Alcohol Biomarkers in Clinical and Forensic Contexts

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 complements the existing spectrum of tests with high specificity (48–89%) and sensitivity (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.

Biomarkers to detect alcohol consumption or harmful alcohol use offer the opportunity to objectively verify the information about alcohol consumption provided by a patient or subject. A distinction is made between direct and indirect biomarkers. Direct biomarkers are created when ethanol is metabolized or reacts with substances in the body. Indirect biomarkers are enzymes or cells which undergo typical changes in response to acute or chronic alcohol consumption. The parameters to be measured or even the exact summons procedure are defined for certain situations, e.g. in transplantation medicine (1) or for assessment of driving eligibility (2). By contrast, in other cases, e.g. in issues related to custody and employment law or during alcohol withdrawal programs, the choice of markers is left at the discretion of the physician or toxicologist.

These biomarkers do not only differ significantly with regard to the period during which they can be detected and the amount of alcohol consumption required for a positive test result, but also with regard to specificity. Medications or diseases may affect the analysis of the biomarkers. Thus, when choosing a biomarker, both the query (moderate alcohol consumption or abstinence) and the summons procedure (point in time at which the date of sample collection is announced) have to be taken into account.

The aim of this study is to provide an overview of the available alcohol biomarkers, their significance and their appropriate use in clinical practice.

Methods

A selective search was performed in the PubMed database for the direct and indirect alcohol biomarkers discussed in this paper, covering the period January 1958 until August 2017. In addition, the authors contributed their analytical and clinical experiences.

Detection and significance of alcohol biomarkers

Ethanol and methanol

Acute alcohol consumption can be detected by determining blood and breath alcohol levels. Ten to twelve hours after the last drink, ethanol can no longer be detected in urine (e1). If subjects are informed about the upcoming test at least 12 hours in advance, they can avoid the detection of ethanol in blood and urine by stopping the intake of alcohol in time.

In this case, detection of methanol offers an additional advantage: Since the affinity of alcohol dehydrogenase to ethanol is substantially higher, methanol from drinks and endogenously produced from pectins (range: 0.35–3.2 mg/L [3]) culminates in serum above a blood ethanol concentration of 0.2–0.5 ‰ (4).

Based on scientific drinking experiments, methanol concentrations of >10 mg/L are regarded as a biomarker of preceding prolonged, continuous alcohol exposure over at least several hours (3). After analysis of drinking experimental data, the concentration of 5 mg/L was interpreted as a threshold to differentiate between patients with and without alcohol dependence (specificity of 98% [5]). Several studies used a concentration of 5 mg/L as a threshold for recent alcohol intake (end of drinking not longer than 1 day ago) (6–8).

Clinical-chemistry parameters

Carbohydrate-deficient transferrin

Excessive alcohol intake of >50–80 g ethanol per day over a period of at least 1 to 2 weeks results in the loss of carbohydrate side chains of transferrin (9, 10). Carbohydrate-deficient transferrin (CDT) is a specific, but not very sensitive biomarker (Table 1). Individuals with moderate consumption or an episodic drinking pattern show CDT levels in the normal range. Furthermore, false-positive results may occur due to rare genetic variations (11).

Table 1

Specificity and sensitivity of alcohol biomarkers in relation to the reported amount drunk

Mean corpuscular volume, gamma-glutamyl transferase, aspartate and alanine aminotransferase

An increase in mean corpuscular volume (MCV) of red blood cells and in the activity of the enzymes aspartate-aminotransferase (AST) and alanine aminotransferase (ALT), especially a ratio of AST/ALT >2, and above all of gamma-glutamyl transferase activity (GGT), may be signs of harmful alcohol consumption and alcohol-induced hepatic injury (12).

These biomarkers have the advantage that they can be measured as part of inexpensive routine testing in clinical-chemistry laboratories (Table 2). Furthermore, it takes several weeks after termination or reduction of alcohol consumption before they return to normal (CDT: 2–3 weeks; GGT: 2–6 weeks; AST/ALT: 2–4 weeks; MCV: 8–16 weeks [12]). A disadvantage of indirect alcohol biomarkers is their low specificity (Table 1). For example, non-alcohol-induced hepatic conditions, medications or liver transplant dysfunction can also increase these levels (e3–e5).

