Review Special Focus Issue: Forensic and clinical toxicology
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Progress in monitoring alcohol consumption and alcohol abuse by phosphatidylethanol
For early diagnosis and therapy of alcohol-related disorders, alcohol biomarkers are highly valuable. Concerning specificity, indirect markers can be influenced by nonethanol-related factors, whereas direct markers are only formed after ethanol consumption. Sensitivity of the direct markers depends on cut-offs of analytical methods, material for analysis and plays an important role for their utilization in different fields of application. Until recently, the biomarker phosphatidylethanol has been used to differentiate between social drinking and alcohol abuse. After method optimization, the detection limit could be lowered and phosphatidylethanol became sensitive enough to even detect the consumption of low amounts of alcohol. This perspective gives a summary of most common alcohol biomarkers and summarizes new developments for monitoring alcohol consumption habits.
Worldwide the harmful use of alcohol results in about 2.5 million deaths every year and is also very costly to the society. Alcohol abuse is a major global contributing factor to death, disease and injury: to the drinker itself through the negative consequences of alcohol dependence, including liver diseases, cancer and injuries; to others through drunken driving and violence [1] . As in most western countries, health problems created by excessive alcohol consumption are still growing, it is important to develop effective methods for reducing alcohol consumption and to develop potent techniques for the early detection of hazardous drinking [2] . The examination of suspected alcohol dependence or abuse comprises symptoms, medical history, self-report forms, special questionnaires (e.g., AUDIT) and alcohol consumption markers [3] . These markers are formed endogenously and are a proof of ethanol intake. Due to the high and widespread ethanol consumption in the population, these biomarkers are gaining more and more importance in clinical and forensic questions, and also in traffic medicine [4] . About 95% of the consumed ethanol is oxidized to acetaldehyde and acetic acid (phase I metabolism), the
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rest experiences nonoxidative transformation (phase II metabolism). Among the formed direct, nonoxidative metabolites are ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanol (PEth) and fatty acid ethyl esters (FAEE). Besides the classical indirect markers carbohydrate-deficient transferrin (CDT), γ-glutamyl transpeptidase (GGT) or mean corpuscular volume (MCV), these direct markers have proven to be promising biomarkers for the detection of alcohol consumption [5,6] . In comparison to the clinically established indirect markers GGT, MCV and CDT, which are widely used to detect alcohol abuse, direct markers are less influenced by nonethanol-related factors, such as diseases, physiological abnormalities or genetic defects, since they are only formed when ethanol is consumed [2,7] . Sensitivity is strongly depending on thresholds and cut-offs, material for analysis (use of blood, urine, hair – which have different detection windows for the direct marker EtG), and utilization in different fields of application, such as abstinence monitoring, detection of social use of alcohol, differentiation between social consumption habits and alcohol abuse according to WHO definitions [8] .
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Alexandra Schröck*,1, Annette Thierauf2, Friedrich Martin Wurst3, Natasha Thon3 & Wolfgang Weinmann1 Institute of Forensic Medicine, University of Bern, Bühlstrasse 20, 3012 Bern, Switzerland 2 Institute of Forensic Medicine, University Medical Center Freiburg, Albertstrasse 9, 79104 Freiburg, Germany 3 Department of Psychiatry & Psychotherapy II, Christian-DopplerHospital, Paracelsus Medical University Salzburg, Ignaz-Harrer-Strasse 79, 5020 Salzburg, Austria *Author for correspondence: Tel.: +41 31 631 3050 Fax: +41 31 631 8580 [email protected] 1
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Review Schröck, Thierauf, Wurst, Thon & Weinmann With the alcohol biomarker PEth, it is possible to differentiate between social drinking and risky drinking habits. For high amounts of alcohol CDT and GGT are as suitable as PEth, but for medium amounts of alcohol PEth is more suitable than CDT [3] . Until 2012, analytical methods for the quantitation of PEth were not sensitive enough to detect single drinking. Recently, PEth was already detected after a single alcohol consumption using LC–MS/MS [9] . By lowering the detection limit of PEth by optimizing the analytical method, it is possible to get a higher sensitivity for PEth even when low amounts of alcohol were consumed. An overview of different kinds of alcohol markers in comparison to PEth is given. Indirect alcohol biomarkers The clinically established traditional markers of alcohol abuse CDT, GGT and MCV, which appear after a heavy long-term consumption of alcohol