Ethyl glucuronide, a new biochemical marker for acute alcohol intake : Studies on possible causes for false-negative or false-positive results

Abstract: In recent years, there has been a growing interest in various biochemical markers for detecting acute and chronic alcohol consumption. Biochemical markers for acute and chronic drinking play important roles in detecting alcohol use, abuse and dependence in hospital settings, work place settings, traffic medicine and in forensic toxicology examinations. The alcohol biomarkers can be distinguished into two main classes: 1) tests that are sensitive enough to detect a single intake of alcohol, such as ethanol, 5-hydroxytryptophol(5HTOL), ethyl glucuronide (EtG) and ethyl sulfate (EtS), and 2) tests that are able to detect chronic heavy drinking, or indicate body organ or tissue damage caused by long-term exposure to alcohol, such as carbohydrate-deficient transferrin (CDT), g -glutamyl transferase (GGT), aspartate and alanine aminotransferase (AST and ALT), and the mean corpuscular volume of erythrocytes (MCV). After consumption of alcoholic beverages, a small portion (<5%) of the ethanol is excreted unchanged in the urine, sweat and breath. The major part (>95%) instead becomes metabolized mainly in the liver in a two-stage oxidation process, first to acetaldehyde by alcohol dehydrogenase and further to acetate by aldehyde dehydrogenase. Another very small part undergoes non-oxidative metabolism to produce the phase II products EtG and EtS. The interest in EtG and EtS as biochemical markers for acute alcohol intake has primarily focused on the observation that the washout rates for these direct ethanol metabolites are much slower than for the parent compound, allowing for a longer detection time (i.e., higher sensitivity). A positive finding of EtG and/or EtS in urine or serum thus provides a strong indication that the person was recently drinking alcohol, even if drinking is denied and also for some time after ethanol itself is no longer detectable. The purpose of this thesis was to evaluate the accuracy of EtG in urine as a biochemical marker for acute alcohol consumption, by studying possible sources of error that can affect the result of the analysis and cause false-negative or false-positive results. Urinary EtG was determined by liquid chromatography-mass spectrometry (LC-MS). The first study demonstrated that glucuronide conjugation of ethanol forming EtG represents a minor elimination pathway (<0.03%) in the human body, and also confirmed that EtG remains detectable in the urine for many hours after the ethanol has been eliminated. Drinking large amount of fluid prior to voiding was found sufficient to markedly lower the urinary concentration of EtG, but this practice did not influence the concentration of ethanol or the EtG/creatinine ratio. Expressing EtG as a ratio to creatinine may thus be recommended in clinical practice, to compensate for urine dilution. In the second study, no significant accumulation of EtG or 5HTOL was observed, upon multiple-dose administration of ethanol at 0.8 g/kg/day for one week to healthy volunteers. Moreover, the detection time in urine for EtG was demonstrated to be longer than for 5HTOL. In the last study, it was found that EtG but not EtS is sensitive to bacterial hydrolysis, particularly in specimens infected by Escherichia coli, which is a well known source of ß-glucuronidase. Accordingly, to reduce the risk for obtaining falsely low or false-negative EtG results, specimens should be stored refrigerated or frozen prior to analysis. Sampling urine in test tubes containing sodium fluoride was also found effective to prevent bacterial hydrolysis of EtG. In conclusion, the present results demonstrated that urinary EtG is a very sensitive and specific biochemical marker for acute alcohol intake. Urine dilution and bacterial hydrolysis were identified as possible causes for falsely low or false-negative EtG results. For routine clinical use, it may therefore be recommended to express urinary EtG as a ratio to creatinine, and to combine EtG and EtS analysis, which is possible by LC-MS.

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