Michel Yegles

Chronic intake of fermented floral nectar by wild treeshrews

For humans alcohol consumption often has devastating consequences. Wild mammals may also be behaviorally and physiologically challenged by alcohol in their food. Here, we provide a detailed account of chronic alcohol intake by mammals as part of a coevolved relationship with a plant. We discovered that seven mammalian species in a West Malaysian rainforest consume alcoholic nectar daily from flower buds of the bertam palm (Eugeissona tristis), which they pollinate. The 3.8% maximum alcohol concentration (mean: 0.6%; median: 0.5%) that we recorded is among the highest ever reported in a natural food. Nectar high in alcohol is facilitated by specialized flower buds that harbor a fermenting yeast community, including several species new to science. Pentailed treeshrews (Ptilocercus lowii) frequently consume alcohol doses from the inflorescences that would intoxicate humans. Yet, the flower-visiting mammals showed no signs of intoxication. Analysis of an alcohol metabolite (ethyl glucuronide) in their hair yielded concentrations higher than those in humans with similarly high alcohol intake. The pentailed treeshrew is considered a living model for extinct mammals representing the stock from which all extinct and living treeshrews and primates radiated. Therefore, we hypothesize that moderate to high alcohol intake was present early on in the evolution of these closely related lineages. It is yet unclear to what extent treeshrews benefit from ingested alcohol per se and how they mitigate the risk of continuous high blood alcohol concentrations.

Measurement of direct ethanol metabolites suggests higher rate of alcohol use among pregnant women than found with the AUDIT—a pilot study in a population-based sample of Swedish women

OBJECTIVES The objective of the study was to investigate whether biomarkers of alcohol consumption would provide additional information to the use of a validated alcohol questionnaire in pregnant women. STUDY DESIGN One hundred three pregnant women were included in the study. The women completed the Alcohol Use Disorders Identification Test (AUDIT) questionnaire, and a urine and hair sample was collected. The urine samples were used for determination of ethyl glucuronide (EtG) and ethyl sulfate and the hair samples for EtG and fatty acid ethyl esters (FAEE). RESULTS Twenty-six women (25.2%) were identified as possible alcohol consumers by the combined use of AUDIT and direct ethanol metabolites. Seven subjects had EtG or FAEE levels in hair highly suspicious of heavy drinking, but only 1 of these were positive according to the AUDIT questionnaire CONCLUSION The combined use of the AUDIT questionnaire and direct ethanol metabolites appear to identify more potential alcohol consumers among pregnant women than does the sole use of the AUDIT questionnaire.

Detection of benzodiazepines and other psychotropic drugs in human hair by GC/MS

For the detection of psychotropic drugs in human hair we collected hair obtained from 21 corpses that died from an overdose of legal or illicit drugs. These persons were known to have taken psychotropic drugs prior to their death as determined by post-mortem toxicological analysis in blood. After washing, cutting hair into segments of 3 cm, pulverization, enzymatic hydrolysis with beta-glucuronidase/arylsulfatase and solid phase extraction, drugs were identified by GC/MS. For quantification of flunitrazepam we used its metabolite amino-flunitrazepam; for oxazepam and lorazepam we used the hydrolysed forms of the corresponding drugs. In the hair of 21 subjects tested we found in 20 cases nordazepam, in 15 cases diazepam, in 15 cases oxazepam and in eight cases flunitrazepam with maximal concentrations of 1.8, 2.2, 3.4 and 9.5 ng/mg hair respectively. In addition to these compounds, in subject 11 to 21 we also analyzed for and detected amitriptyline (seven positive), carbamazepine (eight positive), lormetazepam (three positive) and lorazepam (one positive) and found maximal concentrations of 106.0, 13.5, 29.0 and 4.9 ng/mg hair respectively. The comparison of hair analysis versus post-mortem blood and tissues analysis of all the drugs studied shows that in 40 cases, where a positive result was found in blood, the corresponding drug could also be detected in hair in 37 cases. Our results show that hair testing is complementary to classical post-mortem analysis in forensic toxicology.

Hair ethyl glucuronide levels as a marker for alcohol use and abuse: A review of the current state of the art

BACKGROUND Ethyl glucuronide (EtG) is a minor alcohol metabolite that has been proposed as a stable marker in hair to detect and quantify alcohol consumption over long time periods. METHODS We provide an outline of currently available techniques for EtG hair sample analysis and highlight the pitfalls related to data interpretation. The literature of EtG analysis has been reviewed from January 1980 up to August 2013. In addition, we present an overview of the clinical and forensic studies which have used EtG quantification in hair as a marker for alcohol consumption/abstinence and we provide suggestions for future research. RESULTS EtG is a stable marker in hair that can be used to detect and quantify alcohol consumption over long time periods. This alcohol metabolite remains in hair after complete elimination of alcohol. Currently, there are three main analytical techniques used to quantify EtG in hair: gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). No standardized protocols are yet available for the analysis of EtG levels in hair samples, and the current protocols vary in sample preparation and extraction procedures. Variables such as hair length, cosmetic treatment, gender, and pathophysiological conditions influence the final results and should be taken into account. CONCLUSIONS EtG quantification in hair is a useful tool for the objective detection of alcohol consumption over extended time periods, but care should be taken when interpreting the results.