due to changes from the normal physiologic values, are still widely used [7] . However, these markers have only a moderate clinical sensitivity and specificity, which means that a large portion of high consumers may have normal values and that an elevated value in a significant proportion of cases has a reason other than alcohol [3] . GGT is a membrane-bound glycoprotein enzyme located on the cell surface membrane of most cell types [2,7] . GGT has its highest activity in liver, kidney and bile. An increase of GGT values in serum is a sign of liver or bile damages. GGT is responsible for the extracellular catabolism of gluthatione, and catalyzes the transfer of the γ-glutamyl moiety of glutathione to various peptide acceptors. An increase in serum GGT activity may be due to increased synthesis of GGT as a result of enzyme induction by alcohol or drugs. Serum GGT has been widely used as an index of liver dysfunction and as a marker of alcohol misuse. An increase of GGT activity can be a sign for hepatobiliary diseases. Chronic alcohol intake also increases serum GGT activity. The specificities and sensitivities of GGT vary in different studies, but are usually slightly better than those of other commonly used long-term markers of alcohol intake. Serum GGT is also increased by age, smoking and various other diseases [2,7] . However, analysis of GGT from serum is a standard procedure in clinical chemistry. Key terms Social drinking: A daily alcohol intake ≤60 g of ethanol is classified as moderate, social drinking behavior. Risky drinking: A daily alcohol intake ≥60 g of ethanol is classified as heavy and risky drinking behavior.
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MCV (red blood cell volume) is an often used marker for the detection of excessive alcohol intake. In persons with high alcohol consumption, MCV increases and responds slowly (due to cell renewal within 2 to 4 months) to abstinence. The mechanisms of alcohol-induced increase of MCV are still unknown, but it seems that ethanol and its metabolites, especially acetaldehyde, have hematotoxic effects and may affect the red cell structure and its metabolism. Usually, the sensitivities and specificities of MCV are lower than those of GGT, and even moderate alcohol intake may increase MCV. Furthermore, MCV could also be elevated in patients with liver diseases, vitamin B12 or folic acid deficiency, hematological diseases, reticulocytosis or hypothyroidism, which limits the reliability of MCV for the detection of alcohol consumption [2,7] . CDT is a glycoprotein, which is important for the iron transport in plasma. Human transferrin is composed of 679 amino acids with two potential glycosylation sites, which usually bind two bi- and/or triantennary carbohydrate chains of variable composition, containing four different carbohydrates. Sialic acid is the only charged residue in these saccharide chains, and, when present, bears a negative charge [10] . CDT has a half-life of 7–10 days and is synthesized in the liver. As a result of heavy alcohol intake, the concentrations of desialylated isoforms of CDT in serum are increased. The exact mechanisms remain unknown, but both protein transport and enzyme activities seem to be influenced by ethanol. It is possible to analyze CDT by electrophoretic, chromatographic and immunometric methods. Currently, CDT is considered as the most useful marker for long-term alcohol misuse. A disadvantage of CDT is that its sensitivity varies greatly in different experimental conditions, clinical settings and populations. While alcohol intake above 60–80 g/day correlates well with increase of CDT, the results concerning the effects of moderate drinking are inconsistent. A high level of CDT is not always a sign of alcohol abuse, though. There are other factors such as gender, age, body mass index, smoking, liver disease, anorexia, pregnancy and hyperferremia, influencing the CDT level to obtain ‘false-positive’ results [2,7] . Analytical methods for CDT
For the identification and quantitation of CDT isoforms, the separation from other blood components is required [11] . While in former times the concentration of total CDT was determined, nowadays new analytical methods render a differentiation of CDT subtypes possible. Isoelectric focussing (IEF), capillary electrophoresis (CE) and liquid chromatography (LC) provide a distribution pattern of isoforms. IEF is highly selective and is used as a reference method for
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Progress in monitoring alcohol consumption & alcohol abuse by phosphatidylethanol
