Biomarkers Of Alcohol Consumption Research Articles

Next-generation biomarkers for alcohol consumption and alcohol use disorder diagnosis, prognosis, and treatment: Acritical review.

This critical review summarizes the current state of omics-based biomarkers in the alcohol research field. We first provide definitions and background information on alcohol and alcohol use disorder (AUD), biomarkers, and "omic" technologies. We next summarize using (1) genetic information as risk/prognostic biomarkers for the onset of alcohol-related problems and the progression from regular drinking to problematic drinking (including AUD), (2) epigenetic information as diagnostic biomarkers for AUD and risk biomarkers for alcohol consumption, (3) transcriptomic information as diagnostic biomarkers for AUD, risk biomarkers for alcohol consumption, and (4) metabolomic information as diagnostic biomarkers for AUD, risk biomarkers for alcohol consumption, and predictive biomarkers for response to acamprosate in subjects with AUD. In the final section, the clinical implications of the findings are discussed, and recommendations are made for future research.

Mental Manifestations and Biomarkers of Alcohol Consumption.

The purpose of this study is to examine healthcare usage, morbidities, and alcohol consumption monitoring in patients before a diagnosis of mental manifestations to assist in the early identification of individuals at risk. Biological markers of alcoholism are separated into two groups: those biological variables that highlight with great confidence and validate the presence of a predisposition to alcoholism, also called trait markers, or those that highlight alcohol consumption, called markers of alcoholism ("status markers"). Biomarkers are the true "gold standard" for the diagnosis of alcoholism. They are valuable for tracking evolution and progress during biological and psychological therapy and for highlighting relapse. This review compiles the existing data from research on healthcare utilization, comorbidities, and alcohol consumption monitoring in patients before a diagnosis of mental manifestations to aid in the early identification of individuals at risk. This documentary study took place over three to four months by searching for terms on the Science Direct platform, PubMed, Web of Science, and Google Scholar such as alcoholism, alcohol use disorders, alcohol abuse, and biomarkers. Studies reporting on the development, characteristics, and utilization of blood biomarkers for alcohol consumption were included in the search. The initial search included a number of 11.019 articles that contained the keywords biomarkers and alcohol. Finally, a total of 50 research articles were considered. I am involved in clinical studies, meta-analyses, reviews, and case studies regarding alcohol consumption detection, as well as potential alcohol markers.

A physiologically-based digital twin for alcohol consumption—predicting real-life drinking responses and long-term plasma PEth

Alcohol consumption is associated with a wide variety of preventable health complications and is a major risk factor for all-cause mortality in the age group 15-47 years. To reduce dangerous drinking behavior, eHealth applications have shown promise. A particularly interesting potential lies in the combination of eHealth apps with mathematical models. However, existing mathematical models do not consider real-life situations, such as combined intake of meals and beverages, and do not connect drinking to clinical markers, such as phosphatidylethanol (PEth). Herein, we present such a model which can simulate real-life situations and connect drinking to long-term markers. The new model can accurately describe both estimation data according to a χ2 -test (187.0 < Tχ2 = 226.4) and independent validation data (70.8 < Tχ2 = 93.5). The model can also be personalized using anthropometric data from a specific individual and can thus be used as a physiologically-based digital twin. This twin is also able to connect short-term consumption of alcohol to the long-term dynamics of PEth levels in the blood, a clinical biomarker of alcohol consumption. Here we illustrate how connecting short-term consumption to long-term markers allows for a new way to determine patient alcohol consumption from measured PEth levels. An additional use case of the twin could include the combined evaluation of patient-reported AUDIT forms and measured PEth levels. Finally, we integrated the new model into an eHealth application, which could help guide individual users or clinicians to help reduce dangerous drinking.

Non-invasive tests for alcohol-associated liver disease.

Alcohol consumption is a global phenomenon and a major contributor to alcohol-associated liver disease (ALD). Detecting individuals at risk of ALD has been challenging, with only a small fraction of patients being identified at early stages compared to other chronic liver diseases. In response to this challenge, non-invasive tests (NITs) have become essential tools for the detection of ALD, offering opportunities for early identification and intervention to mitigate the disease burden. Non-invasive alcohol consumption biomarkers are crucial in estimating individuals' recent alcohol intake, providing valuable insights into their drinking patterns. Various NITs have been investigated for the initial screening of asymptomatic individuals at risk of ALD, as well as for identifying specific stages of the disease. These NITs are applied in two main clinical scenarios: population-based stratification for identifying and predicting liver-related symptoms, and diagnosing and prognosticating compensated cirrhosis or advanced chronic liver disease in secondary or tertiary care settings. Moreover, NITs play a significant role in the prognostic assessment of patients with various manifestations of ALD, including alcohol-associated hepatitis, decompensated cirrhosis, and metabolic and alcohol-associated liver disease. These tests guide appropriate treatment decisions and predict outcomes. In this review, various NITs for the early detection and monitoring of alcohol consumption were discussed. Additionally, the evaluation of NITs for screening and predicting ALD and liver complications was addressed comprehensively. Future perspectives of NITs for ALD were explored, alongside a thorough discussion of the opportunities and challenges associated with NITs for ALD screening.

