Biomolecules and Biomarkers Used in Diagnosis of Alcohol Drinking and in Monitoring Therapeutic Interventions

Background: The quantitative, measurable detection of drinking is important for the successful treatment of alcohol misuse in transplantation of patients with alcohol disorders, people living with human immunodeficiency virus that need to adhere to medication, and special occupational hazard offenders, many of whom continually deny drinking. Their initial misconduct usually leads to medical problems associated with drinking, impulsive social behavior, and drunk driving. The accurate identification of alcohol consumption via biochemical tests contributes significantly to the monitoring of drinking behavior. Methods: A systematic review of the current methods used to measure biomarkers of alcohol consumption was conducted using PubMed and Google Scholar databases (2010–2015). The names of the tests have been identified. The methods and publications that correlate between the social instruments and the biochemical tests were further investigated. There is a clear need for assays standardization to ensure the use of these biochemical tests as routine biomarkers. Findings: Alcohol ingestion can be measured using a breath test. Because alcohol is rapidly eliminated from the circulation, the time for detection by this analysis is in the range of hours. Alcohol consumption can alternatively be detected by direct measurement of ethanol concentration in blood or urine. Several markers have been proposed to extend the interval and sensitivities of detection, including ethyl glucuronide and ethyl sulfate in urine, phosphatidylethanol in blood, and ethyl glucuronide and fatty acid ethyl esters in hair, among others. Moreover, there is a need to correlate the indirect biomarker carbohydrate deficient transferrin, which reflects longer lasting consumption of higher amounts of alcohol, with serum γ-glutamyl transpeptidase, another long term indirect biomarker that is routinely used and standardized in laboratory medicine.

An overall strong, positive correlation between the cumulative AUDIT-C score and BrAC reading (r = 0.416, p = 0.001) shows a strong correlation between a qualitative measurement of long-term hazardous drinking and current drinking [60]. A breath test did not show sufficiently sensitivity in identifying self-reported heavy drinking in a cross-sectional sample of alcohol-dependent patients [61].
Relapse is routinely measured in patients under alcohol dependence treatment [62]. Relapse was assessed by ethyl glucuronide (EtG) in urine, breath alcohol tests and self-reports in outpatients undergoing long-term alcohol dependence treatment. The percentage of patients showing alcohol relapse was 1.1% by self-report, 4.4% by breath test (range: 0.06-2.60 g alcohol/L), and 37.7% by EtG measurement (mean concentration 47.2 mg/L, range 0.2-1220 mg/L). A good agreement was observed between self-report and breath test, between self-report and EtG, and between breath test and EtG measurement. However, low discrepancies exist, and a high percentage of alcohol relapse cases (93.2%) were only identified by EtG measurement. In contrast, the breath test identified a few cases of alcohol relapse that were not identified by EtG measurement. In these cases, high alcohol concentrations (mean 1.24 g/L) were found, likely suggesting recent alcohol consumption following abstinence [62]. This study shows that each method has its specific timeframe during which it is useful, and multiple methods should ideally be used together in order to identify immediate, short-term or long-term alcohol consumption.

Factors that Affect Breath Test Results
Variability exists in replicate breath alcohol exhalation profiles for one subject collected over a short time interval. There were no age or gender influences, while the breath exhalation volume and breath exhalation time also lacked significant associations [63]. However, the breathing pattern seems to have an effect. These include measuring too early in the expiratory phase, shallow expiration or hyperventilation, or measuring hyperventilation under conditions of chilly ambient temperature. All of these factors give rise to underestimates of BrAC, compared to reference values, while the expired volume is kept constant [64].
The complex exchange of gasses and water in the blood vessels and the mucosa of the airways may further affect the BAC/BrAC ratio [50,65]. Estimating BAC from BrAC is based on the premise that exhaled air reflects the alveolar air alcohol concentration, which is thought to be in direct equilibrium with the blood in the pulmonary circulation. Several factors determine BrAC, most importantly body and lung physiology [66]. During expiration, humidified air at core body temperature loses heat and moisture as it passes through the cooler airway mucosa. Thus, expired air is cooler and drier than alveolar air, as well as containing fewer soluble gases such as alcohol. In addition, alcohol is further lost in the airways, due to the high solubility of the airway tissue to both water and alcohol. Taken together, these suggest that exhaled alcohol levels reflect the concentration of this molecule in the airways rather than in the alveoli [66]. Increases in breath temperature and BrAC were observed with increasing breath volumes, relative to the values for the forced vital capacity. A breath volume of 10% of the forced vital capacity contains around 80% of the end-expiratory breath concentration, and a breath-concentration plateau occurs at around 70% of the forced vital capacity [67]. As BrAC is a function of time and expired air volume, the more of the available lung volume is expired, the higher the BrAC, such that BrAC depicts a more accurate measurement of BAC when an increasing fraction of the available lung volume is expired and BrAC gets closer to the alveolar alcohol concentration.
Body size and lung capacity are important. As some instruments require a volume of exhaled air as high as 1.5 L, an individual with a smaller lung capacity may need to exhale a greater fraction of the available lung volume compared to a person with a higher lung capacity, resulting in a higher BrAC in the exhaled air [66,68]. Breathing patterns also affect BrAC. Hyperventilating or deep breathing lead to lower BrAC, while holding one's breath leads to higher BrAC than normal breathing. These can be explained by the altered diffusion of alcohol between the expired air and the alveolar mucosa during altered breathing patterns [66,69].
An important analytical observation should always be clear. The technical features of the analyzers are different, as are the procedures and the technical ability of the person following them.

Measuring Blood Alcohol Concentration
Xiao et al. [71] describe a quantitative method of determining the levels of alcohol in whole blood by headspace gas chromatography-mass spectrometry with good specificity and sensitivity (limit of quantification 39.5 ȝg/mL and limit of detection 0.4 ȝg/mL). Headspace gas chromatography/flame ionization detection was also used in a retrospective study to determine BAC in public and private drivers involved in traffic accidents [72]. Headspace gas chromatography/flame ionization detection was also used for post-mortem analysis of blood samples in a large sample of traffic accident victims. This study showed that alcohol was often a contributing factor in traffic accidents (judged to be positive when measured to exceed 0.5 g/kg) [73]. In another study, Sutlovic et al. [74] show that while there is generally good agreement between the BAC measured by headspace gas chromatography/flame ionization detection at different time points following different storage periods in post-mortem blood samples, variability of up to 10% was observed in some instances, which is unacceptable in precise forensic evidence. This was judged to result mainly from alcohol oxidation during storage, showing that storage methods have a great influence on the alcohol content in blood [74].
A recent study by Bielefeld et al. [75] shows that estimating BAC based on the amount of alcohol consumed (using Widmark's equation) may lead to erroneous estimates, as significant differences were found between the estimated BAC (Widmark's equation) and the measured BAC (headspace gas chromatography/flame ionization detection) in a sample of elderly volunteers participating in a drinking experiment aiming to achieve a BAC of 0.6 g/kg (Widmark factors used: 0.7 for males, 0.6 for females). The actual BAC was significantly higher than the estimated BAC in both males and females, and this was found to occur due to a large degree of variation in the calculated individual-specific Widmark factors [75].
Using headspace gas chromatography, Mitchell et al. [76] show that Cmax, Tmax and AUC were different after administration of alcohol (0.5 g/kg body weight), as either beer (5.1% v/v), white wine (12.5% v/v), or vodka/tonic (20% v/v), in a small sample of healthy men. Vodka/tonic (20% v/v) led to the higher Cmax and AUC, along with the lowest Tmax. There were no significant differences between beer (5.1% v/v) and white wine (12.5% v/v). Spirits resulted in higher exposure than beer or wine, while beer was associated with a lower, more delayed exposure [76].