transferrin isoforms. Separation proceeds in a gel containing a pH gradient according to the characteristic isoelectric points (pI) of the transferrin isoforms [11] . The disadvantage of IEF is its inaccuracy and imprecision in quantitative evaluation, which can be overcome by CE or by LC [10] . CE in fused-silica capillaries is an effective tool for the separation of the major transferrin isoforms [12] . A widespread method for CDT determination is microcolumn ion-exchange chromatography followed by immunometric quantitation based on the different charge characteristics of desialyzed transferrins. These recent methods indicate the measurement results as percentage of the total transferrin and allow for the detection of natural variability. Direct alcohol biomarkers In contrast to indirect markers, the direct markers are straightly related to the ethanol metabolism. They are formed in a nonoxidative phase II reaction. Some of them even occur after the uptake of trace amounts of ethanol. Aside from the detection of ethanol in blood, urine or exhaled air, there are the already abovementioned markers EtG, EtS, FAEE and PEth, which can be measured in blood and/or urine and also in hair (EtG) [3] . PEth represents a group of abnormal phospholipid homologs. The structure of PEth is a glycerol molecule with two fatty acid chains in sn-1 and sn-2 position, typically containing 14–22 carbon atoms with 0–6 double bonds, and with phosphoethanol as the head group. So far 48 different homologs were identified [13] . PEth is localized in cell membranes and is formed there from phosphatidylcholine only if ethanol is present. Ethanol acts as a co-substrate in the transphosphatidylation reaction catalyzed by the enzyme phospholipase D (PLD) (Figure 1) [3,14] . Compared with its formation, the elimination rate of PEth is slow. The elimination half-life time of PEth in human blood is approximately 4 days [7] . The concentration of PEth is highly correlated with the amount of ingested ethanol [15] . It is detectable in blood of alcohol abusers even up to 3 weeks after alcohol withdrawal; therefore it is a promising new biomarker for the detection of alcohol abuse [16,17] and for monitoring of abstinence. Analytical methods for PEth
When PEth was first measured in blood samples obtained from alcoholics, thin layer chromatography (TLC) with quantification by image analysis was used [18] . TLC separation was time consuming and the coefficient of variation (CV) was relatively high, however this study showed PEth as a possible marker of alcohol abuse with a high specificity and a long detection
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window. Other authors used mainly high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) [19–21] and nonaqueous capillary electrophoresis (NACE) combined with UV detection [22] . With an LOD of about 0.8 μmol/l (∼560 ng/ml PEth) [21] HPLC-ELSD has an essentially higher sensitivity than TLC, but is also time consuming with run times of about 60 min and ELSD has only been used for the detection of the total sum of PEth homologs. Compared with HPLC methods, NACE has shorter run times and needs less sample volume. NACE with UV detection has a slightly lower LOD of 0.4 μmol/l (∼280 ng/ml PEth) than the HPLC-ELSD methods, but it is also not possible to separate the different PEth homologs present in human blood. Using NACE, the detection times of PEth varied between the measured blood samples fortified with PEth standards and the clinical samples. This seemed to be due to the fact that the fortified samples only contained one PEth homolog (PEth 16:0/18:1). Also an internal standard (IS) was not included in the NACE method [22] . Coupling NACE with ESI–MS enhanced sensitivity (LOD of 0.1 μmol/l, ∼70 ng/ml PEth) and selectivity of this method, and increasing accuracy and precision was obtained by the use of an IS [23] . A more selective and sensitive detection method for PEth is MS with triple quadrupole or TOF analysis [19,24,25] . The mass spectrometric detection methods are advantageous compared with previous HPLC-ELSD or NACE-UV methods, because they have lower LODs and single PEth homologs can be differentiated, whereas with ELSD or UV-detection, it was only possible to detect the sum of PEth homologs with less sensitivity. For our research on PEth, an LC–MS/MS method was developed for quantitation of the PEth homologs 16:0/16:0, 18:1/18:1, 16:0/18:2 and 16:0/18:1 with an LOD of 100 ng/ml. The most abundant homologs in human blood are PEth 16:0/18:1 and PEth 16:0/18:2 [26,27] , with a nearly 10 times higher area ratio of PEth 16:0/18:1 than the area ratio of PEth 16:0/16:0 and 18:1/18:1. For each above-mentioned PEth homolog, the corresponding deuterated IS was synthesized in-house by transphosphatidylation reaction of the corresponding phosphatidylcholines (PC) with D6 -ethanol enzyme-catalyzed by phospholipase D according to a published method with minor modifications [26,28] . To assess the time window of detectability of PEth after single alcohol consumption, the combination of liquid–liquid extraction and LC–MS/ MS was switched to protein precipitation followed by online-SPE-LC–MS/MS. This improved the LOD from 100 ng/ml to 10 ng/ml. Table 1 gives an overview
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O R1
phosphatidic acid
O
O R2
H2O
O O
H
O
P
O
O–
R1 O CH3 +
H3C
O O
O
O
R2
N
H3C
O
O O
R1
phospholipase D
P
O
O–
phosphatidylcholine
R2
EtOH
O O
H3C
phosphatidylethanol
O
O
O
P O–
Figure 1. Enzymatic formation of phosphatidic acid from phosphatidylcholine, in the presence of ethanol phosphatidylethanol is formed.
