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Urine tested positive for ethyl glucuronide and ethyl sulphate after the consumption of "non-alcoholic" beer
Abstract
In abstinence maintenance programs, for reissuing the driving licence and in workplace monitoring programs abstinence from ethanol and its proof are demanded. Various monitoring programs that mainly use ethyl glucuronide (EtG) as alcohol consumption marker have been established. To abstain from ethanol, but not from the taste of alcoholic beverages, in particular non-alcoholic beer has become more and more popular. In Germany, these "alcohol-free" beverages may still have an ethanol content of up to 0.5vol.% without the duty of declaration. Due to severe negative consequences resulting from positive EtG tests, a drinking experiment with 2.5L of non-alcoholic beer per person was performed to address the question of measurable concentrations of the direct metabolites EtG and EtS (ethyl sulphate) in urine and blood. Both alcohol consumption markers - determined by LC-MS/MS - were found in high concentrations: maximum concentrations in urine found in three volunteers were EtG 0.30-0.87mg/L and EtS 0.04-0.07mg/L, i.e., above the often applied cut-off value for the proof of abstinence of 0.1mg EtG/L. In the urine samples of one further volunteer, EtG and EtS concentrations cumulated over-night and reached up to 14.1mg/L EtG and 16.1mg/L EtS in the next morning's urine. Ethanol concentrations in blood and urine samples were negative (determined by HS-GC-FID and by an ADH-based method).
... Studies showed that EtG concentrations above 0.1 mg/l can also be determined in urine after the consumption of food and beverages that contain small amounts of alcohol, such as nonalcoholic beer [24,25] or yeast in combination with sugar [26] and after the use of alcohol-containing mouthwash [21,27] or hand sanitizers [10,28,29]. Because of these results, it is essential to establish cutoff values which exclude both false positive results due to unintentional consumption of alcohol as well as negative results despite prior alcohol intake. ...
... Even EtG concentrations above 0.1 mg/l may be detectable in urine after consumption of food or beverages containing hidden amounts of alcohol or after intensive use of alcohol-containing cosmetics [10,21,[24][25][26][27][28][29]. An excess of the cutoff was determined only for a limited time period which is considerable shorter than 24 h [24,25,28]. ...
... Even EtG concentrations above 0.1 mg/l may be detectable in urine after consumption of food or beverages containing hidden amounts of alcohol or after intensive use of alcohol-containing cosmetics [10,21,[24][25][26][27][28][29]. An excess of the cutoff was determined only for a limited time period which is considerable shorter than 24 h [24,25,28]. ...
Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are commonly used alcohol markers for previous alcohol consumption. Nevertheless, the optimum EtG cutoff for urinary abstinence tests is still being discussed, and no cutoff has been recommended for EtS yet. The aim of this study was to verify cutoffs by investigating EtG and EtS concentrations (cEtG and cEtS) in the urine of healthy persons after drinking small, but realistic amounts of alcohol (one or two glasses of beer or white wine), and to look for the window of detection in strongly alcohol-intoxicated patients who were beginning withdrawal treatment. Very high EtG and EtS concentrations were measured in the first urine samples of patients under withdrawal treatment. However, 24 h later, concentrations decreased considerably, and c
EtG < 0.5 mg/l and c
EtS < 0.1 mg/l were determined in 26.7 % (4/13) and 13.3 % (2/13) of the samples, respectively. Concentrations above 0.1 mg/l (EtG) and 0.05 mg/l (EtS) were measured for 23.5 and 20.5 h after consuming 0.1 l of white wine or 0.33 l of beer, and 24 h after the experiment, 75 % (9/12) of the urine samples were tested negative for EtG and EtS using the following cutoffs: EtG 0.5 mg/l and EtS 0.1 mg/l. In half of the samples, concentrations below 0.1 mg/l (EtG) and 0.05 mg/l (EtS) were detected. Urinary cutoffs for EtG of 0.5 mg/l or higher are not suitable for testing abstinence. Even 0.1 mg/l is not effective to detect the intake of small amounts of alcohol in the context of abstinence tests. For EtS, 0.05 mg/l were found to be a potential cutoff to exclude the repeated intake of alcohol. Yet, further research is required to verify this cutoff. For a limited time period, EtG and EtS concentrations within the range of these cutoffs are also detectable after unintentional consumption of alcohol. Participants of abstinence programs have to be informed about the alcohol content of certain foods and beverages whose consumption is in conflict with strict abstinence.
... EtG in hair has been used as a marker of alcohol abuse. False positives have been reported for both hair [128] and urine [129][130][131]. The concentrations of EtG in OF are considerably lower than those in blood and its detection window is also shorter [132]. ...
... Therefore, concurrent determination of EtS and EtG will improve sensitivity when being used as biomarkers of recent drinking. However, false positives have been reported in the urine of a patient drinking "non-alcoholic" beer [130]. ...
Urine drug testing is one of the objective tools available to assess adherence. To monitor adherence, quantitative urinary results can assist in differentiating “new” drug use from “previous” (historical) drug use. “Spikes” in urinary concentration can assist in identifying patterns of drug use. Coupled chromatographic-mass spectrometric methods are capable of identifying very small amounts of analyte and can make clinical interpretation rather challenging, specifically for drugs that have a longer half-life. Polypharmacy is common in treatment and rehabilitation programs because of co-morbidities. Medications prescribed for comorbidities can cause drug-drug interaction and phenoconversion of genotypic extensive metabolizers into phenotypic poor metabolizers of the treatment drug. This can have significant impact on both pharmacokinetic (PK) and pharmacodynamic properties of the treatment drug. Therapeutic drug monitoring (TDM) coupled with PKs can assist in interpreting the effects of phenoconversion. TDM-PKs reflects the cumulative effects of pathophysiological changes in the patient as well as drug-drug interactions and should be considered for treatment medications/drugs used to manage pain and treat substance abuse. Since only a few enzyme immunoassays for TDM are available, this is a unique opportunity for clinical laboratory scientists to develop TDM-PK protocols that can have a significant impact on patient care and personalized medicine. Interpretation of drug screening results should be done with caution while considering pharmacological properties and the presence or absence of the parent drug and its metabolites. The objective of this manuscript is to review and address the variables that influence interpretation of different drugs analyzed from a rehabilitation and treatment programs perspective.
... Once the alcohol has been ingested, it is degraded to two minor metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), which are excreted through the urine (0.010-0.016% on molar basis) (Høiseth et al., 2008). Both are determined in clinical and forensic laboratories for the surveillance of abstinence, establishing the alcohol consumption in workplace testing and rehabilitation programmes for alcohol dependence (Thierauf et al., 2010a;Thierauf et al., 2011). ...
... Contrarily, EtS has been demonstrated to be stable in manometric respiratory testhigh concentrations of bacteria and EtSfor at least 6 days and up to 28 days in closed bottle testlow EtS and bacteria density from a WWTPs effluent (Halter et al., 2009;Thierauf et al., 2008). Analytical methods have been described for the determination of EtS in biological matrices such as plasma, serum, vitreous humour, placental and foetal tissues, and meconium (Kummer et al., 2013;Morini et al., 2010;Morini et al., 2007;Thierauf et al., 2010a;Thierauf et al., 2011;Thierauf et al., 2008;Thierauf et al., 2010b) either by gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography with pulsed electrochemical detection, capillary zone electrophoresis, or immunochemical test. Otherwise, few bioanalytical methods are available for the determination of EtS in wastewater where a lower detection limit is required Reid et al., 2011;Rodríguez-Álvarez et al., 2014). ...
... Cutoff PEth concentrations were set at <20 ng/ml (compatible with abstinence or low alcohol consumption) and ≥200 ng/ml (indicating chronic excessive alcohol consumption) (Ulwelling and Smith, 2018;Luginbühl et al., 2022). For EtG in urine (uEtG), a cutoff concentration of 100 ng/ml was applied (Thierauf et al., 2010). The half-lives of the two PEth analogs were calculated using Prism 5.03, GraphPad Software. ...
Aims:
Phosphatidylethanol (PEth) is used to monitor alcohol consumption in alcohol use disorder (AUD). In this study, we aim to evaluate the elimination time of PEth with regard to the clinically established 200 and 20 ng/ml cutoffs for PEth 16:0/18:1.
Methods:
Data from 49 patients undergoing treatment for AUD were evaluated. PEth concentrations were measured at the beginning and repeatedly during the treatment period of up to 12 weeks to monitor the elimination of PEth. We evaluated the time in weeks until the cutoff concentrations of <200 and <20 ng/ml were achieved. The correlation between the initial PEth concentration and the number of days until the PEth concentration had dropped below 200 and 20 ng/ml was assessed by calculating Pearson's correlation coefficients.
Results:
The initial PEth concentrations ranged from <20 to >2500 ng/ml. In 31 patients, the time until the cutoff values were reached could be documented. Even after 6 weeks of abstinence, PEth concentrations above the cutoff of 200 ng/ml could still be detected in two patients. A strong significant positive correlation was found between the initial PEth concentration and the time required to drop below the two cutoffs.
Conclusion:
A waiting period of more than 6 weeks after declared abstinence should be granted for individuals with AUD before using only one single PEth concentration to assess the consumption behavior. However, we recommend to always use at least two PEth concentrations for the evaluation of alcohol-drinking behaviors in AUD patients.
... Ethyl sulfate (EtS), a minor metabolite of ethanol, has been widely used as a WBE biomarker for alcohol consumption, 4 and it is also an established biomarker used in clinical and forensic settings. [97][98][99] HD4: Are changes in alcohol consumption reflected in changes in EtS levels? Excretion of EtS was shown to be dose dependent, based on the quantity of ethanol ingested. ...
Background:
Wastewater-based epidemiology (WBE) is rapidly developing as a powerful public health tool. It can provide information about a wide range of health determinants (HDs), including community exposure to environmental hazards, trends in consumption of licit and illicit substances, spread of infectious diseases, and general community health. As such, the list of possible candidate HDs for WBE is almost limitless. Consequently, a means to evaluate and prioritize suitable candidates for WBE is useful, particularly for public health authorities, who often face resource constraints.
Objectives:
We have developed a framework to assist public health authorities to decide what HDs may be appropriate for WBE and what biomarkers could be used. This commentary reflects the experience of the authors, who work at the interface of research and public health implementation.
Discussion:
To be suitable for WBE, a candidate HD should address a public health or scientific issue that would benefit from better understanding at the population level. For HDs where information on individual exposures or stratification by population subgroups is required, WBE is less suitable. Where other methodologies are already used to monitor the candidate HD, consideration must be given to whether WBE could provide better or complementary information to the current approach. An essential requirement of WBE is a biomarker specific for the candidate HD. A biomarker in this context refers to any human-excreted chemical or biological that could act as an indicator of consumption or exposure to an environmental hazard or of the human health state. Suitable biomarkers should meet several criteria outlined in this commentary, which requires background knowledge for both the biomarker and the HD. An evaluation tree summarizing key considerations for public health authorities when assessing the suitability of candidate HDs for WBE and an example evaluation are presented. https://doi.org/10.1289/EHP11115.