Ethyl Glucuronide and Ethyl Sulfate in Urine
EtG (ethyl ȕ-D-6-glucuronide) and ethyl sulfate (EtS) represent direct biomarkers of alcohol use. EtG is a minor non-oxidative metabolite of alcohol that forms in the liver through the conjugation of ethanol and glucuronic acid [77]. This reaction is carried out by members of the uridine 5'-diphospho-glucuronosyltransferase family of enzymes, using uridine diphosphate glucuronic acid as a cofactor. Multiple uridine 5'-diphospho-glucuronosyltransferases carry out this reaction, such that they compensate for polymorphisms in one another [78]. However, conversion of alcohol to EtG was recently shown to be influenced by the interaction between uridine 5'-diphospho-glucuronosyltransferases and nutritional components [79].
EtG can be detected in various body fluids, tissue and hair beginning a few hours after alcohol consumption and it remains detectable for up to 80 h after the complete elimination of alcohol from the body [80][81][82]. It can be detected in the blood for up to 36 h and in the urine for up to 5 days, long after alcohol got eliminated [77,83]. EtS is another minor, direct alcohol metabolite, produced through conjugation with sulfate. This reaction is carried out by cytosolic sulfotransferase enzymes [84]. EtG is primarily used to detect heavy alcohol use. Although only a relatively low amount of alcohol is eliminated by glucuronidation, EtG is an important biomarker that can determine alcohol consumption. The presence of EtG indicates recent alcohol use, even if there is no detectable alcohol in the body [83]. Urine EtG and EtS have been identified as important markers of recidivism in a large sample of drivers charged with DUI in a Canadian study [85].
A recent alcohol challenge study (doses calibrated to achieve blood concentrations of 20, 80 or 120 mg/dL) shows that urine EtG was always detectable at the 100 and 200 ng/mL cut-offs 12 h after ingestion. At 24 h, the sensitivity associated with these cut-offs was low following ingestion of the low does. The sensitivity of the assay was low at 24 h regardless of dose. There was generally good correlation between urine EtG and EtS. This report showed that light drinking can be detected through urine EtG analysis during the first 24 h [86]. Results of recent studies correlating urine EtG and EtS with alcohol consumption patterns are shown in Table 1 [87][88][89][90][91][92][93].
Positive EtG (>0.1 mg/L) and EtS (>0.05 mg/L) can be measured in healthy volunteers drinking 1-2 drinks for up to 24 h. Among patients under withdrawal treatment, the highest urine EtG and EtS levels were obtained at the first sample, and decreased with time and repeated sampling [94]. The kinetics of EtG and EtS formation and elimination were assessed in a small sample of healthy volunteers after consuming 4 or 8 units of alcohol (40 or 80 mL) [95]. Median EtG Cmax was 0.4 ± 0.3 ȝg/mL in serum and 3.5 mg/h ± 1.2 mg/h in urine after 4 units, achieved after 2.0 ± 0.8 h in serum and 3.0 ± 1.0 h in urine (Tmax). The corresponding Cmax values for EtS were 0.2 ± 0.1 ȝg/mL in serum and 1.3 ± 0.6 mg/h in urine, with Tmax 1.0 ± 1.0 h and 2.0 ± 0.5 h, respectively [95]. After 8 units, EtG Cmax was 1.3 ± 0.4 ȝg/mL in serum and 10 ± 3.4 mg/h in urine, with Tmax 4.0 ± 1.8 h and 4.0 ± 2.0 h, respectively. EtS Cmax was 0.6 ± 0.1 ȝg/mL in serum and 3.5 ± 1.1 mg/h in urine, with Tmax 3.0 ± 1.0 h and 3.0 ± 1.0 h, respectively. The EtG/EtS ratio increased as a function of time after alcohol administration in both serum and urine samples for up to 6 h. This occurred to a lesser extent after 8 units of alcohol than after 4 units [95]. A dose-effect relationship between alcohol ingestion and EtG Cmax was observed in another sample of healthy volunteers. EtG levels in urine were higher than in blood or saliva, suggesting that EtG measurements are most sensitive in urine [96].  Urinary EtG correlates well with EtS [87,92,93]. Both EtG and EtS were below the cut-off value among subjects who denied alcohol consumption, and were generally undetectable (<0.1 μg/mL) in subjects who reported only one drink on the day before sampling. EtG (r = 0.448, p < 0.02) and EtS (r = 0.406, p < 0.04) show modest correlation with the number of drinks [87]. In a different study, the minimum EtG and EtS were both found in the same individual, as were the maximum EtG and EtS. Both EtG and EtS levels decreased rapidly in alcohol-dependent patients during withdrawal. EtG and EtS were detectable on day 8 in one patient only, both of which were present in the same individual [93].
EtG and EtS in urine can help either prove or disprove self-reported alcohol consumption patterns in various populations in which abstinence is encouraged. Self-reported past 3 days, alcohol consumption was significantly related to the EtG and EtS concentrations in urine in patients from hepatology clinics (r = 0.94, p < 0.001) [91]. Urine EtG was identified as the strongest marker of alcohol consumption in a sample of liver transplant recipients and liver transplant candidates. Urine EtG levels were strongly correlated with the amount of alcohol consumed (p < 0.001), and as such it can be used to detect alcohol consumption (89.2% sensitivity, 98.8% specificity, 97.1% PPV and 95.4% NPV at cut-off >500 ng/mL). This biomarker was useful in predicting alcohol consumption in subjects with either positive or negative AUDIT results [97].
Urine EtG tested positive in 29.3% of patients undergoing treatment for alcohol use disorder, of which 45.5% admit recent alcohol consumption and 22.7% have positive breath tests [98]. While superior to a breath test in identifying recent alcohol consumption, urine EtG still generally detects only moderate to high alcohol consumptions in the past 2 days prior to sample collected [99].