about the different analysis methods for PEth and their LODs and LOQs. EtG and EtS are direct metabolites of ethanol. They are widely used for clinical and forensic applications. EtG and EtS are detectable in blood for up to 8 h; in urine the detection window is up to 80 h after heavy alcohol consumption. EtG and EtS are formed by a nonoxidative elimination of ethanol, catalyzed by the enzymes uridine diphosphateglucuronosyltransferase or sulfotransferase, respectively, being less than 0.1% of the ethanol intake excreted through this phase II metabolism [6] . Already after consumption of trace amounts of alcohol, EtG and EtS are detectable in Key terms Online SPE: The instrumental setup for online SPE uses a modified HPLC system. The sample is loaded onto a trapping column where the sample is concentrated and washed. Then a switching valve is switched and the mixture is eluted through an analytical column, which separates the sample chromatographically. Society of Hair Testing (SoHT): The Society of Hair Testing (SoHT) was founded in 1995 and has the goal to promote research in hair testing technologies in forensic and clinical sciences.
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urine [31] , and are still present after ethanol has been eliminated. Therefore, the analysis of EtG and EtS is a well-suited method for the detection of recent alcohol exposure and for abstinence monitoring [5,7,32] . However, there is the possibility that EtG can be produced in urine after sampling, if the specimen contains bacteria such as Escherichia coli and ethanol, which might be produced from sugar and yeast, thus causing positive EtG results. Furthermore, EtG, is sensitive to bacterial degradation if the samples are contaminated with bacteria and transported without cooling or if stored improperly, causing ‘false negative’ results [33–35] . For EtS neither postsampling formation nor degradation has been found in urine samples. With the collection of dried urine spots (DUS), it is possible to inhibit bacterial degradation [36] , but for better clinical specificity, a combination of both, EtG and EtS has been recommended. EtG is as well detectable in hair. The detection window of EtG can be several months, depending on the hair length. In Switzerland and Germany, the analysis of EtG in hair is meanwhile used for abstinence monitoring in driving aptitude testing before re-issuing a driver’s license after ‘drunken driving’. The Society of Hair Testing (SoHT) suggested a cut-off of 30 pg/mg to differentiate excessive alcohol
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Progress in monitoring alcohol consumption & alcohol abuse by phosphatidylethanol
consumption from social drinking. For abstinence monitoring, a cut-off of 7 pg/mg EtG in hair was recommended [37,38] . Positive results are always associated with alcohol consumption, but in cases where EtG is not detected alcohol consumption cannot be excluded definitely [39–45] . FAEE are nonoxidative products of ethanol metabolism [46] . They are formed by esterification of fatty acids with ethanol, catalyzed by FAEE synthases; and also carboxylesterases and glutathione transferase have FAEE synthase activity [46,47] . As FAEE are bound to albumin or lipoproteins, they can be transferred between lipid compartments. Elevated levels of FAEE are detectable in blood shortly after alcohol intake and remain elevated in serum for approximately 24 h [7] . In hair, FAEE are suitable long-term markers of high and risky alcohol consumption [48] ; and at least 15 different FAEE have been identified. Thereof ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate have been chosen as ethanol consumption markers [49,50] . For quantitation of FAEE by GC–MS, the sum of these four FAEE has been used [48] . In hair the sum of the
Review
concentrations of the four FAEE with a cut-off value of 0.5 ng/mg reveals a sensitivity of 90% and a specificity of 90% [38] . For detecting prenatal exposure to alcohol, FAEE in meconium have been proposed as biomarkers [7] . Figure 2 gives an overview of the different kinds of biomarkers described in this paper and their detection windows with respect to consumption habits. Recent developments in PEth analysis & applications The clinically established indirect markers GGT, MCV and CDT are widely used to detect alcohol abuse, but these markers are not sensitive and specific enough to identify the extent of alcohol abuse [2,7] , as they can be influenced by other factors such as gender, age, body mass index, smoking and liver diseases. Currently, the most useful indirect marker for long-term alcohol misuse is CDT. It has a wide window of detection, shows a significantly higher clinical specificity than GGT or MCV, but also lacks sensitivity, as CDT is only detectable after high and long-term alcohol abuse. Therefore, slowly eliminated direct markers with improved
Table 1. Overview of LOD and LOQ of PEth with different methods. Procedure
LOD
LOQ
Analytes and IS
Ref.