Feasibility of a telehealth-based contingency management intervention for alcohol use disorders using the phosphatidylethanol (PEth) 16:0/18:1 alcohol biomarker: a pilot randomized trial

ABSTRACT Background: Phosphatidylethanol (PEth) is a blood-based biomarker for alcohol consumption that can be self-collected and has high sensitivity, specificity, and a longer detection window compared to other alcohol biomarkers. Objectives: We evaluated the feasibility and acceptability of a telehealth-based contingency management (CM) intervention for alcohol use disorder (AUD) using the blood-based biomarker PEth to assess alcohol consumption. Methods: Sixteen adults (7 female, 9 male) with AUD were randomized to Control or CM conditions. Control participants received reinforcers regardless of their PEth levels. CM participants received reinforcers for week-to-week decreases in PEth (Phase 1) or maintenance of PEth consistent with abstinence (<20 ng/mL, Phase 2). Blood samples were self-collected using the TASSO-M20 device. Acceptability was assessed by retention in weeks. Satisfaction was assessed with the Client Satisfaction Questionnaire (CSQ-8) and qualitative interviews. The primary efficacy outcome was PEth-defined abstinence. Secondary outcomes included the proportion of visits with PEth-defined heavy alcohol consumption, negative urine ethyl glucuronide results, and self-reported alcohol use. Results: Retention averaged 18.6 ± 8.8 weeks for CM participants. CM participants reported high levels of satisfaction (CSQ-8, Mean = 30.3 ± 1.5). Interview themes included intervention positives, such as staff support, quality of life improvement, and accountability. 72% of PEth samples from CM participants were consistent with abstinence versus 34% for Control participants (OR = 5.0, p = 0.007). PEth-defined heavy alcohol consumption was detected in 28% of CM samples and 52% of Control samples (OR = 0.36, p = 0.159). CM participants averaged 1.9 ± 1.7 drinks/day versus 4.2 ± 6.3 for Control participants (p = 0.304). Conclusion: Results support the acceptability and satisfaction of a telehealth PEth-based CM intervention, though a larger study is needed to assess its efficacy [NCT04038021].

Перспективы использования лабораторных биомаркеров в диагностике употребления алкоголя в пренатальном периоде

Aim. To analyze laboratory diagnostic methods confirming the fact of alcohol consumption in order to determine the possibility of using the most effective methods in screening for the detection of alcohol in the blood of women of reproductive age and pregnant women. Key points. The analysis of domestic and foreign scientific literature was carried out using the keywords: fetal alcohol syndrome, fetal alcohol spectrum disorder, pregnancy, biomarkers of alcohol consumption, phosphatidylethanol, blood test. The review was compiled according to the databases: Scopus, MedLine, The Cochrane Library, CyberLeninka, RSCI, etc. The material was collected on the basis of the literature data of review articles, the selection was carried out depending on the scientific value, with the formation of the final list of references according to the relevance of the analyzed material. This review included 35 articles (36.4% of the total number of records found). All entries included were original articles, of which there were 15 prospective cohort studies, 20 observational-descriptive studies. The studies analyzed phosphatidylethanol (PEth), and other biomarkers in newborn blood in neonatal blood and in pregnant women. In 80% of the included articles, PEth was found to be a more sensitive diagnostic tool than other biomarkers of alcohol use. Conclusion. Pregnant women tend to underreport their alcohol use, which creates a need for sensitive and specific biomarkers. Studies show that PEth has a high specificity in the diagnosis of alcohol use and provides the ability to determine the fact of alcohol consumption in the prenatal period in pregnant women and in the blood of a newborn. In general, the accumulated data allow us to consider the determination of PEth in the blood as a promising marker of episodic alcohol use, both in small and large doses, as well as chronic alcohol intoxication. An urgent task for further research is to study the sensitivity, specificity, threshold values of PEth for various regimens of alcohol consumption and alcoholization. Keywords: alcohol, pregnancy, alcohol use biomarkers, phosphatidylethanol, biochemical methods, blood test.

Alcohol-Associated Liver Disease: Evolving Concepts and Treatments.

