with E. coli and contain ethanol. Given the associated risks for false identification of alcohol consumption and false-negative EtG results due to bacterial degradation, we recommend that measurement of EtG be combined with EtS, or in the future possibly replaced by EtS.

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    • "Freeze-thaw stability was expressed as a ratio of the observed means versus the respective target concentration. Post-collection syntheses of EtG, POPE, and other alcohol biomarkers in specimens exposed to or contaminated with ethanol have been reported under a variety of conditions [52] [53] [54] [55]. In the field, it is reasonable to expect that a UC specimen may be exposed to ethanol either intentionally or unintentionally. "
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    ABSTRACT: In utero exposure to ethanol continues to be a significant public health issue and neonatal healthcare professionals are in need of objective means to identify exposed newborns. The aim of this study was to fully validate two methods for the detection of two direct alcohol biomarkers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPE) and ethyl glu-curonide (EtG), in umbilical cord and apply the assays to a group of authentic specimens. The limits of detections were 2 and 1 ng/g for POPE and ETG and the limits of quantitation were 4 and 3 ng/g, respectively. Inter and intra-day preci-sion and accuracy measurements were within 15%. The assays were applied to 308 authentic specimens where we de-tected POPE in five (1.6%) specimens and EtG in twelve (3.9%) specimens. The mean concentrations were 11.4 ng/g ± 9.4 ng/g and 127.2 ± 227.7 ng/g for POPE and EtG, respectively. This study suggested that umbilical cord was a suit-able specimen type for the identification of newborns exposed to ethanol in the womb and the prevalence of POPE and EtG detected in umbilical cord were consistent with the prevalence of self-reported binge drinking reported by the Na-tional Birth Defect Prevention Study (NBDPS) and Behavioral Risk Factor Surveillance System (BRFSS). Further studies are required to fully describe the association between the observed concentrations of POPE and EtG in umbilical cord to the level of maternal consumption of ethanol.
    Full-text · Article · Nov 2012 · American Journal of Analytical Chemistry
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    • "EtG and EtS are conjugated ethanol metabolites. We have demonstrated [29,30] that EtG is sensitive to bacterial degradation (e.g. in the presence of E. coli) but also that EtG may be formed due to bacterial activity when ethanol is present. In contrast, EtS is not susceptible to bacterial degradation/synthesis making it the preferable biomarker for recent drinking in post-mortem toxicology. "
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    ABSTRACT: Alcohol makes an important contribution to premature mortality in many countries in Eastern Europe, including Estonia. However, the full extent of its impact, and the mechanisms underlying it, are challenging issues to research. We describe the design and initial findings of a study aimed at investigating the association of alcohol with mortality in a large series of forensic autopsies of working-age men in Estonia. 1299 male deaths aged 25-54 years were subject to forensic autopsy in 2008-2009. The routine autopsy protocol was augmented by a more systematic inspection of organs, drug testing, assay of liver enzymes and novel biomarkers of alcohol consumption (EtG, EtS and PEth), together with proxy interviews with next of kin for deaths among men who lived in or close to a major town. 595 augmented autopsies were performed. Of these, 66% were from external causes (26% suicide, 25% poisoning). 17% were attributed to circulatory system diseases and 7% to alcoholic liver disease. Blood alcohol concentrations (BAC) of ≥ 0.2 mg/g were found for 55% of deaths. Interviews were conducted with proxy informants for 61% of the subjects who had resided in towns. Of these, 28% were reported in the previous year to have been daily or almost daily drinkers and 10% had drunk non-beverage alcohols. Blood ethanol and the liver enzyme GGT were only associated with daily drinking. However, the novel biomarkers showed a more graded response with recent consumption. In contrast, the liver enzymes AST and ALT were largely uninformative because of post-mortem changes. The presence of extremely high PEth concentrations in some samples also suggested post-mortem formation. We have shown the feasibility of deploying an extended research protocol within the setting of routine forensic autopsies that offer scope to deepen our understanding of the alcohol-related burden of premature mortality. The most unique feature of the study is the information on a wide range of informative alcohol biomarkers, several of which have not been used previously in this sort of post-mortem research study. We have demonstrated, for the first time, the epidemiological value and validity of these novel alcohol biomarkers in post-mortem samples.
    Full-text · Article · Feb 2012 · BMC Public Health
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    • "Freeze-thaw stability was expressed as a ratio of the observed means versus the respective target con- centration. A concern with the use of ethanol biomarkers is the possibility of post-collection synthesis when specimens have been exposed to or contaminated with ethanol [17] [18] [32] [33] [34] [35] "
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    ABSTRACT: Over the past decade, the use of hair specimens for the long-term detection of the alcohol biomarker ethyl glucuronide has been increasing in popularity and usage. We evaluated the usefulness of fingernail clippings as a suitable alternative to hair for ethyl glucuronide detection. A liquid chromatography-tandem mass spectrometry method for the detection of ethyl glucuronide in fingernail clippings was fully validated and used to analyze the hair and/or fingernail specimens of 606 college-aged study participants. The limit of detection was 2 pg/mg, the limit of quantitation was 8 pg/mg and the method was linear from 8 to 2000 pg/mg. Intra-and inter-assay imprecision studies at three different concentrations (20, 40, 200 pg/mg) were all within 7.8% and all intra-and inter-assay bias studies at these levels were within 115.1% of target concentration. Ethyl glucuronide levels in fingernail (mean = 29.1 ± 55.6 pg/mg) were higher than ethyl glu-curonide levels in hair (mean = 9.48 ± 22.3 pg/mg) and a correlation of the matched pairs was observed (r = 0.552, P < 0.01, n = 529). Evaluating each gender separately revealed that the correlation of male fingernail to male hair was large and significant (r = 0.782, P < 0.01, n = 195) while female hair to female fingernail was small yet significant (r = 0.249, P < 0.01, n = 334). The study results demonstrated that fingernail may be a suitable alternative to hair for ethyl glu-curonide detection and may be the preferred sample type due to the lack of a gender bias.
    Full-text · Article · Jan 2012 · American Journal of Analytical Chemistry
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