Discussion
Urinary EtG and EtS were used to estimate relapse in outpatients treated for alcohol-related problems. Alcohol consumption is higher during the weekend than throughout the week in outpatients [62,100]. A good correlations exists between the quantity of self-reported drinking in the 3 days prior to each sample collection and urinary EtG (r = 0.662, p < 0.001) and EtS (r = 0.716, p < 0.001) levels. No recent drinking was self-reported in patients with samples negative for EtG or EtS [101]. Urinary EtG and EtS can identify long-term alcohol use in the presence of other markers such as phosphatidyl-ethanol (PEth) and/or carbohydrate-deficient transferrin (CDT), or can indicate occasional alcohol use when present on their own [102]. Serial testing led to a significant decline in positive samples over time (p = 0.017) among active duty service members receiving addiction treatment. EtG positivity generally correlates poorly with the AUDIT score [89].
Urinary EtG is an important biomarker for assessing alcohol abstinence in orthotropic liver transplantation [90]. Only 3.6% of potential recipients admitted alcohol consumption in a sample of 141 patients, despite 19.8% being positive for at least one alcohol biomarker (urinary EtG, ethanol, methanol, CDT, aspartate aminotransferase (AST), alanine aminotransferase (ALT), Ȗ-glutamyl transpeptidase (Ȗ-GTP) and mean corpuscular volume (MCV)) at any visit. Of these, urinary EtG was the best predictor of alcohol consumption, and increased detection of alcohol consumption compared to other biomarkers (p < 0.001) [90]. The prevalence of urinary EtG and EtS was higher in patients with alcoholic liver disease than in patients with other liver conditions (20% vs. 5%, p = 0.04) [103]. The presence and levels of urine EtG and EtS are also related to the incidence of failing an ignition interlock device BAC test among drivers convicted of DUI [104].
Furthermore, during post-mortem analysis, 68% of individuals with a history of alcohol abuse were found to have a positive BAC (median 1.15‰, range 0‰-3.3‰). EtG concentrations in urine were significantly higher in individuals with a history of alcohol abuse during post-mortem analysis than in individuals without a documented history of alcohol abuse (339 ± 389 mg/L, p < 0.001) [105].

Factors Affecting Urine EtG
As urine EtG and EtS are short-term biomarkers, false negatives may arise due to low alcohol intake (<3 drinks) or a long period between alcohol intake and sample collection (>16 h) despite self-reported alcohol intake [92]. On the other hand, urine EtG and EtS tests are so sensitive to the presence of alcohol that that they are unable to distinguish between alcohol abstinence and low levels of alcohol consumption, and false positive results may be obtained even from accidental exposure [82,92]. Among subjects with no past 7 days drinking history, positive urine EtG and EtS results likely reflect non-beverage alcohol exposure [91]. Thus, factors unrelated to drinking may further distort results, such that alcohol consumption may be either underestimated or overestimated.
Despite EtG values being slightly lower when measured by enzyme immunoassay, a good correlation was found between enzyme immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (r 2 0.996 in clinical samples and 0.956 in post-mortem samples), with a strong correlation between EtG and EtS (r 2 0.9025, p < 0.001, mean EtG/EtS ratio 3.8, median EtG/EtS ratio 3.5) [106]. A good correlation between urine EtG levels measured by a commercially available immunoassay test and a lab-based mass spectrometry test was further observed in a sample of adults with alcohol dependence [107]. A good correlation (r = 0.96-0.98) was further observed between different liquid chromatography-mass spectrometry (LC-MS) methods of measuring EtG in urine [108].
Using dose-adjusted detection times, decreased renal function led to significantly longer detection times for urinary EtG and EtS compared to healthy subjects (p < 0.01). Cmax values were lower, and the detection time of EtG and EtS was correlated to the degree of renal dysfunction. The implication of this is that individuals with decreased renal function may be wrongly suspected of higher or more recent alcohol consumption [109].
Intensive use of mouthwash (4 times/day for 3¼ days) led to EtG and EtS below 500 ng/mL, thus allowing intentional alcohol use to be distinguished from accidental exposure [110,111]. On the other hand, intensive use of an alcohol-based hand sanitizer (120 times/day for 3 days) led to mean EtG levels of 278 ng/mL, with maximum EtG 2001 ng/mL in one subject. The urine concentration of EtG was highest at the end of each study day. EtS levels were lower than EtG levels (<100 ng/mL in all samples). As such, the presence of EtS may allow intentional alcohol use to be distinguished from dermal exposure [112]. A separate study found that transdermal exposure to hand sanitizer does not affect urine EtG levels. However, inhalation of hand sanitizer may increase urinary EtG levels [113].
Non-alcoholic beers (<0.5% alcohol), sauerkraut and matured bananas lead to urine EtG levels >0.1 mg/L for up to 13, 5 and 3.5 h later, respectively [114]. EtG (0.30-0.87 mg/L) and EtS (0.04-0.07 mg/L) were positive after consuming 2.5 L of non-alcoholic beer. EtG were above the abstinence cut-off of 0.1 mg/L. In one subject, overnight accumulation in urine led to high levels of EtG (14.1 mg/L) and EtS (16.1 mg/L) [115]. EtS was positive in urine in subjects consuming non-alcoholic wine (Cmax 2.15 mg/L). No such relationship was observed for EtG [110]. The consumption of baker's yeast and sugar led to EtG and EtS levels above the 0.1 mg/L cut-off for abstinence (0.67 and 1.41 mg/L, respectively). Alcohol was not detected in urine [116].
Ethanol glucuronidation is increased by cannabinol in a dose-dependent manner, and is decreased by cannabidiol in a noncompetitive manner. Other common drugs of abuse like morphine, codeine, lorazepam, oxazepam, nicotine or cotinine had no significant effects on ethanol glucuronidation [117]. Age, gender, ethnicity and liver disease severity did not significantly affect the association between past 3 days drinking and urine EtG or EtS [91].
As there are several factors that can lead to low positive urinary EtG tests in individuals who deny drinking, additional tests can differentiate between accidental alcohol exposure and the patient hiding alcohol consumption. In a recent analysis, 55.6% of individuals testing positive for low levels of urinary EtG or EtS denied drinking. Among these, negative PEth test results supported the subjects' claim of alcohol abstinence and likely suggests accidental exposure in 70.0%, while positive PEth test results contradicted the subjects' claim in 20.0% [118].
Phospholipase D normally catalyzes the hydrolysis of phospholipids to form phosphatidic acid. However, phospholipase D has a higher binding affinity for ethanol than water, resulting in the preferential production of PEth over phosphatidic acid in the presence of even low quantities of alcohol. PEth comprises a group of phospholipids with a common non-polar phosphoethanol head group and two fatty acid moieties [77,82]. PEth has a half-life of approximately 4 days in blood in alcoholic subjects admitted for detoxification, with no correlation to baseline PEth levels [121]. Results of recent studies correlating blood PEth with alcohol consumption patterns are shown in Table 2 [102,[122][123][124][125][126][127].
In a small sample of healthy volunteers drinking the equivalent of 1 g/kg alcohol for 5 consecutive days after 3 weeks of abstinence, followed by a further 16 days of abstinence after the drinking episode, the maximum BAC was 0.99-1.83 g/kg (mean 1.32 g/kg), reached after 1-3 h (mean 1.9 h) after the start of drinking. The maximum PEth 16:0/18:1 levels, measured by LC-MS/MS were 45-138 ng/mL 1 h after the start of drinking. PEth was detectable in 90.9% of the sample. Blood PEth levels continued to rise over the following days, peaking at 74-237 ng/mL between days 3 and 6 [128]. Trace levels of PEth 18:1/18:1, 16:0/16:0 and 18:1/16:0 (or 16:0/18:1) were detected in a blood sample collected 3 h post-drinking in another social drinker after a single 60 g alcohol dose, with no PEth detected prior to alcohol consumption after 3 weeks of abstinence [129].  Outpatients treated for alcohol-related problems; Range 0-16.5 μmol/L (mean 2.6), with 70% above the quantification limit (0.1 μmol/L) and 55% above the reference cut-off for alcohol abuse (0.7 μmol/L) at initial assessment; PEth-16: Negative PEth levels are found in teetotalers, while positive PEth levels are found in samples belonging to known alcoholic patients [130]. PEth levels generally decreased in subsequent samples in outpatients treated for alcohol-related problems, with a half-life of 3.5-9.0 days (mean 6.1 days, median 7.0 days) [102,131]. Blood PEth remains detectable for up to 14 days after the last drink in alcoholics admitted for alcohol detoxification [132]. Furthermore, PEth 0.9 μM was detected in a subject with a long-term history of alcohol abuse 9 days after the last drink, showing that PEth can be detected after a relatively long time since stopping drinking in subjects with a history of high alcohol consumption [133].
Blood PEth levels were assessed in two cohorts of HIV-positive patients who were expected to remain abstinent while waiting to start antiretroviral treatment. A high rate of alcohol consumption was found in one these populations, both by self-reporting as well as through positive PEth results [125]. In the other study, 37.3% of samples were PEth-positive (>8 ng/mL), despite over half of these individuals denying alcohol consumption. Men and subjects from lower economic classes were found to be more likely to under-report alcohol consumption [126]. PEth results were strongly correlated with AUDIT-C scores and measurements of alcohol consumption, including binge drinking in a sample of injecting drug users. Interestingly, almost 95% of individuals who did not report alcohol consumption actually tested negative for PEth [134].
PEth levels in blood distinguished between heavy drinkers (>60 g/day) and social drinkers in a meta-analysis (mean 3.897 vs. 0.288 ȝmol/L). As such, PEth is a tool used primarily to identify chronic excessive drinking [135]. Currently, there is no uniformly accepted cut-off level to differentiate between social drinkers (<40 g/day for males and <20 g/day for females), at-risk drinkers (40-60 g/day) and chronic heavy drinkers (>60 g/day), although a threshold of 0.7 ȝmol/L is sometimes used to classify alcohol-related problems, with blood PEth < 0.7 ȝmol/L generally consistent with low or moderate alcohol consumption in the two weeks prior to sample collection [131,136].
As the formation of blood PEth is specifically dependent on blood alcohol levels, a strong correlation exists between alcohol consumption and blood PEth levels [131,133]. PEth tests can monitor alcohol consumption, can help identify early signs of harmful alcohol consumption, and can help track cases of alcohol abuse or dependence [131]. PEth has 99% sensitivity for detecting excessive alcohol consumption with a cut-off of >0.22 μM [133].