Table 2

Availability of analysis

While these biomarkers can identify harmful/excessive alcohol consumption, they are not suitable for abstinence monitoring because the regular intake of ethanol in small amounts, but also binge drinking, do not lead to increases in these parameters (9, 13) (Table 3).

Table 3

Use of alcohol biomarkers

Ethyl glucuronide and ethyl sulfate

Apart from oxidative metabolism, the phase II metabolite ethyl glucuronide (EtG; 0.02–0.06% of the ingested alcohol) and ethyl sulfate (EtS; 0.010–0.016%) are created from alcohol to a minor extent (14). They can be detected in blood not long after alcohol consumption (<45 minutes). The time during which EtG can be detected in serum is by up to 8 hours longer compared to ethanol (15). EtS can be detected in serum about twice as long as ethanol (16).

EtG and EtS are also excreted in urine not long (<60 minutes) after alcohol consumption. EtG can be detected in urine for up to about 24 hours even after consumption of small quantities; after excessive consumption, the window of detection is up to 130 hours (17). Therefore, EtG and EtS in urine are the short-term biomarkers with leading sensitivity (Table 1). Sensitivity is dependent on alcohol quantity, time interval between sample collection and alcohol intake as well as the cut-off level of the method applied (6, 18, 19). Since the levels in urine are dependent on diuresis, the intake of larger volume of water results in a steep decrease in EtG and EtS urine levels. This may lead to false-negative test results (e6). Therefore, it is important to interpret EtS and EtG levels based on the urine creatinine levels or to state at least a minimum requirement—usually >20 mg/dL (e7).

However, the disadvantage of the very high sensitivity of this method is that the EtG/EtS levels in urine do not allow to distinguish between a binge drinking event several days ago and a (potentially unintended) minor alcohol intake a few hours before the sample was taken. In isolated cases, positive urine findings have been observed after use of ethanol-containing mouth rinses (26.9 volume percent [vol%]) or highly concentrated ethanol-based disinfection solutions (60–96 vol%) (e8, e9). Likewise, larger quantities (e.g. 2.5 L) of alcohol-free beer can result in positive findings of EtG and EtS in urine for up to 20 hours after consumption; declaration of alcohol content is only required starting from 0.5 vol% (e10). Prior to testing, patients or subjects should be informed about this and should be asked about such behavior.

Phosphatidylethanol

Phosphatidylethanol (PEth) is an abnormal phospholipid which is produced after alcohol exposure in cell membranes of, for example, human erythrocytes (20). PEth is not a single molecule but a group of glycerophospholipids with fatty acid groups of various lengths with various degrees of saturation. So far, 48 PEth species have been identified in human blood samples (e11). The PEth homolog 16:0/18:1 is currently the species most commonly determined in analyses. It accounts for the largest proportion of the PEth homologs produced after alcohol intake and is often simply referred to as PEth (21, 22). In drinking experiments, PEth was detected in blood already after 30 minutes; PEth levels peaked after 90 to 120 minutes (23). With frequent alcohol consumption, PEth accumulates in whole blood.

Most studies on PEth were carried out retrospectively from a clinical or epidemiological perspective on alcoholics in detoxification or rehabilitation facilities or on selected patient populations, such as patients with hepatic disease, pregnant women or HIV-positive (HIV, human immunodeficiency virus) patients. Only a few studies with prospective and/or experimental design assessed healthy subjects with an at most moderate drinking behavior (eTable).

eTable

Overview of the studies on phosphatidylethanol

Since PEth can already be detected in blood after about 1 to 2 hours and for up to 12 days after one-time alcohol intake, this biomarker can be used to determine both current consumption and abstinence (24). After long-term moderate and harmful drinking behavior and also after alcohol relapse, the significance of PEth is somewhat higher than that of CDT and is only second to the detection of EtG in hair (25) (eTable). However, PEth analysis allows to verify faster whether a patient/subject has changed their drinking behavior.

