Biochemical markers of alcohol intake

Biochemical markers of alcohol intake can be separated into two categories: direct markers of ethanol metabolism and indirect markers. The different alcohol markers have varying time windows of detection and are a useful additional tool to detect alcohol intake in alcohol-dependent clients.

by Jane Armer and Rebecca Allcock

Introduction
Alcohol dependence is characterized by craving, tolerance, a preoccupation with alcohol and continued drinking in spite of harmful consequences. The World Health Organization Alcohol Use Disorders Identification Test (AUDIT) is recommended for the identification of individuals that are dependent on alcohol [1]. The prevalence of alcohol use disorders (including dependence and harmful use of alcohol) is 11.1% in the UK compared to 7.5% across Europe [2]. In England, 250 000 people are believed to be moderately or severely dependent and require intensive treatment [3].

Alcohol use is the third leading risk factor contributing to the global burden of disease after high blood pressure and tobacco smoking [4]. In 2012, 3.3 million deaths (5.9% of all global deaths) were attributable to alcohol consumption [2]. It is estimated that the UK National Health Service (NHS) spends £3.5 billion/year in costs related to alcohol and the number of alcohol-related admissions has doubled over the last 15 years [3].

In the UK, one unit equals 10 mL or 8 g of pure alcohol, which is around the amount of alcohol the average adult can process in an hour. The latest UK recommendations are to not regularly drink more than 14 units per week (men and women) and to limit the total amount of alcohol consumed on a single occasion [5].

The most common entry into alcohol treatment services in England is either self-referral or referral by the GP [3]. Services have a limited number of options to determine if an individual in treatment for alcohol dependence is continuing to drink alcohol. They rely on self-report by the individuals in the form of alcohol diaries and breathalyser tests. There is no regular schedule for biochemical markers. If a client is found to be drinking alcohol during the treatment programme, an assessment is done of the amount of alcohol consumed, the pattern of alcohol consumption and how it will impact on their treatment. This is factored into the recovery plan and there is a re-assessment of the support and interventions needed for that client. Possible interventions include cognitive behavioural therapies, pharmacological therapies or in-patient assisted withdrawal. In 2013/14, only 38% of clients in alcohol treatment in England successfully completed their treatment [3].

Monitoring clients in alcohol treatment
Diaries that record alcohol intake are commonly used to monitor the progress of clients. However, this relies on accurate self-reporting of alcohol intake by the client and under reporting is a common problem. Biochemical markers of alcohol intake can provide a more comprehensive assessment of a client’s progress.

Direct markers of alcohol intake
Direct markers of alcohol intake include ethanol, ethyl glucuronide (EtG), ethyl sulphate (EtS), fatty acid ethyl esters (FAEE) and phosphatidylethanol (PEth).

Following the ingestion of ethanol, >95% is metabolized in the liver by alcohol dehydrogenase to acetaldehyde then by aldehyde dehydrogenase to acetic acid [14]. Less than 5% is excreted unchanged in the urine, breath and sweat. A small amount of ethanol is conjugated to form EtG and EtS (Fig. 1). Ethanol is usually only detectable in breath and urine after very recent alcohol consumption and the detection time window depends on the amount of alcohol consumed. In comparison, urine EtG and EtS remain detectable for around 24 hours after moderate alcohol intake and for up to 130 hours in subjects admitted for alcohol detoxification [6, 7]. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for EtG and EtS. An immunoassay is also available for EtG [8, 9].

Many studies have demonstrated the benefit of measuring EtG and EtS in clients in alcohol treatment. Continued alcohol consumption can be detected by the measurement of urine EtG and EtS in clients who do not admit to consuming alcohol and provide a negative breathalyser test. This is due to the increased time window of detection for urine EtG and EtS compared to breath ethanol. This demonstrates the unreliability of self-reporting of alcohol intake and the benefit of biochemical markers to detect clients that are continuing to drink alcohol [10].

As with urine testing for drugs of abuse, it is possible for a client to consume a large volume of water to dilute the sample and produce negative EtG and EtS results. Creatinine should always be measured to check for adulteration and it may be beneficial to report EtG and EtS as creatinine ratios to overcome this problem. Further work is required to define cut-offs for EtG and EtS as creatinine ratios.