Alcohol is a prominent cause of liver disease worldwide with higher prevalence in developed nations. The spectrum of alcohol-associated liver disease (ALD) encompasses a diverse range of clinical entities, from asymptomatic isolated steatosis to decompensated cirrhosis, and in some cases, acute or chronic liver failure. Consequently, it is important for healthcare practitioners to maintain awareness and systematically screen for ALD. The optimal evaluation and management of ALD necessitates a collaborative approach, incorporating a multidisciplinary team and accounting for concurrent medical conditions. A repertoire of therapeutic interventions exists to support patients in achieving alcohol cessation and sustaining remission, with complete abstinence being the ultimate objective. This review explores the existing therapeutic options for ALD acknowledging geographical discrepancies in accessibility. Recent innovations, including the inclusion of alcohol consumption biomarkers into clinical protocols and the expansion of liver transplantation eligibility to encompass severe alcohol-associated hepatitis, are explored.

Validation of the quantification of phosphatidylethanol 16:0/18:1 concentrations in TASSO-M20 devices.

Phosphatidylethanol 16:0/18:1 (PEth), found in whole blood, is a biomarker for alcohol consumption with high sensitivity, specificity, and a long detection window. The TASSO-M20 device is used to self-collect capillary blood from the upper arm and has advantages over finger stick methods. The purpose of this study was to (1) validate PEth measurement using the TASSO-M20 device, (2) describe the TASSO-M20 for blood self-collection during a virtual intervention, and (3) characterize PEth, urinary ethyl glucuronide (uEtG) and self-reported alcohol in a single participant over time. PEth levels in blood samples dried on TASSO-M20 plugs were compared to those in (1) liquid whole blood (N=14) and (2) dried blood spot cards (DBS; N=23). Additionally, the self-reported drinking, positive or negative uEtG results (dip card cutoff ≥300 ng/mL), and observed self-collection of blood with TASSO-M20 devices for PEth levels were obtained over time during virtual interviews of a single contingency management participant. High-performance liquid chromatography with tandem mass spectrometry detection was used to measure PEth levels for both preparations. PEth concentrations from dried blood on TASSO-M20 plugs and liquid whole blood were correlated (0 to 1700 ng/mL; N=14; r2 =0.988; slope=0.951) and in a subgroup of samples with lower concentrations (N=7; 0 to 200 ng/mL; r2 =0.944, slope=0.816). PEth concentrations from dried blood on TASSO-M20 plugs and DBS were correlated (0 to 2200 ng/mL; N=23; r2 =0.927; slope=0.667) and in a subgroup of samples with lower concentrations (N=16; 0 to 180 ng/mL; r2 =0.978, slope=0.749). Results of the contingency management participant indicate that changes in PEth levels (TASSO-M20) and uEtG concentrations were consistent with each other and with changes in self-reported alcohol use. Our data support the utility, accuracy, and feasibility of using the TASSO-M20 device for blood self-collection during a virtual study. The TASSO-M20 device had multiple advantages over the typical finger stick method, including consistent blood collection, participant acceptability, and less discomfort as indicated by acceptability interviews.

Determination of phosphatidylethanol in whole-blood by liquid chromatography-tandem mass spectrometry based on intelligent scheduled time-zone acquisition technology and the application to population level survey

Alcohol intake is an important risk factor for cardiovascular disease， liver disease， and diabetes. The accurate and objective evaluation of alcohol intake is important for disease prevention and intervention， as well as alcohol intake monitoring. Phosphatidylethanol （PEth） is a potential clinical biomarker of alcohol consumption. Monitoring PEth levels can provide an objective and quantitative basis for alcohol intake studies. Unlike other current alcohol biomarkers， PEth can only be produced in the presence of alcohol. Therefore， PEth is highly specific for alcohol intake and not affected by confounding factors， such as age， gender， hypertension， kidney disease， liver disease， and other comorbidities. Because of its long half-life and high specificity for alcohol intake， PEth may be used as a tool for monitoring drinking behavior in the clinical， transportation， and other fields. Given rapid developments in mass spectrometry technology over the past decade， liquid chromatography-tandem mass spectrometry （LC-MS/MS） has become the preferred method for PEth detection. However， most current LC-MS/MS methods focus on the determination of one or several PEth homologs， and the number of PEth homologs that can be determined simultaneously is relatively limited. Moreover， the detection capacity of the available methods remains insufficient， and their analytical sensitivity for some PEth homologs must be further improved. In this study， a novel LC-MS/MS method based on an intelligent scheduled time-zone negative multiple reaction monitoring acquisition technology （Scheduled-MRM） was developed. The technology monitors two ion channels in each PEth to ensure reliable results and can quantify 18 PEth homologs in human whole blood simultaneously. Methanol-methyl tert-butyl ether-water was used as the extraction system. An XBridge C18 column （100 mm×2.1 mm， 3.5 μm） was selected for gradient elution with 2.5 mmol/L ammonium acetate isopropanol solution and 2.5 mmol/L ammonium acetate aqueous solution-acetonitrile （50∶50， v/v） as the mobile phases. Negative electronic spray ionization in scheduled-MRM mode was applied for MS/MS detection. The method was validated to have a linear range of 10-2500 ng/mL with correlation coefficients greater than 0.9999. The limits of detection and quantification were 0.7-2.8 and 2.2-9.4 ng/mL， respectively， and the spiked recoveries ranged from 91.0% to 102.2%. The method was confirmed to be simple， rapid， and precise， and subsequently validated for the measurement of 18 PEth homologs in human blood. Scheduled-MRM can assign a suitable scan time to each ion channel and effectively reduce the number of concurrent ion pairs monitored per unit time. This technology overcomes the problem of insufficient dwell time caused by an excessive number of ion channels， thereby avoiding the redundant monitoring of non-retention times. Scheduled-MRM significantly improved the detection sensitivity， data acquisition quality， and signal response of the proposed method. Whole blood samples from 359 volunteers with regular drinking habits were analyzed using this method. The total PEth concentrations ranged from 51.13 ng/mL to 2.89 μg/mL， with a mean of 363.16 ng/mL. PEth 16∶0/18∶1 and 16∶0/18∶2 were the two most abundant homologs， with mean concentrations of 74.21 and 48.75 ng/mL， accounting for approximately 20.43% and 13.42%， respectively， of the total PEth. Spearman correlation analyses showed that the PEth homologs correlated well with each other， γ-glutamyltransferase， a clinically available biological marker of alcohol， and other clinical biochemical parameters related to liver and kidney function. Overall， the method was demonstrated to be sensitive， precise， and accurate； thus， it may be an effective tool for monitoring alcohol intake in the clinical and other fields.