... Currently available biomarkers have several drawbacks (Seneviratne & Johnson, 2012); for example, commonly used biological markers (biomarkers), ethyl glucuronide (EtG) and ethyl sulfate (EtS), are better predictors of alcohol exposure and reported to be less informative of quantity or patterns of drinking (Helander et al., 2009;Mastrovito & Strathmann, 2020). Some studies report elevated EtG and/or EtS levels following consumption of non-alcoholic beverages such as non-alcoholic beer and wine (Hoiseth et al., 2010;Thierauf et al., 2010) or incidental exposures to alcohol in hand sanitizers or cosmetic hair products (Muller & Iwersen-Bergmann, 2018;Reisfield et al., 2011), questioning their utility for predicting abstinence. ...
Background:
The serotonin transporter (SERT) mRNA was previously reported as a quantitative and pathophysiology-based biomarker of heavy drinking in 5HTTLPR:LL genotype-carriers treated with ondansetron. Here, we further validated the potential use of SERT mRNA for quantitative prediction of recent alcohol consumption (in the absence of treatment) and compared with known biomarkers ethyl glucuronide (EtG) and sulfate (EtS).
Methods:
Binge drinking men and women of European ancestry aged 21-65 years were enrolled in a 12-day, in-patient, randomized, double-blind, crossover study, where three beverage doses (placebo, 0.5g/kg (0.4g/kg), and 1g/kg (0.9g/kg) for men (women)) were administered individually in three four-day periods (experiments), separated by minimum of seven-day washout. Diet, sleep, and physical activity were controlled throughout the inpatient experiments. Twenty-nine participants were randomized to receive beverage doses counterbalancing sequence of treatment and gender within subgroups stratified by SERT genotypes 5HTTLPR:LL+rs25531:AA (LA LA ) vs 5HTTLPR:LS/SS. Peripheral venous blood was collected daily for (1) quantification of SLC6A4 mRNA (primary outcome measure) using qRT-PCR and (2) plasma EtG and EtS levels using tandem mass-spectrometry.
Results:
The association between administered beverage dose and SERT mRNA from completers of at least one four-day experiment (N=18) assessed by a linear mixed model was not statistically significant. Significant positive associations were found with beverage dose and plasma EtG, EtS and EtG/EtS ratio (beta=5.8, SE=1.2, p<0.0001; beta=1.3, SE=0.6, p=0.023; and beta=3.0, SE=0.7, p<0.0001, respectively; C-statistic for discriminating outcomes were 0.97, 0.8, and 0.92, respectively). Additionally, we observed a sequence effect with greater placebo effect on SERT mRNA when administered during the first experiment (p=0.0009), but not on EtG/EtS measures.
Conclusion:
Larger and more innovative studies addressing effects of placebo, race, gender, and response to treatment with serotonergic agents are needed to fully assess SERT, and possibly other mRNA biomarkers of heavy and binge drinking.
... The reported EtG and EtS concentrations were not normalized to creatinine. As a result, direct comparison to previously published data in urine after consuming non-alcoholic beers and drinks containing yeast and sugar or trace amounts of ethanol might not be possible (22)(23)(24). However, the reported results showed that positive EtG and EtS detection in urine was plausible through the consumption of just a single serving of non-alcoholic kombucha beverage. ...
Although kombucha is a popular fermented beverage, the presence of alcohol markers has not been well studied despite being potential indicators of unintentional impairment. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) were measured in oral fluid and urine collected after consumption of regular or hard kombucha. Participants drank within 20 min and provided all urine voids for 12 h, the first urine voids on days 2 and 3, and oral fluid specimens at fixed time points for 48 h. Screening employed liquid chromatography–tandem mass spectrometry (LC–MS-MS; EtS, 25 ng/mL cutoff [oral]; 100 ng/mL cutoff [urine]; EtG, 500 ng/mL cutoff [urine] and immunoassay (IA; EtG, 500 ng/mL cutoff[urine]). After consuming regular kombucha (n = 12 participants), EtS was not detected in oral fluid but both markers were detected by LC–MS-MS in urine specimens within the first 5 voids from 83% of participants with median (range) concentrations of 240 (100–3,700) ng/mL for EtS and 830 (530–2,200) ng/mL for EtG. Neither marker was positive by IA nor LC–MS-MS after day 1. After consuming hard kombucha (n = 7 participants), 2 (2.8%) of the 70 collected oral fluid specimens tested positive for EtS 3 h after consumption; however, 21 (30%) had EtS levels above the limit of detection (LOD, 10 ng/mL) after 0.5–8 h. Both markers were detected in urine specimens from all participants with median (range) concentrations of 3,381 (559–70,250) ng/mL for EtS and 763 (104–12,864) ng/mL For EtG. Urine specimens were negative for EtG and EtS by the end of the 48-hour study.
... As larger studies were conducted to establish the validity of these assays, it also became clear that negative or positive results did not always provide definitive measures of compliance with abstinence or proof of alcohol intake. For example, it was possible to get positive results for EtG and EtS even when drinking non-alcoholic beer (24), sub-intoxicating doses of ethanol (25) or using ethanol-based hand cleansers (26). Likewise, the final concentration of biomarkers may be affected by the bacterial degradation of EtG (27). ...
The aim of the study is to evaluate the contribution of ethanol metabolite detection in postmortem cases by showing the connection between the presence of ethanol metabolites, which are indicators of alcohol consumption, and the detection of potential postmortem ethanol formation in decomposed and diabetic cases. Determination of ethanol consumption before death is often one of the most important questions in death investigations. Postmortem ethanol formation or degradation products in the blood make it difficult to distinguish antemortem consumption or postmortem formation of ethanol and eventually may lead to misinterpretation. Decomposed bodies and diabetic cases are vulnerable to postmortem ethanol formation due to putrefaction, fermentation or other degradations. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are two metabolites of ethanol produced only in the antemortem time interval. In this study, EtG and EtS levels in urine and vitreous humor samples of 27 postmortem cases, including diabetic and degraded bodies were compared to ethanol results of their blood, urine and vitreous humor samples. EtG and EtS in urine and vitreous humor were analyzed by liquid chromatography–tandem mass spectrometry, and ethanol was assayed by routine headspace gas chromatography–flame ionization detector. These cases were devoid of other influences from forensically relevant drugs, so ethanol and/or glucose were among the only positive findings in these cases. The results of this pilot study indicate the postmortem ethanol concentrations do not correlate with the measured EtG and EtS values but are beneficial in rulings of accidental or natural deaths. This preliminary study gives additional data to help distinguish between antemortem ethanol intake and postmortem formation. EtG and EtS were well correlated positively with antemortem ethanol use instead of forming spontaneously in samples from decedents who are decomposing or have a history of diabetic hyperglycemia.
... Pojawiają się także doniesienia, że okazjonalna ekspozycja na alkohol zawarty w produktach codziennego użytku (np. płyny do płukania jamy ustnej, żele do dezynfekcji rąk), a także spożycie drożdży piekarskich z cukrem lub bezalkoholowego piwa mogą spowodować wzrost stężenia EtG, dlatego bardzo ważne jest informowanie o tym pacjentów, u których planowane jest jego oznaczenie [14,38,39]. Śladowe ilości EtG występują naturalnie w winie, przez co możliwe jest zanieczyszczenie zewnętrzne próbki poddawanych analizie włosów. ...
Alcohol ranks as one of the leading behavioral threats to health and life in developed countries. Alcohol abuse triggers serious social and economic effects: it contributes to higher prevalence of work-related and road accidents, as well as absence from work. The diagnosis and treatment of alcoholism still remain very difficult. Hence, the use of objective biochemical markers of alcohol abuse may contribute to earlier detection, more effective therapy and reliable teetotalism control. The aim of this study is to present the sensitive and specific biomarkers of alcohol abuse available in Poland, with particular emphasis on the practical use possibilities. Such tests may be widely used, e.g., in driving license regranting cases involving drivers whose licenses were suspended for driving when intoxicated, for the early detection of persons abusing alcohol in employment-related health controls, for abstinence monitoring during withdrawal treatment, for detecting alcohol consumption in transplant settings, for assessing the prevalence of alcohol drinking in pregnancy, as well as in autopsical examinations. The standardization of biomarkers measurement methods is essential. Moreover, concomitant disorders may pose a significant problem in the proper outcome analysis. Despite these limitations, objective biochemical markers of ethyl alcohol abuse may become helpful tools in medical care. They can play a particular role in occupational medicine diagnostics, contributing to the higher level of safety on public roads and to worker safety. Med Pr. 2021;72(2).
... 015 g/210 l) on a portable breath testing device within (Musshoff et al. 2010). Non alcoholic beers with ethanol concentrations of 3.1 to 3.6 g/l were investigated by Musshoff et al. (2010) and Thierauf et al. (2010) after consumption of 2 to 3 l and 2.5 l, respectively. Both studies revealed positive urine EtG and EtS levels. ...
Abstract Background Traditional beverages such as şalgam, boza, kımız and kefir are frequently being consumed in Turkey. During the production of these beverages fermentation provides long time bio-preservation due to protective effects of ethanol, a product of microorganisms’ metabolism. Especially in traffic controls and in rehabilitation process of alcoholic patients, fermented beverages may cause confusion since it may be linked with false positivity of ethanol and ethanol metabolite tests. The present study aims to assess blood ethanol and urine ethanol metabolite levels after consumption of traditional fermented beverages. Method Twelve participants consumed 300 mL standardized homemade traditional şalgam, boza, kımız and kefir. Blood samples were collected before and after (at 45th min) consumption and urine samples were collected before and after (at 4th hour) consumption from the participants. The samples of these four beverages and the blood samples were analyzed using headspace gas chromatography for ethanol levels, and urine samples were analyzed with liquid chromatography tandem mass spectrometry for ethyl-glucuronide and ethyl-sulphate levels. Results Ethanol levels in şalgam, boza, kımız, and kefir samples, used at the present study, were 80.02 mg/dl, 30.02 mg/dl, 57.83 mg/dl and 6.07 mg/dl, respectively. After consumption of beverages, blood ethanol levels were negative for all analyzed samples. No statistically significant correlation was found between initial and post-consumption ethyl-glucuronide and ethyl-sulphate levels.