By measuring PEth levels, daily alcohol consumption of more than 60 g ethanol can clearly be distinguished from lower alcohol consumption. Thus, PEth can be used to identify individuals with chronic excessive drinking behavior (26). Currently, no cut-offs have been officially set, but several pertinent recommendations exist.

Even though the preanalysis and analysis of PEth is complex, this biomarker can now be determined in routine analysis, thanks to the use of dried blood samples, the availability of deuterated standards and of modern analytic technologies. Since PEth can detect chronic and one-time alcohol consumption, this biomarker is well suited to monitor abstinence and drinking behavior and to identify relapse. Therefore, Germany should follow the example of Sweden and introduce this reliable biomarker into clinical laboratories (27).

Alcohol biomarkers in hair

Ethyl glucuronide in hair

There is a close correlation between level of alcohol consumption and EtG concentrations in hair (28, 29). Measuring EtG in hair offers two advantages:

• The analysis of a proximal 3–6 cm long hair segment allows to retrospectively evaluate a period of several months.
• Short-term reduction in alcohol consumption has no effect on test results.

Thus, EtG analysis can be used to detect chronic harmful alcohol consumption, potentially causing fatty liver disease or liver cirrhosis. In a forensic setting, the use of long-term biomarkers can eliminate the need for repeated short-term checks of a subject/patient.

Based on internationally adopted cut-off concentrations, abstinence from alcohol can be verified (EtG in hair <7 pg/mg) and chronic excessive drinking with a consumption of 60 g or more of ethanol per day can be detected (>30 pg/mg). Concentrations between 7 and 30 pg EtG/mg hair are regarded as a strong indicator of regular alcohol consumption (30).

Fatty acid ethyl esters

Fatty acid ethyl esters (FAEEs) are produced in the presence of ethanol from, for example, triglycerides or free fatty acids by specific FAEE synthases and other enzymes. These products of nonoxidative ethanol metabolism can be detected in blood, tissue and also in hair. The standard substrate for quantitative analysis is ethyl palmitate; in addition, other parameters can be determined (31, 32). Besides EtG, FAEE concentrations can be measured in hair as a plausibility control, but alone they are not suitable to verify abstinence. The ethyl palmitate cutoff concentration for abstinence is 0.12 ng/mg for a 3 cm proximal scalp hair segment. A concentration of 0.35 ng/mg is regarded as a strong indicator of chronic excessive alcohol consumption (30).

For both EtG and FAEEs in hair it applies that occasional alcohol consumption is not ruled out by a negative result. Consequently, alleged abstinence cannot be verified, but at best disproved. It should be noted that 3 to maximal 6 cm long proximal hair segments are recommended as samples. If this is not the case, quantitative results should be interpreted with great caution (30). An average hair growth of approximately 1 cm per month is assumed. Interpretation should take the intradermal hair segment which is not included in the analysis and the cyclical growth pattern of hair into account (33).

Cosmetic hair treatment (tinting, coloring, bleaching, perming, straightening) can significantly reduce the concentrations of the analyte. Ethanol-containing hair care products have no effect on EtG, but may lead to false-positive FAEE results (34–36).

Clinical benefit of alcohol biomarkers

In a clinical setting, alcohol biomarkers are increasingly used to objectively determine alcohol abstinence or to rule out harmful alcohol consumption. The costs of the analyses are billed according to the German Scale of Medical Fees (GOÄ) and reimbursed by health insurances.

Detection of harmful alcohol use (German Clinical Guideline: Screening, Diagnosis and Treatment of Alcohol Use Disorders [e12]), chronic excessive drinking or alcohol dependence, e.g. with craving, loss of control or development of tolerance (compare International Classification of Diseases [ICD] 10 [e13]), should trigger close medical and/or psychiatric monitoring to reduce the risk of organ disease and of further medical, social and psychological problems (e14). Referral to an addiction specialist and/or a physician with additional qualification in addiction primary care would be desirable. Survival rates in patients with alcoholic liver cirrhosis achieving abstinence are significantly better compared to those in patients who continue to consume alcohol (37). In a prospective cohort study, the 1-year and 5-year survival rates increased from 63% to 95% and from 36% to 61%, respectively (38).