Discussion
PEth was associated with ignition interlock devices BAC test failure. Higher PEth levels were found in individuals with a higher risk of interlock BAC failure (1.45 ± 1.17 μmol/L) compared to the low risk group (0.61 ± 0.61 μmol/L) [104]. LC-MS/MS (limit of detection 0.005 ȝmol/L) identified positive alcohol consumption in a higher proportion of driver blood samples with failed interlock blood alcohol than high-performance liquid chromatography (HPLC) (limit of detection 0.25 ȝmol/L). A good correlation was found between the methods. Both methods further identified alcohol consumption in DUI offenders without failed interlock tests. Overall, 88.5% of samples were positive by LC-MS/MS and 71.2% were positive by HPLC [137]. Interestingly, while zero failed interlock BAC tests suggests an absence of drinking and driving behavior, negative PEth suggests absence of drinking. Therefore, interlock BAC tests and PEth tests distinguish between drinking, and drinking and driving behavior [137].
PEth analysis is a measure of sobriety in alcohol-dependent subjects entering detoxification. Using a limit of quantification of 0.22 μmol/L, blood PEth correlates well with alcohol consumption during the 7 days prior to entering alcohol detoxification (range 0.63-26.95 μmol/L at day 1, mean 6.22 μmol/L, median 4.70 μmol/L). The sensitivity of PEth decreases with passing time since admission from 100% at day 1, to 92.5, 76 and 64.3% at days 7, 14 and 28, respectively. Gender does not influence PEth levels [138].

Factors Affecting Blood Phosphatidylethanol
A trend towards higher PEth levels with increasing alcohol consumption levels is reported in a recent review of published data. Blood PEth is generally undetectable in abstinent subjects and low in the general population. PEth is strongly affected by the subject's drinking pattern. The odds of detecting PEth in blood were associated with the average daily alcohol consumption pattern, especially the cumulative amount of alcohol consumed in a period of time (1-2 weeks) [139]. Blood PEth levels do not correlate with the number of heavy drinking days, the number of days during which any alcohol was consumed, or days since the last drink. PEth levels were not correlated with the average number of drinks/week (r < 0.05, p > 0.05). A trend towards an association with days since the last heavy drinking day was however observed, with PEth levels significantly correlated with heavy drinking during the preceding 1-4 days (p < 0.001) but not during the preceding >5 days (p > 0.2) [122,123]. PEth levels were particularly high in DUI subjects (median 0.5, mean 0.7 μmol/L), especially high-risk DUI subjects (median 1.0, mean 1.5 μmol/L), as well as in alcohol clinic outpatients (median 2.9, mean 3.4 μmol/L in lower risk outpatients and median 7.5, mean 7.5 μmol/L in high-risk inpatients) [44]. PEth was not detected in a sample of pregnant women with low, infrequent alcohol consumption [140].
A strong correlation was found between blood PEth and urine EtG and EtS, all of which represent markers of recent alcohol consumption. In contrast, blood PEth was not associated with CDT and Ȗ-GTP levels [104]. PEth is not related to other biomarkers in subjects undergoing alcohol withdrawal [93]. Liver diseases or hypertension do not influence blood PEth levels [139].