... Ñïåöèôè÷åñêèìè ïðÿìûìè ìàðêåðàìè óïîòðåáëåíèÿ ýòàíîëà, âûÿâëÿåìûìè â ìî÷å ÷åëîâåêà â òå÷åíèå íåñêîëüêèõ ñóòîê ïîñëå òîãî, êàê ñàì ñïèðò â áèîëî-ãè÷åñêèõ aeèäêîñòÿõ íå îïðåäåëÿåòñÿ, ÿâëÿþòñÿ ïðîäóêòû åãî íåîêèñëèòåëüíîãî ìåòàáîëèçìà -ýòèëãëþêîðîíèä (EtG) è ýòèëñóëüôàò (EtS) [4,15,29,30,34,46,75]. Íåñìîòðÿ íà òî, ÷òî ñîäåðaeàíèå EtG è EtS â ìî÷å íå ïðåâûøàåò 0,02% è 0,011% ñîîòâåòñòâåííî îò èñõîäíîé äîçû ýòàíîëà [23,30,78], ýòè ìàðêåðû óâåðåííî îïðåäåëÿþòñÿ â ìî÷å ñïóñòÿ íåñêîëüêî ÷àñîâ äàaeå ïîñëå óïîòðåáëåíèÿ «áåçàëêîãîëüíûõ» ïèâà è âèíà, êâàøåíîé êàïóñòû, àïåëüñèíîâîãî ñîêà, çðåëûõ áàíàíîâ èëè èñïîëüçîâàíèÿ ñïèðòîñîäåðaeàùèõ ïîëîñêàíèé äëÿ ðòà [54,63]. Ýòè ìàðêåðû ìîãóò áûòü âûÿâëåíû òàêaeå â âûñóøåííûõ êàïëÿõ ìî÷è [52]. ...
Review summarizes data for a new direction in epidemiology - "Wastewater-based epidemiology", SBE or "Sewage-based epidemiology", SBE. This approach is based on the analysis of content in the wastewater of specific markers of alcohol, nicotine and illegal drugs, excreted with urine. It highlights the key methodological techniques, including sample preparation, analytical methods and calculation method. Are shown comprehensive published data on the consumption of ethanol and nicotine research in tens of cities in Europe, the USA, Canada and Australia. Presents results comparing the analytical data obtained by high performance liquid chromatography and mass spectrometry with results traditional epidemiological studies. Presents the results of assessment the epidemiological risk of the use of ethanol by means of determining the "margin of exposure" (MOE). It sets out information about the possibility of using nicotine metabolites as markers to determine the population size of the population covered by wastewater treatment plants, as well as predictors of illicit drug use.
Ref. 81.
Keywords: Epidemiology; Wastewater; Sewage; HPLC; LC-MS; Ethanol; Alcohol; Nicotine; Tobacco; Margin of exposure.
... However, results are unlikely to be above the cut-offs with mouthwash use at the recommended frequency (Reisfield et al., 2011b). Certain non-alcoholic beers do contain a small amount of alcohol (up to 0.5%) and a study where volunteers consumed 2.5 L of 0.5% beer indicated that this could cause low positive EtG but not EtS results in a urine sample collected the following morning (Thierauf et al., 2010). To remove the possibility of false negative ethanol, EtG and EtS results after the consumption of large amounts of water, it is prudent to also measure creatinine in the urine (Dahl et al., 2002). ...
Aims
The ethanol metabolites ethyl glucuronide (EtG) and ethyl sulphate (EtS) are detectable for longer in urine than breath ethanol or urine ethanol after alcohol intake. This study compared the performance of breath ethanol, urine ethanol, urine EtG and EtS to detect alcohol consumption in clients in community alcohol treatment.
Methods
Clients attending the community alcohol treatment programme were asked to provide an alcohol diary, breathalyser test and urine for ethanol, EtG and EtS measurement (n = 42). Positive results were defined using the detection limits (breath ethanol and urine ethanol) or clinical cut-offs (EtG: 0.26 mg/L and EtS: 0.22 mg/L). The sensitivities and specificities of each marker to detect alcohol intake <24 and 48–72 h prior were calculated.
Results
The sensitivities of each alcohol marker to detect alcohol intake <24 h prior were 57, 71, 100 and 100% for breath ethanol, urine ethanol, urine EtG and urine EtS, respectively. The specificity was 100% for urine ethanol and urine EtS. The EtG specificity could be increased to 100% by using a higher cut-off (0.50 mg/L). The sensitivity of all markers (including EtG and EtS) to detect alcohol intake of ≤10 units 48–72 h earlier decreased to 0%.
Conclusions
In community alcohol treatment clients, urine EtG and EtS showed the optimum diagnostic performance to detect alcohol intake in the previous 24 h. We propose a flowchart to routinely use EtG and EtS for clients in community alcohol treatment.
Short
The ability of breath ethanol, urine ethanol, urine EtG and urine EtS to detect continued alcohol consumption in clients in community alcohol treatment were compared. Urine EtG and EtS showed the optimum diagnostic performance and we propose a flowchart to routinely use EtG and EtS in community alcohol treatment.
... However, this in vitro reaction can be prevented by refrigerating, freezing, or collecting urine samples in NaF containers [59] or using dried urine on filter paper [68] . Cannabinol, ingestion of high amounts of baker's yeast, sauerkraut, non-alcoholic beer, or alcohol containing mouth washes as well as severe kidney disease can cause false positive results [59,[69][70][71] . Recently, Høiseth et al [72] showed that chronic kidney disease increased the detection window of uEtG and uEtS. ...
Alcoholic liver disease is an established, yet controversial, indication for liver transplantation. Although an abstinence period of up to 6 mo prior to transplantation is mandatory, alcohol relapse after transplantation is a common event. In case of recurrence of heavy drinking, graft survival is significantly impaired. Guidelines on detection and surveillance of alcohol consumption in this patient cohort are lacking. This review summarizes the challenge of patient selection as well as the current knowledge on established and novel alcohol biomarkers with special focus on liver transplant candidates and recipients.
... Furthermore, when EtG is used as a clinical research or treatment outcome, the 500 ng/ml cutoff level may underdetect drinking (Anton, 2014;Jatlow and O'Malley, 2010;Jatlow et al., 2014). Therefore, research is needed to build consensus regarding an acceptable EtG cutoff level, as commercial laboratories, immunoassay manufacturers, and foreign regulatory authorities use cutoff levels ranging from 100 to 1,000 ng/ml (Rohrig et al., 2006;Thierauf et al., 2009Thierauf et al., , 2010. ...
... Furthermore, when EtG is used as a clinical research or treatment outcome, the 500 ng/ml cutoff level may underdetect drinking (Anton, 2014;Jatlow and O'Malley, 2010;Jatlow et al., 2014). Therefore, research is needed to build consensus regarding an acceptable EtG cutoff level, as commercial laboratories, immunoassay manufacturers, and foreign regulatory authorities use cutoff levels ranging from 100 to 1,000 ng/ml (Rohrig et al., 2006;Thierauf et al., 2009Thierauf et al., , 2010. ...
... Unintentional ethanol intake from ethanol-based mouthwash (Costantino et al., 2006) and hand sanitizers may generate positive results. Moreover, the drinking of non-alcoholic beer, which contain around 0.4 % ethanol vol/vol, as well as the consumption of foods made with baker's yeast, sugar and water, can lead to ethanol formation by fermentation and, consequently, to increased EtG concentrations in urine (Thierauf et al., 2010a(Thierauf et al., , 2010b. These observations highlight the need for careful interpretation of low-level of EtG in urine. ...
Alcohol abuse is frequently associated with an increased risk of road accidents and violence, and can also lead to serious social and health problems. Therefore, the use of reliable markers to detect excessive punctual and/or chronic consumption of alcohol is necessary to prevent the harmful consequences of alcohol abuse. Ethylglucuronide (EtG) has been proposed as a marker of alcohol consumption in a variety of clinical and forensic contexts. Compared with the indirect markers (e.g. CDT, γ-GT), this minor metabolite of ethanol is very sensitive and specific, and is quantifiable in various biological matrices. It is formed by conjugation of ethanol with uridine 5'-diphosphate glucuronic acid (UDP-GA) via the action of UDP-glucuronosyltransferase (UGT) enzymes. However, the knowledge of the UGTs involved in the glucuronidation of ethanol, and the potential sources of the interindividual variability of EtG production are still not clearly established. The aims of our work were (1) to develop and validate a method for the determination of EtG in different biological matrices by gas chromatography coupled with tandem mass spectrometry, (2) to identify the human UGT isoforms involved in the glucuronidation of ethanol, and then to evaluate qualitatively and quantitatively their specific contribution in the formation of EtG, (3) to study the impact of the co-administration of drugs frequently used by ethanol consumers on the in vitro production of EtG, (4) to study the impact of functional genetic polymorphisms of two UGTs on the hepatic production of EtG, and finally (5) to study the impact of the consumption of cannabis and other drugs on the production of EtG using post-mortem samples. The main results of our study showed that (1) ethanol is primarily glucuronidated by the liver and, to a lesser extent, by kidneys, (2) UGT1A9 and 2B7 were identified as the main human UGTs involved in ethanol glucuronidation, (3) morphine, codeine, nicotine, and cotinine did not modify EtG in vitro formation rate; lorazepam and oxazepam produced a minor, but not significant, increase of EtG formation. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation; cannabinol significantly increased the glucuronidation of ethanol in a concentration-dependent manner, whereas cannabidiol inhibited the glucuronoconjugaison of ethanol by a non-competitive mechanism (CI50 = 1.17 mg / L; Ki = 3.1 mg / L), (4) the SNP c.-900G>A and IVS1+399T>C affecting UGT2B7 and UGT1A9, respectively, seem to increase the in vitro production of EtG, and (5) cannabis and/or drugs consumption (mainly opioids, benzodiazepines, and paracetamol) seem to be associated with ratios of blood concentrations of EtG/ethanol significantly higher than those observed among only alcohol consumers. Taken together, these results show the existence of several factors that could potentially influence the production of EtG, and that should be taken into account when interpreting its concentration in vivo.
... For example, "non-alcoholic" beers are known to contain small but detectable amounts of alcohol. In one study, repeated sampling of several lots of nonalcoholic beers yielded mean ethanol concentrations of 3.1 and 3.2 g/kg, equivalent to 0.41 and 0.42 vol% (Thierauf et al., 2010). Similarly, fruits, fruit juices, and soft drinks are known to contain small amounts of alcohol (Logan and Distefano, 1998). ...
