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.

Detection of alcohol consumption and surveillance of alcohol abstention in patients with alcoholic liver disease (ALD) are at the center of attention of transplant physicians. Liver transplantation (LT) is an established treatment of end-stage ALD, which presents the second most common indication for LT in Western countries[1,2]. Due to the prevalent perception within the population and among treating physicians that ALD is a self-induced disease, and possibly perpetuated by the evident lack of donor organs, this indication is controversial[3]. Therefore, ALD as an indication for LT all the more requires transparent, comprehensible selection criteria. Since alcoholism is a life-long disease and is not cured by LT, optimal selection of patients with a low risk of alcohol relapse as well as continuous monitoring and support after LT are essential.

In order to facilitate optimal patient selection for the life-saving opportunity of LT, patients currently have to undergo a thorough psychological evaluation by an experienced addiction specialist. In addition, an abstinence period of at least 3 up to 6 mo is required in most transplant centers. Nevertheless, alcohol relapse on the waiting list is detected in up to 25%[4-6] and occurs in up to 50% of liver transplant recipients[7,8]. Of these, up to 36% of patients resume heavy drinking[9-11]. Long-term outcome after LT is significantly reduced in the patient cohort with return to excessive alcohol consumption but is similar to other indications despite return to low to moderate alcohol consumption[12,13].

Therefore, our eligibility criteria to list patients need to be refined, and we need more reliable tools to predict the individual risk of relapse to heavy drinking. In addition, surveillance and concomitant psychological support after LT should take a central role. This requires continuous surveillance programs for LT recipients to avoid alcohol relapse as well as tools to maximize rates of early detection of alcohol relapse to prevent graft damage.

Objective direct alcohol parameters could support evaluation by addiction specialists and improve patient selection prior to LT and optimize surveillance and early detection of alcohol consumption in liver transplant recipients.

This review summarizes the challenges of detection and correct assessment of alcohol consumption. It focuses on the current knowledge on alcohol biomarkers in liver disease and their particular value within the liver transplant setting and gives insight into possible future developments.

Correct diagnosis of ALD is critical for individual treatment approaches, but it is sometimes difficult, since specific features are lacking. After ruling out different liver diseases, diagnosis has to be based on patients’ statement on alcohol consumption. Yet, reliable objective alcohol parameters for confirmation have not yet found their way into routine clinical use (see below).

According to the current guidelines of the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD), ALD may diagnosed upon documentation of consumption of > 30 g/d of ethanol as well as the presence of clinical and/or biological signs of liver injury[14,15]. According to EASL guidelines, diagnosis should be based on “clinical” [such as gamma-glutamyl transferase (GGT), carbohydrate deficient transferrin (CDT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), mean corpuscular volume (MCV)], “biological” (serum fibrosis markers), and “ultrasound parameters” (detection of steatosis, grading of parenchymal alterations). Furthermore, liver biopsy might be required for confirmation of diagnosis.

However, all mentioned parameters may be non-diagnostic in the case of mild ALD or early cirrhosis[15,16], and patients are normally asymptomatic until an advanced state of liver disease has been reached. The documentation of regular alcohol intake, however, and especially the assessment of the alcohol amount is difficult. Commonly, patients do not admit alcohol consumption or underreport or do not indicate correct amounts of alcohol intake[17-19]. Furthermore, the amount of alcohol intake and development of liver alterations or severity of liver disease are not strictly linear[20]. Clear diagnosis of alcohol consumption and ALD is complicated by the lack of definite cut-off values of ethanol identified as harmful in certain populations.

In 60% of patients with regular alcohol intake > 60 g/d, hepatic steatosis was found[21,22]. In 29% of a large patient series, liver cirrhosis was detected by liver biopsy in 29% of a large series of patients with alcoholism[23]. Differential diagnosis to non-alcoholic fatty liver disease (NAFLD) (ethanol cut-off: 20 g/d for women, 30 g/d for men) and the assessment of alcohol as an additional hit to the liver are difficult. Even liver biopsy cannot safely discriminate ALD from NAFLD[24,25]. Moreover, in a meta-analysis, daily consumption of > 25 g of ethanol has been associated with an increased risk of liver cirrhosis and its complications[26]. Increased risk of mortality due to liver cirrhosis was recently found even below 25 g/d of ethanol (12-24 g/d)[27]. Thus, patients might be at risk at ethanol levels even below the current public recommendations for alcohol consumption.