Ethyl Glucuronide and Fatty Acid Ethyl Esters in Head Hair
Hair EtG is an important marker of long-term alcohol consumption [141]. A linear correlation between hair EtG levels and the amounts of alcohol consumed in alcohol-dependent individuals was recognized in a recent study [142]. The Society of Hair Testing identifies EtG (cut-off 30 pg/mg in the 0-3 cm proximal segment) and fatty acid ethyl esters (FAEE) (cut-off 0.5 ng/mg in the 0-3 cm proximal segment or 1.0 ng/mg in the 0-6 cm proximal segment) as direct alcohol consumption markers that can be used to determine excessive alcohol consumption. These cut-offs are considered to correspond to chronic excessive alcohol consumption of >60 g/day for several months. The concomitant use of these two molecules is recommended in order to prevent false positive or false negative results with either biomarker. A 3 cm segment of hair corresponds roughly to a 3-month history of drinking pattern [143,144]. Results of recent studies correlating hair EtG with alcohol consumption patterns are shown in Table 3 [130,[145][146][147][148][149][150][151][152][153][154][155][156][157][158].

Ethyl Glucuronide
Hair EtG analysis showed excessive alcohol consumption for approximately 17 months prior to sampling in a murder victim (>170 pg/mL), in a driver whose license was reinstated as result of a lack of regular alcohol consumption (<10 pg/mL), and in another driver whose license was suspended as result of regular alcohol consumption (>30 pg/mL). Hair EtG levels were undetectable or <1.0 pg/mL in 10 teetotalers [159]. In a meta-analysis, the hair EtG concentrations in social drinkers (mean 7.5 pg/mg, 95% CI 4.7-10.2, p < 0.001), heavy drinkers (mean 142.7 pg/mg, 95% CI 99.9-185.5, p < 0.001) and deceased subjects with a known history of chronic excessive drinking (mean 586.1 pg/mg, 95% CI 177.2-995.0, p < 0.01) were higher than the values in teetotalers (<7 pg/mg, with slight overlap with social drinkers) [160]. Alcohol consumption of 16 g/day for 3 months did not lead to hair EtG levels higher than the threshold of 7 pg/mg for alcohol abstinence, while no subject consuming 32 g/day had hair EtG in excess of 30 pg/mg consistent with alcohol abuse [161].

Fatty Acid Ethyl Esters
A recent large study conducted in healthy volunteers assessed the relationship between self-reported daily alcohol intake and FAEEs concentration (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) in hair with the scope of differentiating alcohol abstinence from moderate (<60 g/day) or excessive drinking (60 g/day). Based on the correlations between self-reported daily alcohol intake and FAEEs concentration, this study found that a FAEEs cut-off of 0.5 ng/mg in 3 cm of proximal hair offers the best means of discriminating between social drinking and excessive alcohol consumption [162]. Mean FAEEs levels were 0.87 ng/mg ± 214% in another sample of volunteers, with 0.42 ng/mg ± 114 in non-drinkers or social drinkers, and 1.41 ng/mg ± 186 in alcoholics. FAEEs in hair samples show 59.3% sensitivity and 91.0% specificity for heavy drinking at a cut-off level of 0.675 ng/mg [157].   FAEEs were assessed in a large sample of 1057 autopsy cases (168 social drinkers, 502 alcohol abusers and 387 unknown). Median FAEEs levels were 0.302 ng/mg (range 0.008-14.3 ng/mg) among social drinkers and 1.346 ng/mg (range 0.010-83.7 ng/mg) among alcohol abusers. Based on these findings, the optimal cut-off value for differentiating social drinkers from alcohol abusers was calculated at 1.08 ng/mg [157].
Using cumulative concentrations of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate, FAEEs ranged between 0.11-31 ng/mg (mean 1.77 ng/mg, median 0.82 ng/mg), with 46.3% of samples above the cut-off for heavy drinking (0.5 ng/mg in samples <3 cm and 1.0 ng/mg in samples 3-6 cm in length) among individuals suspected of alcohol abuse in child protection cases. FAEEs were above the cut-off in 23.7% of self-reported abstainers, 43.6% of self-reported moderate drinkers (<60 g/day) and 77.9% of self-reported excessive drinkers (>60 g/day) [163]. Similar findings in a subsequent study show a relatively low reliability of self-reported drinking patterns. FAEEs in hair show 96% specificity and 77% sensitivity for a cut-off of 1.0 ng/mg [164].

Discussion
While EtG in urine can help identify alcohol consumption for a few days after alcohol clears from the blood, hair EtG provides an exposure indicator for long-term alcohol consumption patterns [44,165]. Using the threshold of 30 pg/mg set off by the Society of Hair Testing, hair EtG has a high PPV but a low NPV in a sample of volunteers with a wide range of alcohol consumption patterns. As such, hair EtG is a good tool for identifying alcohol consumption yet it generally fails at correctly identifying abstainers [146].
The presence of EtG in hair (>7 pg/mg) disproved abstinence in 54.5% of a sample of subjects requested to refrain from alcohol consumption [166]. Subjects' false statements often lead to a high number of false positives and thus unreliable sensitivity, specificity, PPV and NPV based on self-reported alcohol history [149]. Positive hair EtG (>7 pg/mg) was detected significantly more frequently among patients with ALD in a large sample of 104 patients scheduled to undergo liver transplantation (32% among ALD compared to 7% among non-ALD patients; p = 0.002) [151].
In contrast, negative results by FAEEs disproved alcohol abuse in 42.3% (<0.2 ng/mg) of subjects suspected of this behavior in a small sample, while showing moderate drinking in 29.5% (0.2-0.5 ng/mg) and proving chronic excessive drinking in 28.2% (0.5 ng/mg) [152]. Double negative or double positive results (EtG >7 pg/mg and FAEEs > 0.2 ng/mg) were found in 72.6% of cases in a small sample of subjects. No linear correlation was found to exist between these two markers [167]. Based on these findings, it is recommended that FAEEs results should only be used to reinforce EtG results due to a high incidence of ambiguous results in classifying individuals according to their alcohol consumption pattern [155,167].
Hair EtG was the only biomarker that can differentiate heavy alcohol consumption from social drinking in a sample of patients whose drinking habits were clinically classified based on their alcohol consumption levels [158]. EtG in hair was the best biomarker for assessing chronic alcohol abuse. Combining EtG with any other biomarker did not improve the diagnostic potential of EtG alone for heavy drinking [147,156].
Hair EtG and FAEEs were in agreement in 75.3% of hair samples belonging to subjects undergoing driving ability examination, workplace testing or in child custody cases, including instances when both biomarkers show abstinence or alcohol consumption [168]. A low to moderate correlation was found between combinations of CDT, Ȗ-GTP, AST, ALT, MCV, EtG and FAEEs in a sample of non-drinkers or social drinkers (<60 g/day) and alcoholics (>60 g/day). Hair EtG shows significant differences between non-drinkers or social drinkers and alcoholics, such that hair EtG can discriminate based on alcohol consumption, using a cut-off value of 60 mg/day [157]. EtG measurements revealed a low level of alcohol consumption during pregnancy in a small sample. Indeed, self-reported alcohol consumption rates were higher than alcohol consumption rates shown by hair EtG levels [169]. The disagreement between self-reports and hair EtG testing can be explained as most women self-reported light drinking (method unable to detect EtG < 5 pg/mg), as well as the fact that hair samples were collected at the end of the pregnancy, while alcohol consumption could have occurred many months before and washed out since [169].