Ethanol-based topical antiseptic hand rubs, commonly referred to as alcohol-based hand sanitizers (ABHS), are routinely used as the standard of care to reduce the presence of viable bacteria on the skin and are an important element of infection control procedures in the healthcare industry. There are no reported indications of safety concerns associated with the use of these products in the workplace. However, the prevalence of such alcohol-based products in healthcare facilities and safety questions raised by the U.S. FDA led us to assess the potential for developmental toxicity under relevant product-use scenarios. Estimates from a physiologically based pharmacokinetic modeling approach suggest that occupational use of alcohol-based topical antiseptics in the healthcare industry can generate low, detectable concentrations of ethanol in blood. This unintended systemic dose probably reflects contributions from both dermal absorption and inhalation of volatilized product. The resulting internal dose is low, even under hypothetical, worst case intensive use assumptions. A significant margin of exposure (MOE) exists compared to demonstrated effect levels for developmental toxicity under worst case use scenarios, and the MOE is even more significant for typical anticipated occupational use patterns. The estimated internal doses of ethanol from topical application of alcohol-based hand sanitizers are also in the range of those associated with consumption of non-alcoholic beverages (i.e., non-alcoholic beer, flavored water, and orange juice), which are considered safe for consumers. Additionally, the estimated internal doses associated with expected exposure scenarios are below or in the range of the expected internal doses associated with the current occupational exposure limit for ethanol set by the Occupational Safety and Health Administration. These results support the conclusion that there is no significant risk of developmental or reproductive toxicity from repeated occupational exposures and high frequency use of ABHSs or surgical scrubs. Overall, the data support the conclusion that alcohol-based hand sanitizer products are safe for their intended use in hand hygiene as a critical infection prevention strategy in healthcare settings.
Copyright © 2015. Published by Elsevier Inc.
... The consumption of baker's yeast and sugar led to EtG and EtS levels above the 0.1 mg/L cut-off for abstinence (0.67 and 1.41 mg/L, respectively). Alcohol was not detected in urine [116]. ...
The quantitative, measurable detection of drinking is important for the successful treatment of alcohol misuse in transplantation of patients with alcohol disorders, people living with human immunodeficiency virus that need to adhere to medication, and special occupational hazard offenders, many of whom continually deny drinking. Their initial misconduct usually leads to medical problems associated with drinking, impulsive social behavior, and drunk driving. The accurate identification of alcohol consumption via biochemical tests contributes significantly to the monitoring of drinking behavior.
A systematic review of the current methods used to measure biomarkers of alcohol consumption was conducted using PubMed and Google Scholar databases (2010-2015). The names of the tests have been identified. The methods and publications that correlate between the social instruments and the biochemical tests were further investigated. There is a clear need for assays standardization to ensure the use of these biochemical tests as routine biomarkers.
Alcohol ingestion can be measured using a breath test. Because alcohol is rapidly eliminated from the circulation, the time for detection by this analysis is in the range of hours. Alcohol consumption can alternatively be detected by direct measurement of ethanol concentration in blood or urine. Several markers have been proposed to extend the interval and sensitivities of detection, including ethyl glucuronide and ethyl sulfate in urine, phosphatidylethanol in blood, and ethyl glucuronide and fatty acid ethyl esters in hair, among others. Moreover, there is a need to correlate the indirect biomarker carbohydrate deficient transferrin, which reflects longer lasting consumption of higher amounts of alcohol, with serum γ-glutamyl transpeptidase, another long term indirect biomarker that is routinely used and standardized in laboratory medicine.
... Üblicherweise erfolgt die Bestimmung durch Tandem- Massenspektrometrie, was relativ zeitaufwendig und teuer ist (). (Skipper 2009, Thierauf, Gnann et al. 2010). ...
Gegenstand dieser Arbeit ist es nachzuvollziehen, ob Ethylglucuronid einer bakteriellen Degradation respektive einer Neusynthese durch E. coli unterliegt und ob die präanalytische Stabilität der Ethylglucuronidkonzentration im Urin durch Zugabe bakterizider Substanzen wie Natriumfluorid und Borat gewährleistet werden kann. Dabei wurde auch der Einfluss unterschiedlicher Temperaturen bei Lagerung der Proben mitbeurteilt. Darüberhinaus wurde untersucht, ob nach Konsum alkoholfreien Bieres oder durch den Verzehr von Bäckerhefe forensisch relevante Ethylglucuronidkonzentrationen im Urin nachgewiesen werden können. Ferner wurde die Nachweismethodik mittels Massenspektrometrie mit der durch Immunoassay verglichen.
... SPE-LC/MS/MS was indicated to be the most reliable method. LC/MS/MS determination methods using dilution for sample preparation have also been carried out with human urine specimens (26)(27)(28)(29)(30)(31). Hegstad et al. (32) determined EtG/ethyl sulfate (EtS) in urine samples of sexual assault victims using ultra-performance LC (UPLC)/MS/MS. ...
A highly specific and selective analytical method using LC/MS/MS for the quantitative determination of ethyl glucuronide (EtG) in urine was developed and fully validated. Since the determination of EtG in urine may be possible days after the elimination of alcohol, it is an indication of alcohol use in alcohol treatment programs and antemortem and postmortem toxicological investigations. Propyl glucuronide (PrG), which increased the selectivity of the method, was used as an internal standard. The method was validated in terms of selectivity, linearity, LOD, LOQ, intraday and interday precision, and recovery. The analyte and PrG in the SPE cleaned up extract were separated on a 150 mm C18 column in 3.3 min with high resolution. The rest of the peaks from the matrix were eluted in 9.0 min. The LOD was 90.8 ng/mL and the LOQ was calculated using the EURACHEM method as 185.0 ng/mL. The intraday and the intermediate precision of the method was calculated using analysis of variance and confirmed with calculation of HorRat values, which were found within acceptable limits. The method provided a reliable solution for monitoring patients under alcohol addiction treatment and was successfully applied to real samples.
... Furthermore, when EtG is used as a clinical research or treatment outcome, the 500 ng/ml cutoff level may underdetect drinking (Anton, 2014;Jatlow and O'Malley, 2010;Jatlow et al., 2014). Therefore, research is needed to build consensus regarding an acceptable EtG cutoff level, as commercial laboratories, immunoassay manufacturers, and foreign regulatory authorities use cutoff levels ranging from 100 to 1,000 ng/ml (Rohrig et al., 2006;Thierauf et al., 2009Thierauf et al., , 2010. ...
Ethyl glucuronide (EtG) is an alcohol biomarker with potential utility as a clinical research and alcohol treatment outcome. Debate exists regarding the appropriate cutoff level for determining alcohol use, particularly with the EtG immunoassay. This study determined the EtG immunoassay cutoff levels that most closely correspond to self-reported drinking in alcohol-dependent outpatients.
Eighty adults with alcohol dependence and mental illness, taking part in an alcohol treatment study, provided urine samples 3 times per week for up to 16 weeks (1,589 samples). Self-reported drinking during 120 hours prior to each sample collection was assessed. Receiver operating characteristic analyses were conducted to assess the ability of the EtG immunoassay to detect self-reported alcohol use across 24- to 120-hour time periods. Sensitivity and specificity of EtG immunoassay cutoff levels was compared in 100 ng/ml increments (100 to 500 ng/ml) across 24 to 120 hours.
Over half (57%) of the 1,589 samples indicated recent alcohol consumption. The EtG immunoassay closely corresponded to self-reported drinking from 24 (area under the curve [AUC] = 0.90, 95% confidence interval [CI]: 0.88, 0.92) to 120 hours (AUC = 0.88, 95% CI: 0.87, 0.90). When cutoff levels were compared across 24 to 120 hours, 100 ng/ml had the highest sensitivity (0.93 to 0.78) and lowest specificity (0.67 to 0.85). Relative to 100 ng/ml, the 200 ng/ml cutoff demonstrated a reduction in sensitivity (0.89 to 0.67), but improved specificity (0.78 to 0.94). The 300, 400, and 500 ng/ml cutoffs demonstrated the lowest sensitivity (0.86 to 0.33) and highest specificity (0.86 to 0.97) over 24 to 120 hours.
For detecting alcohol use for >24 hours, the 200 ng/ml cutoff level is recommended for use as a research and clinical outcome.
Copyright © 2015 by the Research Society on Alcoholism.
... Nutritional and other environmental factors can influence the amount of nonoxidative alcohol metabolites [29]. There are small amounts of FAEE in meconium of neonates without active maternal alcohol consumption, which may originate from endogenous ethanol or from ethanol traces contained in common foods [30][31][32]. In contrast to the analysis of EtG and FAEEs in the patients' hair, there are no established and scientifically tested cutoff values for differentiating the mothers' drinking behavior via meconium, and imagining a reliable, scientifically correct, and ethical way to test the cutoff values in pregnant women is hardly possible [33]. ...
Aim. Identification of women with moderate alcohol abuse during pregnancy is difficult. We correlated self-reported alcohol consumption during pregnancy and patient characteristics with objective alcohol indicators measured in fetal meconium. Methods. A total of 557 women singleton births and available psychological tests, obstetric data and meconium samples were included in statistical analysis. Alcohol metabolites (fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG)), were determined from meconium and correlated with patient characteristics.
Results. We found that 21.2% of the 557 participants admitted low-to-moderate alcohol consumption during pregnancy. Of the parameters analyzed from meconium, only EtG showed an association with alcohol history (P < 0.01). This association was inverse in cases with EtG value above 120 ng/g. These values indicate women with most severe alcohol consumption, who obviously denied having consumed alcohol during pregnancy. No other associations between socioeconomic or psychological characteristics and the drinking status (via meconium alcohol metabolites) could be found. Conclusion. Women who drink higher doses of ethanol during pregnancy, according to metabolite measures in meconium, might be less likely to admit alcohol consumption. No profile of socioeconomic or psychological characteristics of those women positively tested via meconium could be established.
... The drinking of non-alcoholic beer, which contain 0.41/0.42 vol.% ethanol, can lead to increased EtG concentrations in urine [33]. The consumption of baker's yeast with sugar and water can lead to the formation of EtG and EtS reaching relevant concentrations in urine [34]. ...
... The previous publications [4,10,11] all offer different cut-offs (0.5-2 mg/ml) and although a limit of reporting (LOR) of 0.1 mg/ml [13][14][15][16] has previously been advocated, there is much debate surrounding this issue. [7,13,14,[17][18][19][20][21][22] The present study describes the use of DRI-ETG EIA to determine EtG concentrations in 1000 samples. As well as describing the largest sample size to date, the study is the first to incorporate analysis of post-mortem samples (n=800). ...
A commercial enzyme immunoassay for the qualitative and semi-quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post-mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EtG and ethyl sulfate.
Using a cut-off of 0.5 µg/ml and LC-MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post-mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut-off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post-mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade-offs between sensitivity and specificity for LC-MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut-offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC-MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi-quantitative presumptive detection of ethyl glucuronide in urine. Copyright
... Therefore, the hair treatment should always be recorded during the sampling. Positive EtG cases in hair due to food or none-hair cosmetics like mouthwash or hand sanitizers have not yet been reported in contrast to positive EtG urine results [36][37][38][39][40][41]. That can be explained by the small amount of ethanol ingested or absorbed leading to EtG concentrations in urine insufficiently high to produce a positive EtG findings in hair. ...