Blood Cell Responses Following Heavy Alcohol Consumption Coincide with Changes in Acute Phase Reactants of Inflammation, Indices of Hemolysis and Immune Responses to Ethanol Metabolites.

Aberrations in blood cells are common among heavy alcohol drinkers. In order to shed further light on such responses, we compared blood cell status with markers of hemolysis, mediators of inflammation and immune responses to ethanol metabolites in alcohol-dependent patients at the time of admission for detoxification and after abstinence. Blood cell counts, indices of hemolysis (LDH, haptoglobin, bilirubin), calprotectin (a marker of neutrophil activation), suPAR, CD163, pro- and anti-inflammatory cytokines and autoantibodies against protein adducts with acetaldehyde, the first metabolite of ethanol, were measured from alcohol-dependent patients (73 men, 26 women, mean age 43.8 ± 10.4 years) at baseline and after 8 ± 1 days of abstinence. The assessments also included information on the quantities of alcohol drinking and assays for biomarkers of alcohol consumption (CDT), liver function (AST, ALT, ALP, GGT) and acute phase reactants of inflammation. At baseline, the patients showed elevated values of CDT and biomarkers of liver status, which decreased significantly during abstinence. A significant decrease also occurred in LDH, bilirubin, CD163 and IgA and IgM antibodies against acetaldehyde adducts, whereas a significant increase was noted in blood leukocytes, platelets, MCV and suPAR levels. The changes in blood leukocytes correlated with those in serum calprotectin (p &lt; 0.001), haptoglobin (p &lt; 0.001), IL-6 (p &lt; 0.02) and suPAR (p &lt; 0.02). The changes in MCV correlated with those in LDH (p &lt; 0.02), MCH (p &lt; 0.01), bilirubin (p &lt; 0.001) and anti-adduct IgG (p &lt; 0.01). The data indicates that ethanol-induced changes in blood leukocytes are related with acute phase reactants of inflammation and release of neutrophil calprotectin. The studies also highlight the role of hemolysis and immune responses to ethanol metabolites underlying erythrocyte abnormalities in alcohol abusers.

Blood transcriptomic biomarkers of alcohol consumption and cardiovascular disease risk factors: the Framingham Heart Study.

The relations of alcohol consumption and gene expression remain to be elucidated. We examined cross-sectional associations between alcohol consumption and whole blood derived gene expression levels and between alcohol-associated genes and obesity, hypertension, and diabetes in 5531 Framingham Heart Study (FHS) participants. We identified 25 alcohol-associated genes. We further showed cross-sectional associations of 16 alcohol-associated genes with obesity, nine genes with hypertension, and eight genes with diabetes at P < 0.002. For example, we observed decreased expression of PROK2 (β = -0.0018; 95%CI: -0.0021, -0.0007; P= 6.5e - 5) and PAX5 (β = -0.0014; 95%CI: -0.0021, -0.0007; P = 6.5e - 5) per 1g/day increase in alcohol consumption. Consistent with our previous observation on the inverse association of alcohol consumption with obesity and positive association of alcohol consumption with hypertension, we found that PROK2 was positively associated with obesity (OR = 1.42; 95%CI: 1.17, 1.72; P = 4.5e - 4) and PAX5 was negatively associated with hypertension (OR = 0.73; 95%CI: 0.59, 0.89; P = 1.6e - 3). We also observed that alcohol consumption was positively associated with expression of ABCA13 (β = 0.0012; 95%CI: 0.0007, 0.0017; P = 1.3e - 6) and ABCA13 was positively associated with diabetes (OR = 2.57; 95%CI: 1.73, 3.84; P = 3.5e - 06); this finding, however, was inconsistent with our observation of an inverse association between alcohol consumption and diabetes. We showed strong cross-sectional associations between alcohol consumption and expression levels of 25 genes in FHS participants. Nonetheless, complex relationships exist between alcohol-associated genes and CVD risk factors.