‐
0.22 μmol/l (150 ng/ml)
Total sum of PEth homologs, IS: phosphatidylbutanol
[20]†
HPLC–ELSD
0.8 μmol/l (560 ng/ml)
‐
Total sum of PEth homologs, no IS
‐
0.7 μmol/l (500 ng/ml)
Total sum of PEth homologs, IS: phosphatidylbutanol
NACE–UV
0.4 μmol/l (280 ng/ml)
‐
Total sum of PEth homologs, no IS
NACE–MS (QQQ)
0.1 μmol/l (70 ng/ml)
0.4 μmol/l (280 ng/ml)
Single PEth homologs, IS: phosphatidylbutanol
[23]†
0.001 μmol/l (0.7 ng/ml)
Single PEth homologs, IS: phosphatidylbutanol
[25]† [24]†
LC–HRMS/MS (Orbitrap) 0.0005 μmol/l (0.35 ng/ml)
[21] [3] [22]
LC/Q–ToF–MS/MS
0.001 μmol/l (1 ng/ml)
0.001 μmol/l (1 ng/ml)
Single PEth homologs, IS: phosphatidylpropanol
LC–MS (Q)
0.07 μmol/l (5 ng/ml)
‐
Total sum of PEth homologs, no IS
0.02 μmol/l (14 ng/ml)
0.1 μmol/l (70 ng/ml)
Single PEth homologs, IS: phosphatidylpropanol
[27]†
LC–MS/MS (QQQ)
0.02 μmol/l (14 ng/ml) for PEth 16:0/16:0; 0.024 μmol/l (17 ng/ml) for PEth 18:1/18:1
0.064 μmol/l Single PEth homologs, (45 ng/ml) for PEth IS: phosphatidylpropanol 16:0/16:0; 0.077 μmol/l (54 ng/ml) for PEth 18:1/18:1
[29]†
0.01 μmol/l (7 ng/ml)
0.03 μmol/l (21 ng/ml)
Single PEth homologs, IS: corresponding penta-deuterated IS
[26]†
Online-SPE LC–MS/MS (QQQ)
0.014 μmol/l (10 ng/ml)
0.028 μmol/l (20 ng/ml)
Single PEth homologs, IS: corresponding penta-deuterated IS
[30]
[19]
‐: No data available. Mass-spectrometric analyzers: PEth: Phosphatidylethanol; Q: Quadrupole, QQQ: Triple quadrupole or hybride triple quadrupole ion trap used in triple quadrupole mode; QToF: Quadrupole-time-of-flight. † Method was validated
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BAC Single/regular consume Short-term markers
EtG/EtS (blood) EtG/EtS (urine) 6
4
2
Days PEth (blood)
High-risk consume Intermediary long-term markers
CDT (serum) GGT (serum) MCV (blood)
10
6
8
4
2
Weeks
Abstinence/social consume/high-risk consume Long-term markers EtG/FAEE (hair) 8
6
4
2
Months
Figure 2. Overview of the different kinds of biomarkers described in this paper and their detection windows with respect to consumption habits. BAC: Blood alcohol concentration; CDT: Carbohydrate-deficient transferrin; EtG: Ethyl glucuronide; FAEE: Fatty acid ethyl esters; GGT: γ-glutamyl transpeptidase; MCV: Mean corpuscular volume; PEth: Phosphatidylethanol.