Ethyl glucuronide (EtG) in hair has emerged as a useful biomarker for detecting alcohol abuse and monitoring abstinence. However, there is a need to establish a reliable cutoff value for the detection of chronic and excessive alcohol consumption.
One hundred and twenty-five subjects were classified as teetotalers, low-risk drinkers, at-risk drinkers, or heavy drinkers. The gold standard for subjects' classifications was based on a prospective daily alcohol self-monitoring log. Subjects were followed for a 3-month period. The EtG diagnostic performance was evaluated and compared with carbohydrate-deficient transferring (CDT) and the activities of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl-transferase (γGT).
A cutoff of >9 pg/mg EtG in hair, suggesting an alcohol consumption of >20/30 g (at-risk drinkers), and a cutoff of >25 pg/mg, suggesting a consumption of >60 g (heavy drinkers), were determined by receiver operating characteristic analysis. The EtG diagnostic performance was significantly better (P < 0.05) than any of the traditional biomarkers alone. EtG, as a single biomarker, yielded a stronger or similar diagnostic performance in detecting at-risk or heavy drinkers, respectively, than the best combination of traditional biomarkers (CDT and γGT). The combination of EtG with traditional biomarkers did not improve the diagnostic performance of EtG alone. EtG demonstrated a strong potential to identify heavy alcohol consumption, whereas the traditional biomarkers failed to do so. EtG was not significantly influenced by gender, body mass index, or age.
Hair EtG definitively provides an accurate and reliable diagnostic test for detecting chronic and excessive alcohol consumption. The proposed cutoff values can serve as reference for future cutoff recommendations for clinical and forensic use.
... However, determining the threshold urinary concentrations of EtG and EtS that reliably distinguish between deliberate ethanol use and incidental exposure to ethanol-containing consumer products (e.g., hand sanitizers, mouthwashes, and food) has been problematic. There is no uniformly applied threshold; laboratories and regulatory authorities (1) have used thresholds from 100 to 1000 ng/mL (2)(3)(4). Skipper et al. (5) introduced EtG testing in the United States and have suggested that even a 1000 ng/mL threshold may be too low to rule out incidental ethanol exposures. Indeed, we recently reported a maximum urinary EtG concentration exceeding 2000 ng/mL after intensive handwashing with an ethanol-containing hand sanitizer (6). ...
To determine the degree of ethanol absorption and the resultant formation and urinary excretion of its conjugated metabolites following intensive use of high ethanol content mouthwash, 10 subjects gargled with Listerine(®) antiseptic 4 times daily for 3¼ days. First morning void urine specimens were collected on each of the four study days and post-gargle specimens were collected at 2, 4, and 6 h after the final gargle of the study. Urine ethanol, ethyl glucuronide (EtG), ethyl sulfate (EtS), and creatinine were measured. Ethanol was below the positive threshold of 20 mg/dL in all of the urine specimens. EtG was undetectable in all pre-study urine specimens, but two pre-study specimens had detectable EtS (6 and 82 ng/mL; 16 and 83 μg/g creatinine). Only one specimen contained detectable EtG (173 ng/mL; 117 μg/g creatinine). EtS was detected in the urine of seven study subjects, but was not detected in the single specimen that had detectable EtG. The maximum EtS concentrations were 104 ng/mL and 112 μg/g creatinine (in different subjects). Three subjects produced a total of eight (non-baseline) urinary EtS concentrations above 50 ng/mL or 50 μg/g creatinine and three EtS concentrations exceeding 100 ng/mL or 100 μg/g creatinine. In patients being monitored for ethanol use by urinary EtG and EtS concentrations, currently accepted EtG and EtS cutoffs of 500 ng/mL are adequate to distinguish between ethanol consumption and four times daily use of high ethanol content mouthwash.
Rationale
Alcohol use disorder affects 4% to 5% of the world's population. Analysis methods are available for various biological fluids to detect this disorder. Determination of ethyl glucuronide in urine by the liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is frequently used in forensic toxicology. These analyses are known to cause matrix effects.
Methods
The presented study describes the elimination of matrix effects for ethyl glucuronide. This study used two different LC/MS/MS systems containing orthogonal and z‐spray ion sources. Ethyl glucuronide was analyzed in negative polarity in electrospray ionization. A different dilution method was chosen for each study. The methods were developed and validated according to the European Medicines Agency bioanalytical method validation parameters.
Results
The lower limit of quantitation of the developed methods was 0.025 μg/mL for ethyl glucuronide. The calibration curve of ethyl glucuronide was between 0.025 and 100 μg/mL with a correlation coefficient of >0.99 for the two methods.
Conclusions
It was determined that the analyses using the z‐spray ion source were more affected by the matrix effect. The two validated methods involve rapid analysis time and simple sample preparation. Also, the methods were applied to real patients' urine.
Phosphatidylethanol (PEth) can be determined in capillary blood collected as dried blood spots (DBS) and is a promising direct alcohol biomarker for determination of drinking habits. Its use for abstinence monitoring needs to be evaluated. Studies with patients undergoing alcohol withdrawal have shown that elimination of PEth can take up to two months. For the determination of PEth 16:0/18:1, a cutoff of 20 ng/mL has been agreed upon in the major US laboratories. However, it is not yet clear what minimum blood alcohol concentrations (BACs) have to be achieved by a single drinking episode to result in PEth concentrations above this cutoff after previous long-term abstinence. To determine whether low drinking amounts can result in a positive PEth concentration above 20 ng/mL, we recruited 12 participants ("social" drinkers). After four weeks of abstinence, alcohol was consumed at two separate drinking events with target BACs of 0.5 and 0.3 g/kg, resulting in maximum BACs in the ranges of 0.30-0.63 g/kg and 0.10-0.28 g/kg, respectively. Capillary blood was collected at different time points of the drinking experiment and PEth was extracted from dried blood spots (DBS) and analyzed by liquid chromatography-tandem mass spectrometry. Despite drinking doses up to 0.58 g ethanol per kg body weight and reaching BACs of up to 0.63 g/kg, PEth 16:0/18:1 and PEth 16:0/18:2 could not be detected at or above the 20 ng/mL cutoff in any participant at any time after the drinking events. We conclude that after long-term abstinence the cutoff of 20 ng/ml for single alcohol consumption leading to blood alcohol concentrations up to 0.63 g/kg is not exceeded.
Alcohol use disorder (AUD) is a major risk factor for numerous social concerns worldwide. In the Republic of Korea, the court imposes compulsory medication treatment on criminals diagnosed with AUD who are on probation. The purpose of the treatment is to reduce the number of repeat offenses committed by AUD-afflicted criminals. In this study, a method employing liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine ethyl glucuronide (EtG), acamprosate (ACAM), naltrexone (NTX), and 6β-naltrexol (6BNT) in urine was developed and validated to monitor the medication compliance and alcohol use of probationers diagnosed with AUD. A zirconia-based hybrid solid–phase extraction technique was introduced to increase the sensitivity toward target analytes having significantly differing pKa values while minimizing the matrix effect of the urine sample. The pretreated urine samples were analyzed by LC–MS/MS performed in a polarity-switching electrospray ionization mode via a three-period multiple-reaction monitoring method. All the target analytes were separated and detected within 6.5 min with an Xselect HSS T3 column and gradient elution using water containing 0.02% formic acid and methanol as the mobile phase. The lower limits of quantification of EtG, ACAM, NTX, and 6BNT were 10.0, 4.0, 0.4, and 0.2 ng mL⁻¹, respectively. The determination coefficients of each calibration curve were greater than 0.9989. The intra-day accuracy ranged from −5.5 to 5.3% and the precision was ≤ 5.7%, whereas the inter-day accuracy ranged from −5.3 to 4.6% and the precision was ≤ 4.7%. The recovery, matrix effect, and process efficiency were 99.7–107.9%, 80.7–101.8%, and 80.5–108.1%, respectively. The developed method was successfully applied to analyze 107 urine samples obtained from probationers undergoing medication treatment for AUD and to monitor the probationers’ medication compliance and alcohol use. This study also determined the urinary concentrations of EtG, ACAM, NTX, and 6BNT as well as the metabolic ratio of NTX following repeated oral administration of AUD medications.
Ethyl sulfate (EtS) and ethyl glucuronide (EtG) in urine are biomarkers to monitor ethanol consumption. Due to their high polarity, severe matrix effects have been observed during analysis of EtS and EtG in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS), which can lead to a loss of sensitivity and accuracy. In the present study, a novel and simple sample preparation approach based on fast-dried urine spot was established to reduce the matrix effect of EtS and EtG in urine. 20 μL of urine was dropped on the Whatman 903# paper and was subsequently dried by microwave in one minute. After ultrasonic assisted extraction with 500 μL of methanol, the analysis was conducted using an LC-MS/MS system. Limits of detection were 5 ng/mL and linear ranges were 10 ng/mL- 10 μg/mL for both EtS and EtG. Matrix effects were in the range of 99.3% - 107.8% for EtS and 86.7% - 91.0% for EtG at three QC levels. Matrix effects for EtS and EtG were compared between the current method and other sample preparation methods including protein precipitation, and solid-phase extraction. The results showed that this fast-dried urine spot-based extraction method could eliminate matrix effects significantly in analysis of urine EtS and EtG by LC-MS/MS.
Laboratory tests vary widely in their utility and each test has unique advantages and disadvantages. For the detection of ethanol use and abuse, a variety of direct and indirect markers are available. Alcohol biomarkers provide objective measures for numerous areas of testing including clinical trials, alcohol abuse, postmortem assessment, and drugs of abuse screening. Because the utility of alcohol biomarkers vary depending on the context in which the results will be used, knowing the analogous distribution of results is of value. Herein we report distributions of ethanol in blood, phosphatidylethanol in blood, ethyl glucuronide in urine, and ethyl sulfate in urine for results reported in the last twelve months by our laboratory. Positivity rates were higher for directed analyses when compared to broad screening or panel tests with the highest overall positivity for ethyl glucuronide and ethyl sulfate. The distribution of results for ethyl glucuronide and ethyl sulfate were higher in clinical testing scenarios compared to forensic and a significant correlation between ethyl glucuronide and ethyl sulfate was found consistent with previous reports. Phosphatidylethanol was rarely ordered for forensic use while distributions between routine clinical and clinical trial use were similar. Approximately 21% of all phosphatidylethanol results were in the moderate to chronic alcohol use category. These results provide a summary of four commonly used direct markers for alcohol use with positivity rates and overall quantitative distributions. These data supply insights broken out by various disciplines where applicable providing a concise comparison of results for these markers.
Background:
Biomarkers of alcohol consumption are important not only in forensic contexts, e.g., in child custody proceedings or as documentation of alcohol abstinence after temporary confiscation of a driver's license. They are increasingly being used in clinical medicine as well for verification of abstinence or to rule out the harmful use of alcohol.