Alcohol markers for detection of alcohol consumption and prediction of alcohol relapse need to fulfill certain features in the setting of ALD and LT. Only patients with a low risk of relapse are eligible for transplant. Patients in whom alcohol relapse is detected within the waiting period may be excluded from the life-saving opportunity of LT. Since alcoholism is a life-long disease that is not cured by LT, LT recipients should receive close surveillance to provide the possibility of an early detection in the case of relapse. LT recipients with alcohol relapse are at risk for the development of alcoholic hepatitis and re-cirrhosis in the liver graft. Nevertheless, in case of alcohol relapse, patients may be denied liver re-transplantation.

Optimal alcohol biomarkers in the setting of LT should not be influenced by liver alterations, commonly present kidney dysfunction, changes in body composition (low fat and muscle mass in case of end-stage cirrhosis, increased body water in case of ascites), immunosuppressive medication, or multiple drug therapy and should be cheap and easily accessible. In LT candidates who get evaluated for LT or are on the waiting list, the markers need to be highly specific in order not to deny mistakenly a life-saving LT due to false positive alcohol tests. In LT recipients in whom early detection of relapse is the focus to preserve graft function, high sensitivity is of special interest.

To date, national and international guidelines for alcohol biomarker screening of patients within the transplant setting are still lacking. Many transplant centers in Europe and the United States routinely use ethanol (EtOH) and CDT as direct markers of alcohol consumption as well as GGT and MCV as indirect markers.

The recommendations of the National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism[28] for ALD patients state: “For screening purposes in primary care settings, interviews and questionnaires have greater sensitivity and specificity than blood tests for biochemical markers, which identify only about 10% to 30% of heavy drinkers. Nevertheless, biochemical markers may be useful when heavy drinking is suspected but the patient denies it.” GGT, MCV, and CDT are recommended, nevertheless, to show certain limitations within the transplant setting (see paragraphs on indirect and direct alcohol biomarkers).

After ingestion, ethanol is absorbed via the oral, gastric, and small intestinal mucosa. About 2%-10% of ethanol are excreted via urine, sweat, and exhalation air without modification. EtOH detoxification starts within the stomach and is facilitated by alcohol dehydrogenase (ADH) (sigma-ADH, about 5% of ethanol metabolism). The major part of EtOH is metabolized to acetaldehyde within the liver by ADH, catalase, and the so-called microsomal ethanol oxidizing system (MEOS; via cytochrome P450 CYP2E1; especially in case of > 50 g/d alcohol intake or chronic alcohol intake). Acetaldehyde is further metabolized to acetic acid via acetaldehyde dehydrogenase. Acetic acid is metabolized via the citrate cycle and the mitochondrial respiratory chain and exhaled as CO₂. A very small amount (< 0.1%) of ethanol undergoes conjugation reactions with glucuronic acid in the presence of membrane-bound mitochondrial uridine diphosphate glucuronyl transferase to produce ethyl glucuronide (EtG) and with sulfate to produce ethyl sulfate (EtS), which are excreted in the urine[29-31].

State markers such as MCV[32] and the liver function tests GGT, ALT, and AST are widely used for routine screening of chronic alcohol consumption. These indirect markers of alcohol consumption may yet be elevated not only in case of ALD but all other forms of acute or chronic liver disease or vitamin B12 and/or folic acid deficiency. In a recent study on 210 non-excessive drinkers, 272 excessive drinkers, and 76 patients with alcoholic cirrhosis investigating the kinetics of alcohol markers during abstinence, the diagnostic accuracy of GGT to detect excessive drinkers was moderate [area under the curve (AUC) 0.68] and was associated with the amount of alcohol consumed within the past 30 d, which is in contrast to the AST/ALT ratio[33]. In this patient cohort consisting of ALD patients only, GGT reached a sensitivity and specificity of 49.6%, and 83.9%, respectively, for the detection of excessive alcohol consumption[33]. In contrast, in a setting of 141 liver transplant candidates and recipients, different markers were analyzed for their value in screening the consumption of any amount of alcohol, and the specificities of GGT, MCV, and AST were rather low[17]. Besides low specificity for chronic alcohol intake and detection of alcohol relapse[17], these markers have also low sensitivity for recent intake of excessive alcohol amounts[34]. When using these markers, we have to be aware of certain limitations, especially within a patient collective with already significant liver disease or on multiple drug therapy[35,36].