specificity and sensitivity seem to be highly desirable in clinical and forensic issues [3] . One such biomarker that has gained increasing interest over the last few years is PEth, which is only formed after ethanol intake. Thus, the specificity of PEth is close to 100% whereas that for GGT and CDT is only about 40%. The two most
abundant PEth homologs in human blood are PEth 16:0/18:1 and PEth 16:0/18:2 [26,27] . Studies [3,15] showed that the sensitivity of PEth is much better for the detection of alcohol consumption than CDT. Up to now PEth was not sensitive enough for the detection of small amounts of alcohol, but by optimizing
250
PEth (ng/ml)
200
150
100
50
0 0
100
200
Time (h)
Test person 3
300
400
500
Test person 7
Figure 3. Phosphatidylethanol concentrations after repeated drinking on five subsequent days. PEth: Phosphatidylethanol. Data taken from [51] (two examples shown).
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Progress in monitoring alcohol consumption & alcohol abuse by phosphatidylethanol
the analysis method the LOD could be lowered. With the new LOD of 10 ng/ml (instead of 100 ng/ml), first drinking studies were performed to define the detection window of the PEth homolog PEth 16:0/18:1 after ingestion of a single amount of alcohol, which led to a blood alcohol concentration (BAC) of 1‰. In a first study with cumulative drinking up to a BAC of 1‰ on five subsequent days, Gnann et al. [9] showed that PEth values of alcoholics (PEth values > 700 ng/ ml) cannot be reached (Figure 3) . Thus, it is possible to differentiate with the alcohol biomarker PEth between social drinking and risky drinking. In the most recent pilot study, two volunteers (male and female) took an individual calculated single dose of alcohol, which led to a BAC of 1‰ after an abstinence period of 2 weeks. In the week after the experiment, one blood and one urine sample was taken every day, in the second week samples were taken every 2 days. The test persons stayed abstinent throughout these weeks. At the start of the study, blood samples were negative for PEth. After drinking alcohol, which led to a BAC of 1‰, formation of PEth was observed over several hours, as long as ethanol was present in blood. After elimination of ethanol, PEth decreased with a half-life time of approximately 4 days, and was detectable in blood for up to 10 days [30] . Conclusion The study of Gnann et al. [9] and the described pilot study with a single drinking event show the potential of PEth for abstinence monitoring, since PEth could be detected even after a single drinking event for several days. Until now, positive PEth levels have only been found when ethanol was consumed, demonstrating the specificity of PEth. In general, the risk of sample manipulation or dilution after urine collection or the instability of the analyte due to bacterial contamination and degradation are of major concern when using urine as test specimen for alcohol markers or drugs of abuse. In hair analysis, hair type, color, length and treatment (by hair cosmetics or heat) may have a strong influence on alcohol markers in hair. In contrast, blood samples do not have these disadvantages and are often taken in clinical settings also for other markers. Sampling of venous blood is a welldefined process. For high-throughput analysis blood can be used, while hair need laborious sample preparation and a high degree of automation has not yet been achieved for hair sample work-up. Due to the high specificity of PEth as marker for alcohol consumption and due to its rather slow elimination compared with EtG or EtS, PEth is predesti-
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Key term Dried blood spots (DBS): A simple and cost-effective sampling method is the collection of venous or capillary blood on filter cards. This method is used since years for neonatal metabolic screening, and started to be of interest for therapeutic drug monitoring.