Factors Affecting Hair Ethyl Glucuronide and Fatty Acid Ethyl Esters
Accidental exposure to ethanol can produce results over 1.0 mg/kg in alcohol abstainers [170]. The use of hair biomarkers is generally not suitable to determine absolute abstinence as EtG and FAEEs levels are susceptible to environmental factors and by non-beverage alcohol [171]. This is exemplified in a case report in which a female subject showed sporadically low levels of both FAEEs and EtG. These findings could be interpreted as either low alcohol consumption or abstinence in the presence of environmental factors and use of hair products [171].
Herbal hair tonics may contain EtG. As such, external sources of EtG should be considered, especially if subjects deny alcohol consumption [170,172]. Hair coloring was found to have no effect on EtG levels in vitro, while both bleaching and perming decrease EtG levels, largely as a result of chemical degradation [164,173]. Bleaching and dyeing decrease hair EtG levels by up to 20%-40%, likely due to EtG oxidation by H2O2 [164]. Similarly reduced hair EtG levels in alcohol-dependent patients who bleached or colored their hair compared to those with uncolored/unbleached hair were also found in another study [174]. Thermal hair straightening also reduced EtG levels in vitro compared to untreated strands [175]. A single application of cleansing shampoos was not associated with hair EtG loss [176]. Hair spray had no influence on EtG levels, suggesting that external alcohol does not increase hair EtG levels [164]. On the other hand, the use of hair spray is associated with elevated hair FAEEs levels, likely resulting from alcohol in the product. Bleaching and dyeing have no significant effects on hair FAEEs. Due to the contrasting effects of different hair products on EtG and FAEEs, using FAEEs and EtG assessment concomitantly can help protect against false positive FAEEs or false negative EtG [164]. Use of ethanol-containing hair lotions may give rise to false positive FAEEs results [162].
Using dose-adjusted detection times, decreased renal function led to higher levels of hair EtG compared to healthy subjects, although the correlation between hair EtG and the degree of renal dysfunction was weak (p = 0.08) [177].
The sample length generally has little effect. Assuming constant alcohol consumption over time, the percentage of hair samples with EtG content >7 pg/mg was constant regardless of sample length in a large library, suggesting there is no substantial washout of EtG from the hair strand over time. However, samples <3 cm in length show unusually high EtG levels, suggesting EtG incorporation from sweat following recent alcohol consumption [178,179]. While using a longer length of hair may offer a long-term assessment of alcohol consumption, different drinking patterns over time may confound results [179].
The method used for sample size reduction influences the amount of EtG that can be extracted from hair samples. For example, milling produced markedly higher percentages of extractable EtG than cutting (137%-230%), regardless of the extent of sample pulverization [180]. A different study showed that extensive sample pulverization increased the amount of extractable EtG compared to cutting or weak pulverization. This study argues that while the Society of Hair Testing provides cut-offs for different drinking patterns, inter-laboratory variability may arise owing to different sample preparation methods [181]. In addition, the method used for washing the sample further determined the amount of extractable EtG and FAEEs [182].
Age, gender and body mass index did not significantly affect the ability of hair EtG to predict alcohol drinking patterns [142,147,183]. However, a relatively high degree of discordance between EtG and FAEEs was found in a sample of females selected for alcohol abuse monitoring. This suggests that these two biomarkers should be used together, particularly among females, where use of hair products may alter hair EtG and FAEEs levels. Using a combination of hair EtG and FAEEs led to the lowest rate of false-negative and false-positive [157,184,185]. Interestingly, seasonal differences were found, corresponding to hair growth patterns during winter, spring, summer and autumn. As such, the highest EtG levels in hair were found during the winter and the lowest during the summer [183]. The subject's weight did not play a significant effect in FAEEs incorporation in hair. Furthermore, FAEEs incorporation in head hair and non-head hair was similar. However, FAEEs can leach out during hair washing [163].

Ethyl Glucuronide in Other Hair Matrices
A strong correlation was found between head hair EtG levels and self-reported alcohol consumption in a study (r = 0.8921, p < 0.0001). Among subjects with negative head hair EtG, negative results were also found in beard, chest, axillary, stomach, arm and leg hair. In contrast, pubic hair was positive for EtG in 45.4% of subjects in which head hair was negative. Positive EtG were found in all matrices in subjects in which head hair was positive. Axillary hair generally has lower EtG levels than head hair, while pubic hair has higher levels [186].
Based on pair comparisons, EtG levels in hair were not significantly different between scalp and either of chest, arm or leg. Good correlations were found between scalp hair and chest, arm or leg hair for samples classified as negative (75%-100% association in scalp hair EtG < 7 pg/mg) and samples classified as chronic excessive drinking (73%-100% association in scalp hair EtG > 30 pg/mg). The correlation was poor for social drinkers (EtG 7-30 pg/mg). Chest, arm and leg hair show >78% sensitivity and >75% specificity for drinking behavior as assessed by scalp hair EtG. EtG levels were low in axillary hair, likely a result of degradation by deodorants or leaching through sweat. EtG levels were high in pubic hair, likely due to incorporation from urine. Chest hair appears to be the best alternative to head hair, although sample size differences exist. When comparing EtG levels in different hair samples belonging to the same individual, one must take into account the time frame represented by the sample length, according to the telogen phases of each sample area [145,154]. In another study, EtG became detectable in daily shaved beard after as little as 9 h following alcohol consumption, and fell below the limit of detection after 8-10 days. Peak levels were reached between days 2-4. This study shows the usefulness of an assay that utilizes a small sample of hair [187].