Phosphatidylethanol in whole blood: A complementary biomarker to ethyl glucuronide in hair

Establish the importance of interpreting the analyses of two alcohol use biomarkers, with a different detection window, when monitoring long-term alcohol consumption of an individual. In many medico-legal cases, such as liver transplantation and traffic incidents, there is a need to provide a proof of alcohol abstinence. Long-term alcohol consumption, particularly proving abstinence, remains a challenge. Therefore, various alcohol consumption biomarkers are used to gain information about the drinking behaviour of an individual. Ethyl glucuronide in hair (hEtG) is a reliable long-term alcohol use biomarker due to its long detection window, depending on the length of hair, allowing for a retrospective evaluation of alcohol use (Crunelle. Drug and Alcohol Dependence 2014:1–11). Phosphatidylethanol 16:0/18:1 (PEth) in whole blood is a direct alcohol biomarker that has gained increasing interest, due to its detection window of two to four weeks. (Ulwelling. Journal of Forensic Science 2018;63(6):1634–1640) This study explores the usefulness of analysis of PEth as a complementary biomarker to hEtG to evaluate alcohol use more accurately over the last weeks to months. Hair and whole blood samples were obtained over a three-year period in a medico-legal context, from the University Hospital Antwerp and analysed by Toxicological Centre University of Antwerp and the National Institute investigation of Criminalistics and Criminology (NICC), Belgium. Individuals ( n = 952) were assessed for a 3- or 6- month period of alcohol abstinence. Hair samples were taken from the posterior head region (0–3 or 0–6 cm). The sample preparation for hair analysis consisted in solid-phase extraction. At NICC, analysis was performed on an LC-MS/MS (Kummer. Journal of Analytical Toxicology 2015;39(1):17–23). At the Toxicological Centre, the extract was analysed on a GC-MS/MS (Cappelle. Forensic Science International 2015;249:20–24). Whole blood was collected on the same day as the hair sample and analysed for PEth using liquid-liquid extraction. In both laboratories, the analyses were performed on a LC-MS/MS. (Dumitrascu, Drug Testing 2021;13:1219; Van Uytfanghe, Talanta 2021;223:121694). PEth and hEtG results were classified into three groups, according to thresholds for abstinent/low, moderate, or excessive alcohol consumption (Society of Hair Testing, 2016; Van Uytfanghe. Talanta 2021;223:121694). Spearman correlation test (rs = 0.692, P = 0.001) showed a significant and moderate correlation between the two biomarkers. It was found that 341 individuals were classified abstinent according to the hair analysis (hEtG < 5 pg/mg) out of which 82% were also classified abstinent according to the whole blood analysis (Peth < 20 ng/mL). Furthermore, 329 individuals were classified as heavy drinkers through hair analysis (hEtG > 30 pg/mg), of which 57% presented a PEth concentration > 200 ng/mL, confirming the classification. Fifty-two individuals (15%) were categorised as abstinent/low alcohol consumption through hEtG analysis, but as moderate drinkers through PEth analysis (20–200 ng/mL). This might be explained by recent, moderate alcohol consumption. While PEth can be detected if alcohol was consumed in the past two to four weeks, EtG had insufficient time to be incorporated into the cut hair sample. Out of 329 individuals classified as heavy drinkers based on hEtG analysis, 45 were categorised abstinent through PEth analysis, suggesting these subjects stopped drinking two to four weeks prior to sample collection. A moderate agreement of Kappa reliability analysis (k = 0.444) between PEth and hEtG confirms that, for a more accurate overview of the drinking behaviour of an individual, both biomarkers are needed. This study demonstrates the necessity of analysing two alcohol use biomarkers with different detection windows, when investigating alcohol use retrospectively. PEth in whole blood is a complementary biomarker to hEtG, as it captures the last two to four weeks of alcohol consumption, which would be missed when only analysing for hEtG.