Currently, a range of direct alcohol biomarkers are used that detect EtOH or EtOH metabolites in different body components. We can distinguish between short- and long-term alcohol markers (Table 1).

Phosphatidylethanol (PEth) is a phospholipid formed only in the presence of EtOH via a transphosphatidylation catalyzed by phospholipase D in the cell membrane of peripheral blood cells[84-86]. It is a long-term alcohol parameter that becomes positive after repeated consumption of ≥ 50 g/d EtOH over a period of 2 to 3 wk measured by HPLC in whole blood and can be detected up to > 2 wk (probably up to 6 wk in some cases[87]) after cessation of alcohol consumption[88,89]. Using LC-MS/MS, elevated PEth levels can be found after a single day of excessive alcohol intake[90]. A recent study also reported PEth to be a valuable tool for the detection of moderate alcohol consumption[91]. Novel assays even allow qualitative and quantitative measurement of PEth in dried blood spots[92,93]. PEth was described to bear higher sensitivity than CDT, GGT, or MCV (sensitivity of 99% vs 40% to 77%)[94]. Another study found an excellent diagnostic accuracy (AUC = 0.974, sensitivity 94.5%, specificity 100%) superior to that of CDT, GGT, or MCV in patients with excessive alcohol consumption taking part in a detoxification program[95]. Helander et al[96] showed the superiority of PEth over CDT for monitoring alcohol consumption in an outpatient alcohol detoxification program. In 43% of these patients alcohol consumption was detected only by PEth, in 38% by PEth plus CDT, and in 21% by EtG/EtS only. False positive or high test results can occur if samples are stored at room temperature[92]. PEtH levels are not affected by sex or age[97], and in contrast to CDT, PEth is not influenced by the presence or severity of liver disease[87,98]. Although already first described in the 1980s to be synthesized in peripheral blood[84,85], PEth still has not found its way into routine diagnostics. Data on LT candidates and recipients are currently lacking.

Platelet monoaminoxidase-B (MAO-B) protein levels showed promising accuracy in detection of heavy drinking measured by immunoblotting or enzyme-linked immunosorbent assay (AUC = 0.72)[99]. If combined with CDT, its performance can be improved (AUC = 0.84). Formerly investigated as a genetic trait marker of alcohol dependence, MAO-B presents an interesting target. Protein levels, in contrast to MAO-B enzymatic activity, are not impaired by smoking[100]. This marker, although promising, needs to be further validated.

A recent study investigating a study cohort of 53 excessive drinkers, a control group of 49 individuals, and a validation cohort of 40 excessive drinkers and 40 controls discovered four novel proteins for the detection of excessive alcohol intake by serum proteomic analysis[101]. These proteins were AT-rich interactive domain-containing protein 4B, phosphatidylcholine-sterol acyltransferase, hepatocyte growth-factor like protein, and ADP-ribosylation factor 6. They are indirect markers of alcohol consumption and are superior to routinely used markers such as AST, ALT, GGT, MCV, and CDT (AUC = 0.70 to 0.86 vs 0.21 to 0.67). They might be involved in inflammation processes, cellular organization, protein transportation, and cell proliferation mechanistically linked to alcohol consumption and metabolism. However, to date it remains unclear whether the secretion and excretion of these proteins are influenced by liver function.

Alcohol biomarkers significantly improve detection of alcohol consumption. To account for various drinking patterns, we always examine several markers in combination to cover different time periods. In the setting of LT, sensitivity and specificity are of special concern, since a false positive alcohol test may deny a patient the opportunity of a life-saving LT. During the evaluation process for LT, hEtG seems to be an attractive marker, since a time frame of up to 6 mo is covered and false positive results are unlikely. However, data demonstrating improved relapse rates after LT are missing, and optimal cut offs in this setting are unclear. For surveillance of patients on the waitlist for LT, a combination of uEtG, PEtH, and GGT seem to be the most appropriate tools. In LT recipients, a combination of uEtG, GGT, and CDT or PEtH might be valuable. Long-term follow-up after LT seems to be well covered by additional hEtG concentrations. Currently, MeOH does not seem to have advantages over the mentioned parameters. Importantly, routinely assessed alcohol biomarkers prior to and after LT may help guide treatment decisions for LT and allow early referral for psychological support and alcohol detoxification programs.