False negative EtG results can be caused by the presence of Escherichia coli in urine as glucuronidase is present with high activity in most strains. False positive EtG and EtS results have also been reported following use of ethanol based mouthwash or hand gels and after the consumption of non-alcoholic beers (up to 0.5% alcohol). Due to the risk of positive results due to unintentional alcohol exposure, particularly for urine EtG, it is important that clinical cut-offs used are clearly defined and LC-MS/MS methods that measure both EtG and EtS are preferred [11]. In the USA, the Substance Abuse and Mental Health Administration (SAMHSA) have suggested that EtG results >1.0 mg/L are consistent with alcohol intake and that results between 0.1 and 1.0 mg/L should be interpreted with caution. It is accepted that further work is required to clearly define cut-offs for EtG and EtS and that other biomarkers may be useful when interpreting borderline positive results in the range 0.10–0.50 mg/L [12].

Methods for the measurement of EtG and FAEEs in hair have been developed allowing a longer term assessment of alcohol intake. Hair analysis is most suitable for subjects where longer term abstinence needs to be demonstrated such as in patients awaiting liver transplantation. EtG cut-offs have been suggested by the Society of Hair Testing for chronic excessive alcohol consumption (30 pg/mg) and abstinence assessment (7 pg/mg). However, results may be influenced by hair products and this needs to be taken into account when interpreting results.

PEth is formed from ethanol and phosphatidylcholine in cell membranes. The reaction is catalysed by phospholipase D and occurs in the cell membranes of erythrocytes; therefore, PEth is found in the red blood cell fraction of blood rather than in serum or plasma. PEth is a group of phospholipids with varying carbon lengths and LC-MS/MS methods to detect the major forms of PEth in whole blood have been developed. A single dose of ethanol does not produce a measurable amount of PEth and it has been demonstrated that approximately 50 g of ethanol/day (6.25 UK units) is required to provide a positive PEth result. In comparison to serum carbohydrate deficient transferrin (CDT; see ‘Indirect markers of alcohol intake’ below), urine EtG and urine EtS, PEth demonstrated the highest sensitivity for regular alcohol consumption in clients in alcohol treatment and was found to be positive twice as often as CDT [13]. Further work is required to understand how PEth can be used optimally in combination with other alcohol markers in clients in treatment for alcohol dependence [14].

Indirect markers of alcohol intake
The indirect markers include mean corpuscular volume (MCV), gamma glutamyl transferase (GGT) and CDT. These markers increase following significant alcohol intake over a prolonged time period and are not useful for detecting a single alcohol ‘binge’. MCV and GGT are not specific markers of alcohol intake.

CDT refers to altered glycoforms of transferrin as a result of alcohol-induced changes in the carbohydrate composition of transferrin. The main component of serum transferrin is tetrasialotransferrin, which makes up approximately 80% of the total. Normal samples usually contain approximately 15%, 4–5%, 1–1.5% and 1% of pentasialotransferrin, trisialotransferrin, disialotransferrin and hexasialotransferrin, respectively. An alcohol consumption of at least 60 g/day (7.5 UK units) for 2 weeks is required to increase the disialotransferrin [15]. CDT may also be increased if genetic variants are present and in advanced liver disease. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has recently proposed a reference measurement procedure for CDT and more studies assessing the diagnostic performance of CDT to detect alcohol dependence are now needed using methods harmonized to the international reference measurement procedure.

Table 1 summarizes the time window of detection and limitations of the alcohol markers discussed.

Conclusions
Currently, the assessment of clients in alcohol treatment relies largely on self-reporting and limited biochemical testing, which makes assessment of a client’s progress challenging. There are a number of available biochemical markers that could improve the detection of alcohol use in clients with alcohol dependence and ultimately lead to initiation of early intervention and altered treatment strategies. This in turn could improve the numbers successfully completing treatment. A combination of short-term and longer term biochemical markers is likely to be the most useful approach depending on the treatment setting. The advantage of the breathalyser test over biochemical markers that require laboratory analysis is the immediate availability of the result which allows an immediate intervention for a client with a positive result. Laboratory tests need to be available in a timely manner and with appropriate and well-defined cut-offs. The clinical benefit of alcohol markers in improving the number of clients that successfully complete their treatment for alcohol dependency has not yet been demonstrated. Randomized controlled trials comparing outcomes with or without the use of biochemical markers are required.

References
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The authors
Jane Armer*¹ BA MSc FRCPath and
Rebecca Allcock² BSc MSc FRCPath
¹Department of Blood Sciences,
East Lancashire Hospitals NHS Trust,
Blackburn, UK
²Department of Clinical Biochemistry,
Lancashire Teaching Hospitals NHS
Foundation Trust, Preston, UK

*Corresponding author
E-mail: jane.armer@elht.nhs.uk