During alcohol withdrawal or quit drinking programs, analysis of EtG in urine can be used to detect relapses (39, 40). In the field of liver transplantation, alcohol biomarkers are regularly used to verify alcohol abstinence prior to listing a patient for transplantation, to monitor patients on the waiting list or to detect relapses after transplantation in time. The German Medical Association’s guideline on maintaining waiting lists for patients prior to liver transplantation require that EtG in urine is measured before including a patient in the waiting list and during the waiting time for an organ (1). In case a patient tests positive for EtG in urine, he or she will be confronted with the test result by the treating physician and in addition once again referred to the psychologist or psychiatrist of the transplantation program who are specialized in alcohol disease. Experience shows that in this situation, many patients admit to the earlier denied alcohol consumption and are more open to addiction treatment. If this is not the case, the further procedure is decided by the local interdisciplinary transplantation conference: Either the patient is not listed/removed from the transplant waiting list or their status is only temporarily inactivated during further follow-up. It should be noted that this decision is not solely based on the detection of EtG in urine, but always takes all findings, including the medical history, psychological/psychiatric assessments, other laboratory/alcohol parameters, and, if indicated, liver histology and the patient’s prognosis into account. To reduce the risk of a false-positive EtG test, e.g. due to the intake of alcohol-containing food, a cut-off concentration of 0.5 mg/L instead of 0.1 mg/L is used in transplantation medicine (1, 6, 18). Furthermore, patients are first informed in detail that they must stay away even from small amounts of alcohol in food, such as desserts or sauces.

Alcohol biomarkers have proven to be a very useful approach to monitor patients after liver transplantation who had first been diagnosed with alcoholic cirrhosis. Using these biomarkers, alcohol relapse can be better detected compared to medical consultation, patient-reported information and elevated liver enzyme levels (6, 7). The aim is to motivate the patient to remain abstinent to prevent irreversible damage to the implant. A recently completed study has provided evidence of the diagnostic advantage of the alcohol biomarker PEth in the transplantation situation (25). The case studies in the Box and eBox illustrate the benefit of alcohol biomarkers in transplantation medicine.

Box

Case study 1

eBox

Case study 2

Likewise, alcohol biomarkers are generally used to determine the etiology of fatty liver disease or liver cirrhosis. However, it should be kept in mind that alcohol is often only a co-factor in the development of liver disease; thus, it is crucial to perform a diagnostic evaluation of other potential causative factors too.

Conclusion

The advantage of the clinical use of alcohol biomarkers is that the actual alcohol consumption of a patient can be assessed. This insight should be used to support a patient in such a way that damages resulting from harmful alcohol use are prevented.

The simple and cost-effective EtG urine test is better suited than ethanol-based tests to confirm alcohol abstinence during withdrawal programs, in patients on the transplant waiting list, in a forensic setting, and during primary care follow-up. By combining it with CDT and in the future also with PEth testing, where indicated, harmful alcohol consumption during the preceding 1 to 2 weeks can be detected too. Even though EtG in hair is the most telling test to confirm chronic harmful alcohol consumption, this biomarker is typically only determined in special situations, e.g. to help answer forensic questions.

Finally, it is important to emphasize that alcohol biomarker test results should never be interpreted in isolation, but always in the context of medical history, clinical findings and the patient’s mental and physical state of health.

Conflict of interest
The authors declare that no conflict of interest exists.

Manuscript received on 4 October 2017; revised version accepted on
19 February 2018

Translated from the original German by Ralf Thoene, M.D.

Corresponding author
PD Dr. rer. nat. Hilke Andresen-Streichert
Uniklinik Köln
Institut für Rechtsmedizin
Arbeitsbereich Toxikologie und Alkohologie
Melatengürtel 60/62, 50823 Köln, Germany

►Supplementary material
For eReferences please refer to:
www.aerzteblatt-international.de/ref1818

eMethods, eBox, eTable::
www.aerzteblatt-international.de/18m0309

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