Ethyl Glucuronide and Ethyl Sulfate in Other Matrices
EtG and EtS were analyzed in blood in several studies. EtG Cmax in blood was 0.36 mg/L (range 0.28-0.41 mg/L) in healthy volunteers receiving 0.5 g/kg alcohol and 1.06 mg/L (range 0.80-1.22 mg/L) in healthy volunteers receiving 1.0 g/kg alcohol. EtG levels peaked after 3.5 h with 0.5 g/kg and after 5.5 h with 1 g/kg in blood (Tmax) [96]. EtG and EtS were 11.0 mg/L and 3.7 mg/L, respectively, in a blood sample collected >8 h after alcohol consumption in a driver involved in a traffic accident [188]. The EtG concentration in blood ranged from 460-6250 ng/mL (mean 2179 ng/mL, median 1885 ng/mL) and that of EtS from 200-2720 ng/mL (mean 1157 ng/mL, median 1020 ng/mL) in traffic offense cases. In dried blood spots, the EtG concentration ranged from 428-6690 ng/mL (mean 2126 ng/mL, median 1885 ng/mL) and that of EtS from 161-2680 ng/mL (mean 1177 ng/mL, median 1085 ng/mL) [189]. A recent study conducted among individuals injured in traffic accidents or at work showed that EtG or EtS in blood could be detected in up to 17% of the sample, including individuals with negative BAC [190].
A good correlation was observed between EtG levels in nails and self-reported alcohol consumption. The sample size was too small to allow for the calculation of specificity and sensitivity [191]. EtG in nails showed excellent specificity for detecting any alcohol consumption in another study [192]. EtG in saliva was detectable in only one subject at a dose of 0.5 g/kg alcohol. In subjects receiving 1.0 g/kg alcohol, EtG Cmax in saliva was 0.032 mg/L (range 0.013-0.059 mg/L). EtG levels peaked after 3.5 h [96].
In post-mortem toxicology, the presence of EtG or EtS in urine or blood, coupled with positive alcohol levels, supports ante-mortem alcohol consumption. Alternatively, the absence of EtG or EtS in blood excludes ante-mortem alcohol consumption in alcohol-positive individuals. Alcohol was likely synthesized post-mortem in such individuals, including diabetics. The presence of non-oxidative ethanol metabolites such as EtG and EtS points towards ingestion, thus distinguishing between the two [193][194][195].
EtG concentrations were significantly higher in individuals with a history of alcohol abuse during post-mortem analysis in vitreous humor (4.2 ± 4.8 mg/L, p < 0.001), in serum (6.9 ± 8.9 mg/L, p < 0.01) and in cerebrospinal fluid (1.7 ± 2.7 mg/L, p < 0.01) compared to individuals without a documented history of alcohol abuse [105]. EtG levels in teeth, assessed by LC-MS/MS, can also estimates alcohol use [196].

Carbohydrate-Deficient Transferrin
Transferrin is a liver glycoprotein made up of a polypeptide chain, two metal ion-binding sites and two N-linked glycan chains. CDT refers to transferrin isoforms lacking one or two complete or incomplete glycan chains, the most common of which are asialotransferrin, monoasialotransferrin and diasialotransferrin. CDT quantification generally refers to diasialotransferrin measurements [197]. Chronic alcohol consumption interferes with the glycosylation of several glycoproteins, including transferrin. Moderate to heavy drinking (50-80 g alcohol/day) for several days decreases the carbohydrate content of transferrin, thus giving rise to free sialic acid and sialic-acid deficient transferrin. CDT is thus a biomarker of moderate to heavy alcohol consumption. CDT levels return to normal within approximately 2 weeks of drinking cessation [82,104,198]. As such, CDT is a useful indirect marker for both initial screening as well as relapse [82]. The diagnostic usefulness of CDT is the same when using absolute or relative values [199]. CDT is generally expressed as the percentage of CDT divided by the amount of total transferrin. Furthermore, the disialotransferrin glycoform provides the most accurate representation of alcohol intake. Glycation of serum transferrin in vivo has no influence on CDT levels [102,200,201].
Early studies assessed CDT levels in hospital populations with either suspected alcohol abuse or with conditions not related to alcohol consumption. Each patient's self-reported alcohol consumption was characterized as <60 g/day or >60 g/day, while alcohol intoxication at the time of admission was assessed by breath test. CDT had 70% sensitivity and 98% specificity of identifying alcohol consumption of >60 g/day with a cut-off of 2.4%, regardless of the presence or etiology of liver diseases. The higher incidence of positive CDT results among patients with alcoholic liver diseases than with liver diseases of other etiology suggests continued high alcohol consumption in the former [202]. Furthermore, there were no significant differences between subjects or between groups over time in small samples of healthy male social drinker volunteers receiving 20, 40, 60 or 80 g alcohol/day over a 21 day period, suggesting that CDT is not a good marker for short-term alcohol consumption, even at 80 g/day [203]. Results of recent studies correlating CDT with alcohol consumption patterns are shown in Table 4 [77,90,93,102,105,147,157,158,166,191,[204][205][206][207][208][209][210][211][212][213][214].