Ethyl Glucuronide Determination: Head Hair versus Non-Head Hair

INTRODUCTION In previous studies, hair analysis of ethyl glucuronide (EtG), a non-volatile, water-soluble, direct metabolite of ethanol, was shown to be adequate for the detection of social and chronic excessive alcohol consumption. As in some cases scalp hair is not available, the analysis of hair from alternative anatomical sites becomes of interest. AIMS In this study, hair samples from head, beard, chest, armpit, stomach, pubis, arms and legs from 32 subjects were analyzed when available, in order to compare the EtG concentrations and to study if the cut-offs used for head hair could be used for non-head hair. METHODS EtG was determined by GC/MS in negative chemical ionization mode using EtG-d5 as internal standard, after extraction by solid phase extraction using Oasis MAX columns and pentafluoropropionic anhydride (PFPA) derivatization. RESULTS The results showed that in the cases of negative findings in head hair (EtG < 7 pg/mg), in 7 out of 12 cases negative results could also be found in non-head hair. The five others were positive, due to a positive EtG finding in pubic hair. In 20 cases of positive EtG results for head hair, in all cases positive results could also be found in non-head hair. CONCLUSIONS In conclusion, although preliminary results indicate a clear trend regarding the accordance between EtG results in head hair and non-head hair, interpretation of non-head hair results remains to be carefully done for pubic hair, for which often higher concentrations have been found.

The influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair.

Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE), non-volatile, direct metabolites of ethanol have been shown to be suitable markers for the evaluation of social and chronic excessive alcohol consumption. Previous investigations have shown that the regular use of hair-care products with high alcohol content lead to an increase of FAEE concentration and consequently gave false-positive results for the determination of FAEE in hair. In this study we investigated the influence of a long-term hair treatment with EtOH containing lotion, on the EtG concentrations in hair. In this study 7 volunteer subjects (classified as either rare, social or heavy drinkers) treated the right side of their scalp every day during a one or two month period with a commercial hair tonic (Seborin), which contains 44.0% ethanol (vol%). Collection of hair specimens from both sides of the scalp was done one day before hair treatment, one week and one month after treatment (for 5 subjects also after two months of treatment). A hair segment of 3 centimeters (cm) was cut and then washed with water and acetone, and then pulverized. EtG was quantified by GC/MS after pulverization and 2h of ultrasonication in water, extraction by solid phase extraction using Oasis MAX columns and derivatization with HFBA. Measurements were done in negative chemical ionization mode using EtG-D5 as internal standard. Comparison of EtG concentration in the treated and in the non-treated hair specimens did not show any increase at the different dates of collection for the 7 subjects. In conclusion, these results show that there is no indication for an increase of EtG after use of ethanol containing hair cosmetics.

Assessment of Alcohol Use Among Methadone Maintenance Patients by Direct Ethanol Metabolites and Self‐Reports