Methods:
This review is based on pertinent publications that were retrieved by a selective literature search in PubMed concerning the direct and indirect alcohol markers discussed here, as well as on the authors' experience in laboratory analysis and clinical medicine.
Results:
Alongside the direct demonstration of ethanol, the available markers of alcohol consumption include the classic indirect markers carbohydrate-deficient transferrin (CDT), gamma-glutamyltransferase (GGT), and mean corpuscular volume (MCV) as well as direct alcohol markers such as ethyl glucuronide (EtG) and ethyl sulfate (EtS) in serum and urine and EtG and fatty acid ethyl esters (FAEE) in hair. Phosphatidylethanol (PEth) is a promising parameter that com - plements the existing spectrum of tests with high specificity (48-89%) and sensi - tivity (88-100%). In routine clinical practice, the demonstration of positive alcohol markers often leads patients to admit previously denied alcohol use. This makes it possible to motivate the patient to undergo treatment for alcoholism.
Conclusion:
The available alcohol biomarkers vary in sensitivity and specificity with respect to the time period over which they indicate alcohol use and the minimum extent of alcohol use that they can detect. The appropriate marker or combination of markers should be chosen in each case according to the particular question that is to be answered by laboratory analysis.
Research findings from a new direction in epidemiology - Wastewater-based epidemiology (WBE), or Sewage-based epidemiology (SBE) are reviewed. This approach is based on determination in wastewater of specific markers of alcohol, nicotine and illegal drugs excreted in urine. Major methodical techniques, including sample preparation, analytical and calculation methods are examined. A body of findings from research literature on studying ethanol and nicotine consumption in dozens of cities in Europe, United States, Canada and Australia is presented. Outcomes of comparing analytical data obtained with the use of high-performance liquid chromatography and mass spectrometry with the results of conventional epidemiological studies are reviewed. Epidemiological risk assessment for ethanol consumption using the margin of exposure (MOE) approach is presented. Evidence of the possibility to use nicotine metabolites as population markers for determining number of people covered by sewage disposal, as well as predictors of illicit drug use is discussed.
In Belgium, the analysis of indirect biomarkers such as carbohydrate deficient transferrin (CDT%), gamma-glutamyltransferase (GGT), aspartate aminotransferase/alanine aminotransferase (AST/ALT) and mean corpuscular volume (MCV), is currently used to monitor the alcohol consumption in cases of fitness to drive assessment. To estimate how the use of direct ethanol markers (e.g. ethylglucuronide (EtG), ethylsulfate (EtS) and phosphatidylethanol species (PEths)) could improve the current process, three quantitative methods (EtG in hair; EtG and EtS in urine; and PEth 16:0/18:1, PEth 18:1/18:1 and PEth 16:0/16:0 in blood, venous (V) and capillary (C) dried blood spots (DBSs)) were developed, validated and tested. Fifty volunteers, for whom fitness to drive had to be assessed and for whom a blood analysis for indirect biomarkers was requested, were included in the study. The sampling and analysis of hair, urine and C-DBS were added to the process currently used. Hair EtG and C-DBS PEths are more sensitive to detect alcohol abuse than the currently used indirect biomarkers and allow to disprove an abstinence period. EtG and EtS in urine form a relevant parameter to detect recent alcohol intake (even one single alcohol consumption) during the days (up to 5 days) prior to the sampling and can thus be used to disprove strict abstinence. The three analyses tested here provide different levels of information and can be used separately or combined. The combined use of the three strategies allows better inference about the evolution of the alcohol consumption prior to the sampling. Moreover, the exclusive use of non- or minimally invasive sampling (hair, urine and C-DBS) allows this to be performed directly during the fitness to drive assessment by regular staff members. In conclusion, the three approaches that were evaluated in this work offer the potential to improve the Belgian driver’s licence regranting process.
Monitoring of alcohol consumption by living persons takes place in various contexts, amongst which workplace drug testing, driving under the influence of alcohol, driving licence regranting programs, alcohol withdrawal treatment, diagnosis of acute intoxication or fetal alcohol ingestion. The matrices that are mostly used today include blood, breath and urine. The aim of this review is to present alternative sampling strategies that allow monitoring of the alcohol consumption in living subjects. Ethanol itself, indirect (carbohydrate deficient transferrin, CDT%) as well as direct biomarkers (ethyl glucuronide, EtG; ethyl sulphate, EtS; fatty acid ethyl esters, FAEEs and phosphatidylethanol species, PEths) of ethanol consumption will be considered. This review covers dried blood spots (CDT%, EtG/EtS, PEths), dried urine spots (EtG/EtS), sweat and skin surface lipids (ethanol, EtG, FAEEs), oral fluid (ethanol, EtG), exhaled breath (PEths), hair (EtG, FAEEs), nail (EtG), meconium (EtG/EtS, FAEEs), umbilical cord and placenta (EtG/EtS and PEth 16:0/18:1). Main results, issues and considerations specific to each matrix are reported. Details about sample preparation and analytical methods are not within the scope of this review.
Background:
Detection of alcohol consumption in clients undergoing treatment for alcohol dependence can be difficult. The ethanol metabolites ethyl glucuronide and ethyl sulphate are detectable for longer in urine than either breath ethanol or urine ethanol. Our aim was to develop a liquid chromatography-tandem mass spectrometry method for urine ethyl glucuronide and ethyl sulphate for use in a routine clinical laboratory and define clinical cut-offs in a large population who had not consumed alcohol for at least two weeks.
Methods:
Urine samples were diluted in 0.05% formic acid in HPLC grade water and then directly injected onto a Waters Acquity ultra high performance liquid chromatography coupled to a Waters TQ Detector. Eighty participants were recruited who had not consumed alcohol for at least two weeks to define cut-offs for urine ethyl glucuronide and ethyl sulphate. Samples and alcohol diaries were also collected from 12 alcohol-dependent clients attending a treatment programme.
Results:
The assay was validated with a lower limit of quantitation of 0.20 mg/L for ethyl glucuronide and 0.04 mg/L for ethyl sulphate. Accuracy, precision, linearity and recovery were acceptable. Cut-offs were established for ethyl glucuronide, ethyl sulphate and ethyl sulphate/creatinine ratio (≤0.26 mg/L, ≤0.22 mg/L and ≤0.033 mg/mmol, respectively) in a non-drinking population. The validated cut-offs correctly identified clients in alcohol treatment who were continuing to drink alcohol.
Conclusions:
A simple liquid chromatography-tandem mass spectrometry method for urine ethyl glucuronide and ethyl sulphate has been validated and cut-offs defined using 80 participants who had not consumed alcohol for at least two weeks. This is the largest study to date to define cut-offs for ethyl glucuronide, ethyl sulphate and ethyl sulphate/creatinine ratio.
A criminal who has been convicted of offences attributable to his drug use can be given instructions to refrain from any alcohol consumption and to undergo appropriate checks. However, the ability to monitor court-ordered sobriety is limited. So far, compliance is only checked randomly. A new approach is a continuous (transdermal) alcohol monitoring by an ankle-worn device. A look at the U.S. shows that the electronic monitoring af alcohol consumption has been tested, with positive results. This article presents an overview of the impact of a continuous transdermal alcohol monitoring on abstinence monitoring and how this method can be embedded in the German System.
Alcohol and Its Biomarkers: Clinical Aspects and Laboratory Determination is a concise guide to all currently known alcohol biomarkers, their clinical application, and the laboratory methods used to detect them. Pathologists can use this resource to understand the limitations and cost factors associated with each method for determining certain alcohol biomarkers. In addition, interferences in these determinations are discussed, so that clinicians can understand the causes of falsely elevated biomarkers and pathologists and laboratory scientists can potentially eliminate them. The book focuses on the analytical methods used to detect alcohol in blood and urine, the limitations of alcohol determination using enzymatic methods, and the differences between clinical and forensic alcohol measurement. Chapters also cover cutting-edge alcohol biomarkers for potential use.
Background:
Alcohol-related disorders are common, expensive in their course, and often underdiagnosed. To facilitate early diagnosis and therapy of alcohol-related disorders and to prevent later complications, questionnaires and biomarkers are useful.
Methods:
Indirect state markers like gamma-glutamyl-transpeptidase, mean corpuscular volume, and carbohydrate deficient transferrin are influenced by age, gender, various substances, and nonalcohol-related illnesses, and do not cover the entire timeline for alcohol consumption. Ethanol (EtOH) metabolites, such as ethyl glucuronide, ethyl sulfate, phosphatidylethanol, and fatty acid ethyl esters have gained enormous interest in the last decades as they are detectable after EtOH intake.
Results:
For each biomarker, pharmacological characteristics, detection methods in different body tissues, sensitivity/specificity values, cutoff values, time frames of detection, and general limitations are presented. Another focus of the review is the use of the markers in special clinical and forensic samples.
Conclusions:
Depending on the biological material used for analysis, ethanol metabolites can be applied in different settings such as assessment of alcohol intake, screening, prevention, diagnosis, and therapy of alcohol use disorders.
Alcohol misuse is associated with significant morbidity and mortality. Although clinical history, examination, and the use of self-report questionnaires may identify subjects with harmful patterns of alcohol use, denial or under-reporting of alcohol intake is common. Existing biomarkers for detecting alcohol misuse include measurement of blood or urine ethanol for acute alcohol consumption, and carbohydrate-deficient transferrin and gamma-glutamyl transferase for chronic alcohol misuse. There is a need for a biomarker that can detect excessive alcohol consumption in the timeframe between 1 day and several weeks. Ethyl glucuronide (EtG) is a direct metabolite of ethanol detectable in urine for up to 90h and longer in hair. Because EtG has high specificity for excess alcohol intake, it has great potential for use in detecting "binge" drinking. Using urine or hair, this noninvasive marker has a role in a variety of clinical and forensic settings.
© 2014 Elsevier Inc. All rights reserved.
Ethyl sulfate (EtS) is a minor metabolite of ethanol, usually being present along with ethyl glucuronide in both blood and urine. At present, there have been few studies on sulfotransferases (SULTs) catalyzing EtS formation. Moreover, inhibition by nutritional components on EtS formation, e.g., polyphenols that are extensively sulfonated, has not been addressed at all. Firstly, the incubation procedure was optimized with regard to buffer, substrate concentration, and incubation time. Recombinant SULT enzymes including SULT1A1, 1A3, 1B1, 1E1, and 2A1 were screened for their activity towards ethanol; subsequently, respective kinetics was investigated. The inhibitory potential of resveratrol, quercetin, and kaempferol being abundant in beer and wine was studied thereafter. Analysis was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) using deuterated EtS as the internal standard. All enzymes are involved in the sulfonation of ethanol; respective kinetics followed the Michaelis-Menten model. Among the five SULTs under investigation, SULT1A1 displayed the highest activity towards ethanol followed by SULT2A1. Polyphenols significantly reduced the formation of EtS. Results revealed multiple SULT isoforms being capable of catalyzing the transfer of a sulfo group to ethanol; nevertheless, the relevance of SULTs' polymorphism on the sulfonation of ethanol needs further appraisal. Nutritional components such as polyphenols effectively inhibit formation of EtS; this observation may partly serve as an explanation of the highly inter-individual variability of EtS findings in both blood and urine.