Formation of ethyl glucuronide by beta-glucuronidase

Substantial amounts of endogenous and xenobiotic compounds are transformed to ß-D-Glucuronides in the human body. The conjugation reaction, which requires uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid as a cosubstrate is catalyzed by UDP-glucuronosyltransferases (UGTs). Beta-glucuronidase (GUSB), which is widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants, opposes this reaction: ß-D-glucuronides are hydrolyzed to ß-D-glucuronic acid and the corresponding aglycon in a configuration retaining manner. This study investigated GUSB activity in the presence of ethanol. Phenolphthalein-ß-D-glucuronide (PHPH-G), 4-nitrophenol-ß-D-glucuronide (4NP-G), morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide were used as substrates. Enzymatic reactions were carried out at 37 °C in buffer solution fortified with different amounts of ethanol (0–70% by volume) using GUSB from different sources ( E. coli , H. pomatia , bovine and human recombinant). The enzymatic reactions were stopped after 60 minutes and all samples were tested for ß-D-ethyl glucuronide (EtG) by a validated LC/MS/MS assay. In incubations of PHPH-G and 4NP-G, the release of aglyca was additionally measured by colorimetrical methods. It was found that EtG, which is a minor metabolite of ethanol but one of the most important direct biomarkers of alcohol consumption in man today, builts up from all investigated ß-D-glucuronides by GUSB activity in the presence of ethanol. The glucuronyl transfer reaction, which was neither observed in absence of ethanol nor in absence of GUSB, is detectable but minor at ethanol concentrations which are commonly observed in blood and tissues after consumption of alcoholic beverages. At higher ethanol concentrations (> 5% by volume), the transfer reaction is predominant and the majority of glucuronyl from ß-D-glucuronides is transferred to ethanol. Different types of donor substrates yielded different amounts of EtG in the presence of the same GUSB-activity and different types of GUSB yielded different amounts of EtG from the same donor substrate. The detection of EtG in several settings independent from human UGT-metabolism (e.g., EtG post collection synthesis in E.coli contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the observed mechanism. A contribution of GUSB activity to EtG kinetics in man is suspected and should be further investigated since high concentrations of ethanol (oral intake of alcoholic beverages) and high concentrations of ß-D-glucuronides (hepatobiliary secretion) are present in the gut, where the main GUSB activity is localized. Finally, the findings could be of interest in context of EtG hair analysis, since the prerequisites for in vitro formation of EtG in this study may also be found on head. There is an additional biochemical path and another source of EtG which deserves further attention in context of alcohol biomarker applications.

Quantification of phosphatidylethanol combining dried blood spots sampling with Orbitrap-based LC-HRMS platform: Method validation and proficiency testing

Phosphatidylethanols (PEth) are phospholipids specifically synthetised by an enzymatic reaction in presence of ethanol. PEth is a mid-term direct biomarker for alcohol consumption or abstinence, leading to a growing interest for forensic toxicology purposes. Blood deposition onto a filter paper card as dried blood spots (DBS) allows PEth stabilisation which is otherwise easily degraded at room temperature. The goal of this study was to develop and validate a procedure for the quantification of PEth 16:0/18:1, the most abundant analogue, in DBS samples benefiting from the selectivity brought by Orbitrap-based high-resolution mass spectrometry (HRMS). Calibration samples and quality controls (QCs) are volumetrically spotted (10 μL) onto filter paper card (Whatman 903 protein saver) either using HemaXis DB10™ device, or using laboratory micropipette. The whole DBS is manually punched out and the PEth is then directly extracted using an in vial approach with 100 μL of PEth 16:0/18:1-D5 in methanol. Analyses are then performed by LC-HRMS in PRM mode using a QExactive Orbitrap platform. Validation was carried out on with respect to the Food and Drug Administration (FDA) criteria, using three different QC concentrations (39, 87 and 350 ng/mL) over three non-consecutive days. To assess the robustness of our method, an interlaboratory proficiency testing was carried out using 3 different commercial standards at 4 levels (50, 100, 200, and 300 ng/mL) of quality controls (QCs) provided by ACQ Science (Germany). The method was fully validated according to guidelines set forth by the FDA. Calibration curve was built using a weighting factor of 1/x. Trueness was measured between 100.1 and 103.6% for the tested concentrations. Repeatability and intermediate precision were found to be lower than 5%. Linearity was evaluated between the lower limit of quantification of 20 ng/mL and the upper limit of quantification of 2000 ng/mL with an R2 greater than 0.998. Recovery (RE) and matrix effect (ME) evaluated at 2 concentrations ranged from 76 to 99% and 8 to 10%, respectively. During the proficiency test, precision was evaluated below 8.0%, while the accuracy ranged from 93.8% to 105.9% and from 101 to 104.5% when compared with the expected concentration and the mean measured value of all the labs participating respectively. The validation results demonstrate that the use of Orbitrap-based PRM quantification present an interesting alternative over triple quadrupole platforms considered as the gold standard for LC-MS/MS quantitative approaches. Indeed, multiple reaction monitoring (MRM) and PRM provide comparable performance in terms of sensitivity, linearity, dynamic range, precision and repeatability. Moreover, the use of PRM provides access to the full fragmentation spectra without any preanalytical considerations. The proficiency testing results confirm the suitability of PRM mode showing great quantification performances. In addition, those results and especially the repeatability exhibit the precision of the volume control provided by the use of HemaXis™ device. This tool enables the standardisation of the DBS collection at the fingertip in a non-hospital environment either by minimally-trained medical staff or by the patient himself. A rapid, sensitive, and robust method was developed and fully validated for the detection and quantification of PEth 16:0/18:1 in DBS samples. The results of the validation process and the proficiency testing confirm that the use of DBS associated with HRMS PRM-based analyses does not hinder the analytical performances, while providing full information over the MS-MS spectra. The development of analytical tools enabling the use of DBS microsampling that can be performed in almost every environment opens new opportunities toward clinical and forensic toxicological applications.

