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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: 7.77). 11/2007; 53(10):1855-7. DOI: 10.1373/clinchem.2007.089482
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ABSTRACT 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.
    American Journal of Analytical Chemistry 11/2012; 3(12):800-810. DOI:10.4236/ajac.2012.312106
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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.
    American Journal of Analytical Chemistry 01/2012; 3(01):83-91. DOI:10.4236/ajac.2012.31012
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    • "c o m / l o c a t e / f o r s c i i n t post-mortem tissue [18]. In vitro investigations did not only show degradation of EtG [19], but also possible formation of this ethanol metabolite due to microorganisms [20]. EtS has proven stable in most studies and was degraded only under test conditions with very high bacteria counts in a manometric respiratory test [21]. "
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    ABSTRACT: To clarify the circumstances of death, the degree of inebriation is of importance in many cases, but for several reasons the determination of the ethanol concentration in post-mortem samples can be challenging and the synopsis of ethanol and the direct consumption markers ethyl glucuronide (EtG) and ethyl sulphate (EtS) has proved to be useful. The use of a rather stable matrix like vitreous humor offers further advantages. The aim of this study was to determine the concentrations of ethanol and the biomarkers in the robust matrix of vitreous humor and to compare them with the respective levels in peripheral venous blood and urine. Samples of urine, blood from the femoral vein and vitreous humor were taken from 26 deceased with suspected ethanol consumption prior to death and analyzed for ethanol, EtS and EtG. In the urine samples creatinine was also determined. The personal data, the circumstances of death, the post-mortem interval and the information about ethanol consumption prior to death were recorded. EtG and EtS analysis in urine was performed by LC-ESI-MS/MS, creatinine concentration was determined using the Jaffé reaction and ethanol was detected by HS-GC-FID and by an ADH-based method. In general, the highest concentrations of the analytes were found in urine and showed statistical significance. The mean concentrations of EtG were 62.8mg/L (EtG100 206.5mg/L) in urine, 4.3mg/L in blood and 2.1mg/L in vitreous humor. EtS was found in the following mean concentrations: 54.6mg/L in urine (EtS100 123.1mg/L), 1.8mg/L in blood and 0.9mg/L in vitreous humor. Ethanol was detected in more vitreous humor samples (mean concentration 2.0g/kg) than in blood and urine (mean concentration 1.6g/kg and 2.1g/kg respectively). There was no correlation between the ethanol and the marker concentrations and no statistical conclusions could be drawn between the markers and matrices.
    Forensic science international 02/2011; 210(1-3):63-8. DOI:10.1016/j.forsciint.2011.01.036 · 2.12 Impact Factor
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