This study compared the characteristics of two direct alcohol biomarkers, ethyl glucuronide and ethyl sulfate. Both biomarkers were analyzed from urine specimens submitted by 58 active duty service members at Walter Reed National Military Medical Center's Addiction Treatment Service. These 58 individuals, as a result of serial testing, submitted a total of 374 urine specimens for laboratory analysis. Of 374 specimens, the paired tests were most often negative (n = 295, 78.9%).The paired tests were both positive less frequently (n = 38, 10.2%). In an interesting development ethyl sulfate produced more positive results than ethyl glucuronide (n = 32, 8.6%).
A method for the quantification of ethyl glucuronide (EtG) and ethyl sulphate (EtS) in human urine is developed and fully validated according to international guidelines. Protein precipitation is used as sample preparation. During the development of the method on an UPLC-ESI-MS/MS system using a CSH C18 column, special attention was paid to reduce matrix effects to improve assay sensitivity and to improve detection of the second transition for EtS for specificity purposes. The method was linear from 0.1 to 10μg/mL for both analytes. Ion suppression less than 24% (RSD<15%) was observed for EtG and no significant matrix effect was measured for EtS. The recovery was around 80% (RSD<14%) for both compounds. This method provides good precision (RSDr and RSDt<10%) and bias (<15%) for internal and external quality control samples. The reproducibility of the method was demonstrated by the successful participation to proficiency tests (z-score<0.86). This method was finally used to analyze urine samples obtained from twenty-seven volunteers whose alcohol consumption during the 5 days before sampling was monitored. Concentrations between 0.5 and 101.9μg/mL (mean 10.9, median 1.4) for EtG and between 0.1 and 37.9μg/mL (mean 3.6, median 0.3) for EtS were detected in urine samples of volunteers who declared having consumed alcohol the day before the sampling. EtG and EtS concentrations in urine were highly correlated (r=0.996, p<0.001). A moderate correlation between the number of drinks the day before sampling and the concentration of EtG (r=0.448, p<0.02) or EtS (r=0.406, p<0.04) was observed. Using a cut-off value at 0.1μg/mL for EtG and EtS, this method is able to detect social alcohol consumption approximately 24h after the intake, without showing any false positive result.
Background:
Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer.
Methods:
EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use.
Results:
False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented the inhalation of the sanitizer vapor, the formation of propyl glucuronides and thus false-positive DRI(®) EIA EtG screening results, proving that propyl alcohols are almost exclusively taken up by respiration.
Conclusions:
The widespread use of propanol-containing products such as hand sanitizers may lead to sufficient uptake of propyl alcohols and excretion of significant amounts of propyl glucuronides to yield false-positive DRI(®) EIA EtG screening results. Thus, positive EtG immunoassay results have to be controlled by mass-spectrometry, in clinical cases at least if ethanol intake is denied by the patient.
Alcohol is associated with significant morbidity and mortality. Subjects abusing alcohol can be identified through clinical history, examination or self-report questionnaires. A range of biomarkers is available for detecting alcohol misuse, but there is still a need for a marker that can detect alcohol consumption in the time window between one day (ethanol) and one week (gamma-glutamyl transpeptidase and carbohydrate-deficient transferrin). Ethyl glucuronide is a direct metabolite that can be detected in urine for up to 90 h and has the potential to become a useful marker of 'binge' drinking. As a non-invasive marker, it could have a role in a variety of clinical and forensic settings.
We studied whether ethanol is sulphonated in humans with the perspective of using the urinary excretion of ethyl sulphate after ethanol consumption as a biomarker for SULT (sulphotransferase) activity. We developed a sensitive and selective HPLC-MS/MS method for determining ethyl sulphate in urine. Ten volunteers received a low dose of ethanol (0.1 g/kg of body mass). In general, excretion of ethyl sulphate was maximal in the first or second hour after dosage. Within 8 h, 2.5-6.8 micromol of ethyl sulphate was excreted. A 5-fold increase in the dose of ethanol led to an increase in the amount of ethyl sulphate excreted within 8 h (28-95 micromol) and the presence of this metabolite in urine for at least 24 h. Since ethyl sulphate was still being excreted for a substantial period after the elimination of ethanol, it might be used as a medium-time biomarker for preceding ethanol consumption. We have expressed previously all human SULT forms identified in Salmonella typhimurium. Ethanol sulphonation was studied in cytosolic preparations of these strains. The highest activities were observed with SULT1A2, 1B1 and 1C2, followed by 1A3. Activities were markedly lower with SULT1E1, 1A1 and 2A1, and were negligible with SULT1C1, 2B1a, 2B1b and 4A1. If the expression levels in tissues are additionally taken into account, SULT1A3 might be the predominant form for the sulphonation of ethanol in vivo, although a robust estimate requires further studies. With this limitation, urinary ethyl sulphate excretion appears very promising as a biomarker for SULT activity in vivo.
Physicians recovering from substance-related disorders are usually allowed to return to practice if they agree to remain abstinent from drugs, including alcohol, and to undergo random urine testing. Over 9000 physicians are currently involved in such monitoring programs in the US. To date, it has been difficult to adequately monitor abstinence from alcohol due to the short half-life of alcohol and no other highly specific marker. Ethyl glucuronide (EtG), a direct metabolite of alcohol, offers an extended window for assessment of drinking status (up to 5 days). Our aim was to assess the potential value of EtG testing in abstinence-based monitoring programs.
Urine samples were obtained from 100 participants in a physician monitoring program and additional samples were subsequently obtained 'for cause', 'to verify positive urine alcohol, when drinking was denied' and 'in high risk individuals'. All participants had signed contracts agreeing to remain abstinent from mood-altering drugs, including alcohol, and had agreed to random urine testing. EtG was determined using LC/MS-MS in addition to standard testing. The main outcome measure were urine specimens positive for EtG versus those positive based on standard testing for alcohol and other drugs.
Among the initial 100 random samples collected, no sample was positive for alcohol using standard testing; however, seven were positive for EtG (0.5-196 mg/l), suggesting recent alcohol use. Subsequent EtG testing was performed clinically during the course of monitoring. Of the 18 tests performed to date, eight of eight tests performed 'for cause' were positive for EtG but negative for all other drugs including urine alcohol. All eight were confirmed positive by self reported drinking by the patient when confronted regarding the positive test result. Of six tests performed to 'confirm a positive urine alcohol' two were positive for EtG and confirmed positive by self reported drinking. For the other four samples, especially as two are from a diabetic, in vitro fermentation of ethanol is discussed.
These data suggest that physicians in monitoring programs have a higher rate of unrecognized alcohol use than previously reported. Incorporation of EtG testing into alcohol abstinence monitoring can strengthen these programs.
The dielectric properties of water solutions of ethanol and sugar are investigated in the microwave region with the objective of setting up a method for the quality control of the fermentation process of alcoholic beverages. Alcoholic fermentation is the process by which carbohydrates, in particular sugar, are converted by the yeast into alcohol. During that process several other by-product compounds are produced, including a significant amount of carbon dioxide. The fermentation stage is of fundamental importance in the production of alcoholic beverages because some of the by-products' components have a considerable effect on the flavour, aroma, and other characteristic properties of the beverages. The on-line monitoring of the fermentation process can thus be very useful for controlling the timing and the development of the process in order to correct it earlier if deviations from "normality" occur. Dielectric spectroscopy is shown to be suitable for such a task, being able to discriminate between the initial water-sugar mixture and the final water-alcohol solution and making it possible to detect the production of carbon dioxide during fermentation. A case-study consisting of the monitoring of the primary fermentqtion of beer by dielectric spectroscopy is presented and discussed.
Recent studies show that ethyl glucuronide (EtG) can be decomposed by bacteria; whilst so far no degradation of ethyl sulphate (EtS) has been observed. In the present study, in vitro experiments with bacterial colonies were performed. Bacteria (Escherichia coli, Klebsiella pneumoniae, Clostridium sordellii) were isolated from autopsy material (liver, heart blood, urine, ascites, pericardial fluid, pleural fluid) tested for beta-glucuronidase activity, and three bacterial strains were added to nutrient-deficient medium containing EtG and/or EtS and incubated at 36 +/- 1 degrees C. Samples were taken after various intervals up to 11 days, and EtG and EtS were determined by electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). EtG was degraded by E. coli and C. sordellii--complete degradation occurred in the range of 3-4 days--and these bacteria exhibited beta-glucuronidase activity. EtS was not affected within 11 days of incubation.
An 8-μm thick Cuprophane hollow-fibre membrane not only had a higher ultrafiltration coefficient than an 11-μm thick membrane but was more permeable and selective for ethanol dialysis and extract removal from beer. Transmembrane pressures above 40 Pa did not affect alcohol removal but did prevent further extract removal and therefore improved the final beer quality. To achieve selective removal of alcohol, beer flow rates must be above the critical velocities but remain below values which would require too great a membrane area.
Summary A system for production of non-alcohol beer is described. A limited fermentation is carried out with immobilized cells ofSaccharomyces cerevisiae in a packed bed reactor. In the reactor, combined stress factors such as low temperature (2–4°C) and anaerobic conditions limit cell metabolism. Of the available sugars only a small amount of glucose is metabolized, resulting in low concentrations of ethanol (S. cerevisiae strain W34 grows at low temperatures and a mininum growth temperature of -2 °C was found, indicating that a further reduction of temperature during production will not inhibit growth. The characteristics of the system allow its use in very different applications. Potential applications of the immobilized system are discussed.
Ethyl glucuronide (EtG) is used commonly as a marker for the detection of non-compliance of patients in alcohol withdrawal therapy in psychiatric hospitals in Europe and in work-place monitoring programmes in the United States. With the increased use of this new marker, questions related to an unintentional uptake of ethanol resulting in detectable EtG concentrations have been discussed. The aim of this study was to determine the concentration ranges of EtG and ethyl sulphate (EtS) after the consumption of very small amounts of ethanol (1 and 3 g), which are more likely to be incidental than intended.
Drinking experiments with ethanol amounts of 1 and 3 g, respectively, were performed on a total of 31 volunteers. EtG and EtS analysis in urine was performed by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and creatinine concentration was determined using the Jaffé reaction. Furthermore, data obtained from this experimentation were then compared to data from literature.