BACKGROUND Heavy alcohol consumption may accelerate the progression of hepatitis C (HCV)-related liver disease and/or limit efforts at antiviral treatment. As most of the patients in methadone maintenance treatment (MMT) suffer from hepatitis C infection, this study was conducted to identify the alcohol intake among these patients at a Swiss Psychiatric University Clinic by self-reports and direct ethanol metabolites as biomarkers of ethanol consumption. PATIENTS AND METHODS A convenience sample of 40 MMT patients (15 women, 25 men; median age 39 years) of the total 124 patients was asked and consented to participate in this study. This sample was not different in age, gender distribution, and rate of hepatitis C infection from the total sample. The Alcohol Use Disorders Identification Test (AUDIT) and self-reported ethanol intake during the previous 7 days were assessed. In addition, ethyl glucuronide (EtG) in urine, and fatty acid ethyl esters (FAEEs) and EtG in hair were determined using LC-MS/MS and gas chromatograph/mass spectrometer. The limit of quantitation for UEtG, HEtG, and FAEEs were 0.1 mg/l, 2.3 pg/mg, and 0.1 ng/mg, respectively. RESULTS Fourteen participants reported abstinence from alcohol for the previous 7 days. AUDIT scores were > or =8 in 15 male and >5 in 5 female participants. Direct ethanol metabolites were as follows (median, min, max, standard deviation): UEtG (19 positives; 9.91, 1.38 to 251, 62.39 mg/l); the values of HEtG were 17.65, 0 to 513, 105.62 pg/mg [in 2 cases no material, 8 abstinent (up to 7 pg/mg), 15 social drinkers (up to 50 g per day), and 15 excessive users (>50/60 g/d)]. For the 13 cases, where enough material for additional determination of HFAEEs was available, the values were 0.32, 0 to 1.32, 0.44 ng/mg. Among the 30 HEtG-positive participants, 20 had not reported the corresponding ethanol intake using question 1 (frequency) and 2 (quantity) of the AUDIT. Of the 14 participants reporting no alcohol intake during the previous 7 days, 4 were UEtG-positive. HEtG and AUDIT correlated significantly (r = 0.622, p < 0.0001), but this was not the case for UEtG and self-reported ethanol intake during the previous 7 days. CONCLUSION (1) HEtG identified 20 cases of daily ethanol intake of more than 20 g, that would have been missed by the sole use of question 1 (frequency) and 2 (quantity) of the AUDIT. (2) Using the total score of the AUDIT, HEtG confirmed 10 more cases positive for alcohol intake. (3) Episodic heavy drinking is with 22.5% more frequent than in general population, and (4) of the 14 participants who reported no alcohol intake during the previous 7 days, 4 were UEtG positive. Improved detection of alcohol consumption, which is hazardous or harmful in the context of HCV and opiate dependence, would allow for earlier intervention in this population which is at particular risk of liver disease and fatal respiratory-depressed overdose. The combined use of self-reports and direct ethanol metabolites seems promising.

Ethanol metabolites: their role in the assessment of alcohol intake.

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.

Abstinence monitoring of suspected drinking drivers: ethyl glucuronide in hair versus CDT.

Objective: Ethyl glucuronide (EtG) determinations in the hair of self-reported teetotalers were reviewed and compared with carbohydrate-deficient transferrin (CDT) blood tests (by immunochemistry and high-performance liquid chromatography [HPLC]). Methods: A retrospective study was carried out on 154 people whose fitness to drive had to be assessed because of the suspicion of relevant alcohol problems. Results: EtG was detected in 55 percent of the hair samples and abstinence thus disproved. In two thirds (67%) of these cases, alcohol consumption was even shown to be excessive (EtG values > 30 pg/mg). Of the EtG-positive subjects 54 and 82 percent had CDT values within the reference range by immunochemistry and HPLC, respectively. Thirty-nine percent of the EtG-negative subjects had increased immunochemical CDT values; in contrast, 96 percent had HPLC CDT values within the normal range. Conclusions: EtG analysis in hair is a useful tool for assessing fitness to drive in suspected drinking drivers; compared to CDT values it provides a direct and unequivocal marker for reliable abstinence monitoring over a period of several months, depending on the length of the hair.

Hair ethyl glucuronide as a biomarker of alcohol consumption in alcohol-dependent patients: Role of gender differences

BACKGROUND Ethyl glucuronide (EtG) is a minor alcohol metabolite that accumulates in hair and is proposed as a stable marker for the detection of chronic and excessive alcohol consumption above a cut-off level of 30pg/mg hair. A correlation between drinking behavior and EtG hair concentrations is observed, but large variability exists. AIMS To investigate the correlation between alcohol consumption and hair EtG concentrations in alcohol dependent patients, and the effect of gender differences as a factor for the variability on this correlation. METHODS EtG was measured by gas chromatography coupled to mass spectrometry in the hairs (first 3cm) of 36 alcohol dependent patients (25 males/11 females) starting and alcohol detoxification program. Factors that possibly influence EtG content in hair (except age and gender) were excluded. Detailed retrospective alcohol consumption was obtained over the last 3 months using the Timeline Follow Back interview. RESULTS Median total alcohol consumption over 3 months was 13,050g pure alcohol (range 60-650g/day). Hair EtG concentrations varied between 32 and 662pg/mg. There was a statistically significant linear and positive correlation between hair EtG and amounts of alcohol consumed (Pearson r=0.83; p<0.001), in both males (Pearson r=0.83; p<0.001) and females (Pearson r=0.76; p=0.007). CONCLUSIONS There is a linear correlation, with no significant effect of gender, between hair EtG concentrations and amounts of alcohol consumed in alcohol-dependent individuals. Analysis of EtG in hair can be applied to estimate retrospective alcohol consumption in both male and female alcohol dependent subjects using the same cut-off.