Management of Alcohol-Related Liver Disease and Its Complications.

Alcohol-related liver disease (ALD) is a major healthcare/economic burden and one of the leading causes of liver transplantation. New epidemiological studies that detail the course of the disease are needed since, despite its high prevalence, it is still a stigmatised condition with underlying pathology. Alcoholic hepatitis, as the highest expression of ALD, has high morbidity. Current treatments have suboptimal results with the exception of liver transplantation. Epidemiological studies must also be developed to improve prevention and implement early diagnosis policies. It is essential to develop multidisciplinary health models that allow the liver transplantation candidate to be approached in a holistic way, both for indication and follow up. The implementation of alcohol consumption biomarkers (ethyl glucuronide, phosphatidylethanol) can assist in diagnosing and supporting recovery. There are several initiatives with new therapies that must be validated to establish their effectiveness and indication.

Alcohol Consumption and Its Influence on the Clinical Picture of Puumala Hantavirus Infection.

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome. Characteristic clinical findings include acute kidney injury (AKI), thrombocytopenia, and capillary leakage. Smoking increases the risk of severe AKI, but it is not known whether alcohol consumption predisposes patients to a more severe infection. Liver and pancreatic enzymes, as well as biomarkers of alcohol consumption (gamma-glutamyl transferase, GGT; carbohydrate-deficient transferrin, CDT; GGT-CDT combination; and ethyl glucuronide, EtG), were measured from 66 patients with acute PUUV infection during hospitalization and at the convalescence phase. Alcohol consumption was present in 41% of the study population, 15% showing signs of heavy drinking. Alcohol use did not affect the severity of PUUV induced AKI nor the overall clinical picture of the infection. Liver enzyme levels (GGT or alanine aminotransferase, ALT) were elevated in 64% of the patients, but the levels did not associate with the markers reflecting the severity of the disease. Serum amylase activities at the convalescent stage were higher than those at the acute phase (p < 0.001). No cases with acute pancreatitis were found. In conclusion, our findings indicate that alcohol consumption does not seem to affect the clinical course of an acute PUUV infection.

Comparison of automated determination of phosphatidylethanol (PEth) in dried blood spots (DBS) with previous manual processing and testing.

Phosphatidylethanol (PEth) is a sensitive and specific biomarker of alcohol consumption in the prior 2-3 weeks. Standard, manual PEth testing using dried blood spots (DBS) is a multi-step time-consuming process. A novel, automated processing and testing method has been developed to decrease DBS processing and testing time. We conducted automated testing, using regioisomerically pure PEth reference material, on randomly selected DBS, which had previously been tested via manual methods and then stored for 3-6 years at-80°C, to compare the results (PEth 16:0/18:1 homologue). We chose samples for re-testing using categories found in the literature as follows: 1) PEth <20ng/mL; 2) PEth 20-200ng/mL; 3) PEth >200-1000ng/mL; 4) PEth >1000ng/mL. We calculated agreement between the categories using the weighted kappa statistic (n=49 DBS). We quantified agreement between continuous measures using the intraclass correlation coefficient (ICC), and further described the relationship between variables using Spearman correlation. The median PEth result was 155ng/mL (interquartile range [IQR]: 1-1312ng/mL) via automated methods and 98.8ng/mL (IQR: 10.2-625.0ng/mL) via manual methods. The weighted kappa comparing the automated to manual PEth results was 0.76 [95% Confidence Interval (CI): 0.66-0.86]. The ICC was 0.69 (95% CI: 0.54-0.79), and the Spearman correlation was 0.98 (95% CI: 0.95-0.99). While the new methods yielded somewhat higher PEth values, we found good to excellent agreement between clinically relevant PEth categories. Automated DBS processing and testing using new reference standards are promising methods for PEth testing.

Non-Invasive microRNA Profiling in Saliva can Serve as a Biomarker of Alcohol Exposure and Its Effects in Humans.