Discussion
CDT generally correlates well with an individual's drinking pattern, especially during the preceding 30 days. In a sample of drinkers involved in traffic accidents, CDT in plasma was correlated with the total number of drinks consumed in the past month (r = 0.38, p = 0.003) and the total number of heavy-drinking days in the past year (r = 0.48, p < 0.001) [122]. Similar associations were also shown elsewhere [209,215].
Serum CDT can differentiate between heavy drinkers and non-drinkers, and between heavy drinkers and social drinkers (p < 0.0005 for both), but not between social drinkers and non-drinkers (p = 0.063) [158]. Little variation in CDT levels was seen for alcohol consumption below a threshold of 2 drinks/day (6-10 drinks/week), past which point a significant increase was observed (11-20 drinks/week) [215]. CDT in serum was the best biomarker for detecting an average consumption of >40 g/day compared to <40 g/day in a large Russian population with high levels of alcohol consumption (67% sensitivity and 71% specificity). CDT did not detect hazardous drinking patterns (<60% sensitivity) [216]. CDT results correlate with AUDIT questionnaires results [217], but lack sufficient sensitivity to detect binge drinking [140,218]. CDT and BAC were significantly correlated in drivers involved in car accidents with BAC >0.5 g/L, suggesting chronic alcohol abuse in this population [219].
Median serum CDT levels measured by HPLC were 0.84% among abstinent or light drinkers (<210 g/week for men and <140 g/week for women) in a study. CDT levels were significantly higher in drivers applying for license regranting after a rehabilitation programme (median 0.90%, IQR 0.80-1.10, 3% of sample positive for CDT), as well as in drivers involved in car accidents with BAC > 0.5 g/L (median 1.20%, IQR 0.90-2.00; 27% of sample positive for CDT) compared to controls (p < 0.001) [219]. The incidence of CDT-positive subjects (>1.7%) was 7.5% in a sample of 562 individuals applying for driving license regranting with self-reported alcohol abstinence [220]. CDT levels were not different between first time and recidivist male DUI subjects, using a cut-off of 2.7%. This suggests that CDT cannot be used to predict recidivism among DUI subjects [221].   During post-mortem analysis, positive CDT was found in 60.0% of samples with positive BAC. Of these, 30% had BAC >1‰ (positive CDT in 100.0%), and 70% had BAC <1‰ (positive CDT in 42.8%). Positive CDT was found in 66.7% of individuals with severe liver disease [214].
CDT can be further used in populations required to remain abstinent, such as liver transplant patients and pregnant women. Serum CDT assayed by double antibody radioimmunoassay had 92% sensitivity and 98% specificity for detecting alcohol relapse in a sample of subjects who underwent orthotopic liver transplant for alcoholic cirrhosis [222]. In a sample of orthotropic liver transplantation candidates with alcoholic liver cirrhosis, only 30.2% admitted drinking in the past 6 months, yet 61.9% were positive for at least one alcohol biomarker (hair EtG, urine EtG, BAC, methanol or CDT). Of patients denying alcohol consumption in the preceding 6 months, 8.3% showed positive blood CDT (5 mg/L). As serum CDT is a poor biomarker for low level alcohol consumption, self-reported abstinence in these individuals can be disproved by other, more sensitive methods [149]. CDT had low sensitivity in a sample of patients with a self-reported history of sustained heavy alcohol consumption, as CDT results may be confounded by such factors as cirrhosis and obesity, especially among females [223]. In another sample of liver transplant patients, alcohol consumption was self-reported by only 3.6% of subjects, yet almost 20% were shown to consume some alcohol with the help of biomarkers (urinary EtG, BAC, methanol, CDT, ALT, AST, Ȗ-GTP and MCV) [90].
In a sample of pregnant women, 12.3% continued drinking during pregnancy, with 4.8% reporting infrequent binge drinking. Self-reported drinking during pregnancy was associated with AUDIT scores. However, none of the subjects reporting drinking during pregnancy tested positive for CDT in serum by HPLC (<1.7% disialotransferrin). This reflects relatively infrequent and low alcohol consumption during pregnancy [140].
The fate of CDT in patients beginning alcohol withdrawal treatment was also assessed. A wide range of CDT values were recorded on the day of admission in subjects undergoing alcohol withdrawal. The relative concentration of CDT (%CDT) at study entry were higher in alcohol-dependent males than females (5.67% ± 0.74% vs. 3.22% ± 0.37%, p = 0.027), although daily alcohol consumption was comparable (197.0 ± 17.14 g/day vs. 159.4 ± 21.19 g/day). CDT levels decline rapidly within the first 4 days of treatment, and significant differences can be seen upon completion of detoxification. The percentage of patients with CDT >2.6% generally declined over the treatment period. However, 34.5% of patients continued to have CDT >2.6% for up to 6 weeks into the study, suggesting that CDT data needs to be interpreted with care when abstinence is required [93,212,224]. Disialotransferrin was found to have a half-life of 8.5-15 days (mean 12.6 days, median 13.9 days) [102].

Factors Affecting CDT
CDT levels were significantly associated with the body mass index (p = 3.71 × 10 í9 ), female gender (p = 2.30 × 10 í9 ) and smoking (p = 8.28 × 10 í8 ), but not with age [215,219]. The usefulness of CDT is reduced in overweight or obese subjects, as CDT levels are lower in these compared to lean individuals consuming comparable amounts of alcohol [225,226]. In contrast, CDT levels are higher in smokers compared to non-smokers consuming comparable levels of alcohol [225].
Measurements of CDT have been proposed for the diagnosis of alcoholic liver disease. However, CDT elevations can occur in sepsis, anorexia nervosa, and airway diseases [232]. Lower values of sensitivity have also been reported with iron overload [233]. Although CDT is usually unaffected by the presence of liver disease, false positive results have been reported in patients with primary biliary cirrhosis and with severe non-alcohol-related hepatic failure [234,235]. Therefore, individuals with suspected primary biliary cirrhosis who are positive for CDT should be evaluated further by using mitochondrial autoantibodies to pyruvate dehydrogenase [236]. As a result, the utility of CDT testing depends upon the clinical picture and other biochemical tests [237].
CDT performs better in non-cirrhotic than in cirrhotic patients based on self-reported alcohol consumption in the past 15 days [207]. CDT in serum was analyzed by capillary electrophoresis and by nephelometry in a sample of healthy controls (<25 g alcohol/day), abstinent patients with liver disease, alcoholic patients with liver disease, and individuals consuming varying amounts of alcohol. Among abstinent individuals, CDT levels were higher in those with liver diseases compared to controls (0.9% vs. 0.5%, p = 0.046). Furthermore, CDT levels were higher in individuals consuming >60 g alcohol/day than those consuming <60 g alcohol/day (p = 0.034). CDT levels were slightly lower when assessed by capillary electrophoresis, but a good correlation was found between this method and nephelometry [238]. Hepatitis C virus seropositivity is associated with significantly decreased baseline CDT levels in patients undergoing treatment for alcohol dependence [239]. Absolute CDT values are not affected by liver disease, yet the relative values are. Relative CDT values are highest in patients with alcoholic hepatitis, and lowest in primary biliary cirrhosis patients [213]. In a different study, CDT levels assessed by high-performance liquid chromatography (Helander HPLC) were associated with binge drinking behavior in adolescents with alcohol abuse, while no differences were found between binge drinkers and non-binge drinkers when CDT levels were assessed by immunonephelometric assay (N Latex) [240].
Genetics also play an important role. A CDT indicative of alcohol abuse (2.47%) was achieved after fewer drinks in a sample of Korean subjects who experience facial flushing after drinking, associated mainly with acetaldehyde accumulation, compared to non-flushers [204]. CDT measurements are also influenced by CDT hereditary syndrome [241][242][243][244][245]. Transferrin CD variants may further complicate results, particularly when quantified by liquid chromatography methods [246]. CDT measurements in serum were affected by a T139M transferrin variant. The presence of this variant in a subjects suspected to suffer from alcoholism led to unquantifiable CDT levels by HPLC and capillary zone electrophoresis and low levels for isoelectric focusing. CDT was accurately quantified by immunoassay [247].

Conclusions
Based on the various laboratory tests employed, the array of findings can delineate the specific amount and the period when alcohol was consumed in individuals with alcohol problems. However, many factors can influence the analytical performance of the tests. This review provides clinicians with tests that will accurately detect heavy drinking despite denial by the patient. The role of the laboratory is to promote assay standardization and aid in results interpretation with the intent of guiding the medical professional toward the proper use of a specific laboratory test in a specific time frame in order to meet the clinical need. Clinical management of pharmacotherapy with drugs of use in patients denying drinking is challenging due to inter-individual variability in alcohol metabolism. Therefore, initiating any therapy in this population requires an interdisciplinary team that includes clinicians, the laboratory, and the patient.