The maximum concentration of EtG normalized to creatinine after the uptake of 1 g and 3 g of ethanol was found to be 0.32 mg/l and 1.53 mg/l, respectively, and 0.15 mg/l and 1.17 mg/l for EtS; these peak concentrations are considered to be positive by many laboratories testing urine for ethanol conjugates in work-place testing progammes.
Production of non-alcoholic beer using Saccharomyces cerevisiae has been studied. Non-recombinant mutant strains with a defect in the synthesis of tricarboxylic-acid-cycle enzymes were used and applied in both free and pectate-immobilized form, using both batch and packed-bed continuous systems. After fermentation, basic parameters of the beer produced by five mutant strains were compared with a standard strain of brewing yeast. Results showed that the beer prepared by mutant yeast cells was characterized by lower levels of total alcohols, with ethanol concentrations between 0.07 and 0.31% (w/w). The organic acids produced, especially lactic acid, in concentrations up to 1.38 g x l(-1) had a strong protective effect on the microbial stability of the final product and thus the usual addition of lactic acid could be omitted. Application of the yeast mutants appears to be a good alternative to the classical methods for the production of non-alcoholic beer.
The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.
Ethyl sulfate (EtS)--a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others--was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M - H]- m/z 125 and product ions m/z 97 [HSO4]- and m/z 80 [SO3]-. Pentadeuterated EtS (D5-EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M - H]- m/z 130, product ions m/z 98 [DSO4]- and m/z 80 [SO3]-). After addition of D5-EtS and D5-EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers' urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs.
A liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) method for the determination in urine samples of two ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), was developed and validated. Pentadeuterated EtG was used as internal standard for both EtG and EtS. In addition to the surviving ions, two MS/MS reactions were monitored for each analyte, with the deprotonated molecule as precursor ion: m/z 221 --> 75, m/z 221 --> 85 (EtG), and m/z 125 --> 97, m/z 125 --> 80 (EtS). Sample pretreatment, though very simple and rapid (1:50 water dilution and centrifugation of 50 muL of urine), was found to contain the occurrence of matrix effects. The method was accurate and precise over the linear dynamic range (0.05-10 mg/L). The analytes were stable in frozen urine for at least 1 month. The assay was applied to several authentic urine samples from social drinkers and to alcoholic beverages.
While ethanol is primarily metabolized to acetaldehyde and acetic acid via alcohol dehydrogenase, a minor but increasingly important pathway in the field of forensic science involves the conjugation of glucuronic acid to form an ethyl glucuronide (EtG) metabolite. The kinetics of ethyl glucuronide formation were examined in human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferases (UGTs). The metabolite exhibited a relatively slow rate of formation in a human liver microsome mix of 75.4 pmol/(min/mg). Further investigation identified multiple UGT isoforms to be responsible for catalyzing the addition of glucuronic acid to ethanol, with UGT1A1 and 2B7 being the two most prevalent isoforms. Co-incubation with bilirubin or 3'-azido-3'-deoxythymidine (UGT1A1 and 2B7 inhibitors, respectively) inhibited the greatest amount of ethyl glucuronide formation, though other UGT inhibitors also showed some effect. Enzyme kinetics were performed in human liver microsomes and recombinant UGT enzymes. The apparent Km (Km app) and Vmax values were determined to be 0.17+/-0.08 mM and 75.98+/-5.63 pmol/(min/mg) (human liver microsomes), 0.03+/-0.01 mM and 25.22+/-3.45 pmol/(min/mg) (UGT1A1), and 0.11+/-0.04 mM and 52.03+/-9.8 pmol/(min/mg) (UGT2B7). Thus, it appears that multiple UGTs are responsible for the formation of ethyl glucuronide and that any functional differences in the enzymology underlying ethyl glucuronide formation would most likely be masked by a combination of other enzymatic pathways.
beta-D-ethylglucuronide (EtG) is a stable Phase II metabolite of ethanol which can be detected in urine samples several days after elimination of ethanol. It is a useful diagnostic parameter for monitoring abstinence of alcoholics in alcohol withdrawal treatment. For this purpose, determination in urine is mainly performed by LC-MS, LC-MS/MS, or by GC-MS. For the mass spectrometric identification and detection of controlled substances in more sensitive fields such as forensic toxicology, workplace drug testing, doping analysis, and veterinary organic residue control, official guidelines have been released requiring a chromatographic separation and a minimum of two mass spectrometric transitions of the analyte. However, for detection of EtG none of the published LC-MS/MS methods could fulfill the minimum requirements of any of these guidelines. Therefore, an existing LC-MS/MS method has been modified by monitoring further MS/MS transitions instead of only one (deprotonated molecule [M - H](-)/product ions: m/z 75, 85, 113, and 159 optional) with the aim of withstanding administrative or court scrutiny in forensic or workplace drug testing cases. Full method validation has been performed in accordance to guidelines of the German Society of Toxicology and Forensic Chemistry (GTFCh) and requirements of ISO 17025. One application field in the United States is a workplace monitoring program to detect surreptitious alcohol use among recovering health professionals, who by contract had agreed on total abstinence after drug and alcohol withdrawal therapy.
Two studies were performed to evaluate the effect of alcohol containing mouthwash on the appearance of ethyl glucuronide (EtG) in urine. In the first study, 9 volunteers were given a 4-oz bottle of mouthwash, which contained 12% ethanol. They gargled with all 4 oz. of the mouthwash at intervals over a 15-min period. All urine samples were collected over the next 24 h. Of 39 provided urine samples, there were 20 > 50 ng/mL, 12 > 100 ng/mL, 5 > 200 ng/mL, 3 > 250 ng/mL, and 1 > 300 ng/mL. The peak concentrations were all within 12 h after the exposure. In the second study, 11 participants gargled 3 times daily for 5 days. The first morning void was collected. Sixteen of the 55 submitted samples contained EtG concentrations of greater than 50 ng/mL. All of them were less than 120 ng/mL. These studies show that incidental exposure to mouthwash containing 12% ethanol, when gargling according to the manufacturer's instructions, can result in urinary EtG values greater than 50 ng/mL. All specimens were negative for ethanol. The limits of detection and quantitation for the EtG testing were 50 ng/mL.
Aims:
Current biological state markers remain suboptimal with regard to sensitivity and specificity for monitoring recent alcohol consumption in various settings. Furthermore, these biomarkers can be influenced by age, gender and a variety of substances and non-alcohol-associated diseases and do not cover fully the time axis for alcohol intake. Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable, direct metabolite of ethanol that can be detected in various body fluids, tissues and hair. Shortly after the consumption even of small amounts of ethanol, EtG becomes positive. It can detect ethanol intake up to 80 hours after the complete elimination of alcohol from the body, covering a unique and important time spectrum for recent alcohol use. EtG seems to meet the need for a sensitive and specific marker to elucidate alcohol use not detected by standard testing.
Design, setting, participants, methods and findings:
The literature was reviewed with a focus on possible diagnostic and therapeutic applications, currently available methods and future perspectives. To date, more than 4000 samples of body fluids, tissues and hair from approximately 1500 individuals have been assessed.
Conclusions:
The data suggest that EtG is a useful tool in numerous settings, including alcohol and drug treatment (to detect lapse/relapse and for motivational feedback), in safety sensitive work settings where use is dangerous or in other settings where alcohol use may be risky (e.g. such as driving, work-place, pregnancy or monitoring physicians or other professionals who are in recovery and working) or for resolving forensic questions. If the question of recent alcohol consumption has to be answered in a binary way (yes/no), such as for determining lapses. the use of EtG in urine is among the preferred tests. The use of this marker alone and complementary with other biological state markers and self-reports is expected to lead to significant improvement in treatment outcome, therapy efficacy and cost reduction.
The direct ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), are of increasing importance for clinical and forensic applications, but there are only few studies on the kinetics of EtG in serum and none on EtS. In this study, 13 volunteers (social drinkers) drank ethanol in the form of white wine to reach a blood alcohol concentration of 0.51 +/- 0.17 g/kg, and blood and urine samples were analyzed for EtG and EtS simultaneously by chromatography-tandem mass spectrometry (LC-MS/MS). Mean peak serum EtG and EtS concentrations were 2.9 +/- 1.3 and 2.8 +/- 1.6 micromol/l, respectively, and were reached between 4.0 +/- 0.9 h after the start of drinking (3.0 +/- 0.5 h for EtS). The mean time differences between reaching maximum blood ethanol levels and serum metabolite levels were 2.3 +/- 0.9 h for EtG and 1.2 +/- 0.5 h for EtS. In the last blood samples collected (10-11 h after the start of drinking), 11 (of 13) volunteers were still positive for EtG in serum, whereas only 2 were positive for EtS. In the serum of one female person, no EtS was detectable at any time; however, it was excreted in the urine in (low) concentrations. Ethanol was detectable in the serum for up to 8.6 h after the start of drinking, whereas EtG and EtS were detectable up to more than 5.8 h (EtG) and 4.0 h (EtS), respectively. Mean peak urinary concentrations were 401 +/- 232 micromol/l for EtG and 266 +/- 153 micromol/l for EtS, and mean peak levels were reached 6.2 +/- 0.9 h (EtG) and 5.3 +/- 1.2 h (EtS) after the start of drinking. Maximum concentrations of EtG and EtS in serum showed a wide interindividual variation and could not be correlated to the maximum blood ethanol concentrations. Correlations (p < 0.001, Kendall's Tau b) were found when comparing pairs of parameters, but mostly involved areas under the curve (AUC) of metabolites or of ethanol; one correlation linked the peak concentrations of EtG and EtS in urine.
A 37-year-old female subject had been convicted of driving under the influence of alcohol, and 19 months later, claimed abstinence after supervised disulfiram treatment. Our aim was to elucidate the value of direct ethanol metabolites as measures of abstinence. Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) in hair, phosphatidylethanol in whole blood and EtG and ethyl sulphate in urine were measured. The results were compared with self-report of alcohol consumption and traditional blood biomarkers for chronically elevated alcohol consumption as carbohydrate deficient transferrin (CDT), gamma glutamyl transpeptidase, mean corpuscular erythrocyte volume, aspartate aminotransferase and alanine aminotransferase. EtG was found in distal parts of hair only, whereas the proximal parts were negative. Furthermore, FAEE concentrations were found in the typical distribution over the hair length and showed values typical for either moderate social drinking or abstinence. CDT was above cut-off in 9 out of 16 analyses with a decreasing tendency and the lowest values in the last 2 months before the end of sampling. The data suggest that in addition to traditional markers, a combination of direct ethanol metabolites can be useful in the expert assessment of judging driving ability. A careful individual interpretation of the results for the different markers, however, is an absolute necessity.
Genetic engineering of brewing yeast to reduce the content of ethanol in beer
- Nevoigt