Alcohol Use Disorder (AUD) is one of the most prevalent mental disorders worldwide. Considering the widespread occurrence of AUD, a reliable, cheap, non-invasive biomarker of alcohol consumption is desired by healthcare providers, clinicians, researchers, public health and criminal justice officials. microRNAs could serve as such biomarkers. They are easily detectable in saliva, which can be sampled from individuals in a non-invasive manner. Moreover, microRNAs expression is dynamically regulated by environmental factors, including alcohol. Since excessive alcohol consumption is a hallmark of alcohol abuse, we have profiled microRNA expression in the saliva of chronic, heavy alcohol abusers using microRNA microarrays. We observed significant changes in salivary microRNA expression caused by excessive alcohol consumption. These changes fell into three categories: downregulated microRNAs, upregulated microRNAs, and microRNAs upregulated de novo. Analysis of these combinatorial changes in microRNA expression suggests dysregulation of specific biological pathways leading to impairment of the immune system and development of several types of epithelial cancer. Moreover, some of the altered microRNAs are also modulators of inflammation, suggesting their contribution to pro-inflammatory mechanisms of alcohol actions. Establishment of the cellular source of microRNAs in saliva corroborated these results. We determined that most of the microRNAs in saliva come from two types of cells: leukocytes involved in immune responses and inflammation, and buccal cells, involved in development of epithelial, oral cancers. In summary, we propose that microRNA profiling in saliva can be a useful, non-invasive biomarker allowing the monitoring of alcohol abuse, as well as alcohol-related inflammation and early detection of cancer.

Phosphatidylethanol, ethyl glucuronide and ethanol in blood as complementary biomarkers for alcohol consumption

Alcohol biomarkers can monitor both recent and long-term drinking and provide information about drinking habits as a complement to self-reporting. Ethyl glucuronide (EtG) and phosphatidylethanol (PEth) are the most sensitive available biomarkers for this purpose. The present study aimed to collect data on both PEth and EtG in the same blood sample, in addition to ethanol, in order to evaluate the combined use of these biomarkers. Venous EDTA blood samples (n = 1149) sent to the laboratory as part of a clinical routine service for measuring PEth were investigated. PEth and EtG concentrations were analyzed using liquid chromatography–mass spectrometry methods and ethanol with an enzymatic method. Of the 1149 samples, 95 were positive for ethanol (range 0.11–3.12 g/L), 454 for EtG (1.0–9739 ng/mL), 635 for PEth (0.014–6.0 µmol/L), 534 for PEth ≥ 0.050 µmol/L, and 315 for PEth ≥ 0.30 µmol/L. EtG and PEth concentrations seemed largely independent as the coefficient of determination (r2) between PEth and EtG concentrations was 0.15. However, when the EtG concentrations were evaluated for different subgroups depending on ethanol or PEth concentrations a statistically significant difference between successive higher concentrations was observed. EtG and PEth are independent measures of recent alcohol drinking reflecting different time windows. Their combined measurement in the same blood sample is possible and will provide valuable information regarding recent alcohol consumption as a complement to self-reporting.

Comparison of serum calprotectin, a marker of neutrophil activation, and other mediators of inflammation in response to alcohol consumption.

Previous studies have indicated that heavy alcohol intake stimulates inflammation and impairs the body's ability to regulate inflammation. The aim of this study was to compare changes in neutrophil calprotectin and a wide spectrum of other inflammatory mediators in response to heavy alcohol drinking. Serum calprotectin (a marker of neutrophil activation), suPAR, CD163, and pro- (IL-6, IL-8, TNF-α) and anti-inflammatory (IL-10, TGF-β) cytokines were measured from 61 alcohol-dependent subjects (46 men, 15 women, mean age 43.6±11.0 years) at the time of admission for detoxification and after 8±2 days of abstinence. These biomarkers were also measured from age- and sex-matched healthy controls representing abstainers or light drinkers. The clinical assessments included detailed clinical interviews on the amounts and patterns of alcohol consumption and assays for biomarkers of alcohol consumption (GGT, CDT, MCV, GGT-CDT) and liver function (AST, ALT). The subjects with alcohol use disorder showed significantly higher concentrations of serum calprotectin (p<0.0005), suPAR (p<0.01), CD163 (p<0.01), IL-6 (p<0.0005), IL-8 (p<0.0005), TNF-α (p<0.001), and IL-10 (p<0.0005) than healthy controls. These inflammatory mediators, except for CD163, remained elevated after the 8 ± 2-day period of supervised abstinence, which resulted in significant decreases in the biomarkers of alcohol consumption and indices of liver status. The AUC (0.855) for serum calprotectin in differentiating between the heavy drinkers and healthy controls was equal or equivalent with those of the conventional biomarkers of alcohol consumption (GGT:0.835 or CDT:0.803). The data indicate that neutrophil calprotectin is released in response to heavy alcohol intake in a sensitive manner and may be associated with perpetuation of inflammation in patients with alcohol use disorder. Serum calprotectin may also prove to be a useful biomarker for inflammatory activity in alcohol-consuming patients.