nated for abstinence monitoring and also for detection of problematic alcohol consumption habits. For the detection of problematic drinking, a threshold of 1 μmol/l (∼700 ng/ml) PEth in blood (for PEth 16:0/18:1) has been proposed. Still there are further experiments needed, because up to now, ‘normal’ ranges of PEth, in other words, concentrations typical for social drinking over a long time period have not been established for large collectives. With lowered detection limits, it is now the starting point to establish a cut-off for abstinence monitoring, not including PEth formation from ‘hidden alcohol in nutrition’ – and for further evaluation of the threshold between social drinking and risky drinking habits and alcohol abuse. Future perspective The newest developments in PEth analysis and controlled drinking studies show the potential of PEth in abstinence monitoring. Until now, positive PEth levels were only found when ethanol had been consumed prior to blood sampling. However, further investigations with larger numbers of persons are necessary, to determine whether physiological abnormalities can be detected, and to determine cut-off levels for drinking habits according to WHO categories (abstinence, social drinking and risky alcohol consumption) [8] . For routine analysis of PEth, there is the possibility to speed up the method with UHPLC and by the use of well plate formats [52] . Another promising development might be the analysis of PEth in dried blood spots (DBS) [53,54] . Studies on DBS showed good robustness and the advantage that neoformation of PEth in the presence of BAC does not occur after drying blood on filter paper for DBS sampling [30,54] . Capillary blood sampling is used since many years for neonatal screening [55] and has additionally been applied for neonatal screening of prenatal alcohol exposure [56,57] . However for routine application, this method still needs to be evaluated. Capillary blood sampling would mean easier and faster sample collection, as sampling can also be done by nonmedical personnel. Acknowledgement We would like to thank S. Lanz (IRM Bern) for technical support in setting up the online SPE-LC–MS/MS system, and the team of FTC Bern for measuring blood alcohol concentrations.
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Review Schröck, Thierauf, Wurst, Thon & Weinmann Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employ-
ment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary • Worldwide the harmful use of alcohol results in about 2.5 million deaths every year, and health problems created by excessive alcohol consumption are still growing in most western countries. • Therefore it is important to develop potent techniques for the early detection of hazardous drinking. Besides self-report forms, special questionnaires (e.g., AUDIT), this can be done by alcohol consumption markers.
Different kinds of alcohol biomarkers • Indirect markers, such as CDT, GGT and MCV, appear after a heavy long-term consumption of alcohol due to changes from the normal physiologic values and are widely used, but have only a moderate clinical sensitivity and specificity. • Direct markers are only formed after ethanol consumption. Some of them even occur after the uptake of trace amounts of ethanol. Aside from the detection of ethanol in blood, urine or exhaled air, the markers EtG, EtS, FAEE and PEth can be measured in blood and/or urine and also in hair.
Phosphatidylethanol • The direct alcohol biomarker phosphatidylethanol (PEth) represents a group of abnormal phospholipid homologs. PEth can be used to differentiate social drinking from risky drinking.
Perspective • Drinking studies show the potential of PEth in abstinence monitoring. • Cut-off levels for abstinence, social drinking and risky alcohol consumption have to be determined. PEth might also be detected in dried blood spots, as this method showed good robustness and PEth is more stable in dried blood spots than in whole blood. http://whqlibdoc.who.int/hq/2000/who_msd_ msb_00.4.pdf, pp. 50–54 (2000).
References Papers of special note have been highlighted as: • of interest; •• of considerable interest
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SOHT. Use of alcohol markers in hair for Abstinence Assessment 2012. http://www.soht.org/images/pdf/Use%20 of%20Alcohol%20Markers%20in%20Hair%20for%20 Abstinence%20Assessment%202012.pdf, (2012).
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Auwarter V, Sporkert F, Hartwig S, Pragst F, Vater H, Diefenbacher A. Fatty acid ethyl esters in hair as markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers, and teetotalers. Clin. Chem. 47(12), 2114–2123 (2001).
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Progress in monitoring alcohol consumption and alcohol abuse by phosphatidylethanol
For early diagnosis and therapy of alcohol-related disorders, alcohol biomarkers are highly valuable. Concerning specificity, indirect markers can be influenced by nonethanol-related factors, whereas direct markers are only formed after ethanol consumption. Sensitivity of the direct markers depends on cut-offs of analytical methods, material for analysis and plays an important role for their utilization in different fields of application. Until recently, the biomarker phosphatidylethanol has been used to differentiate between social drinking and alcohol abuse. After method optimization, the detection limit could be lowered and phosphatidylethanol became sensitive enough to even detect the consumption of low amounts of alcohol. This perspective gives a summary of most common alcohol biomarkers and summarizes new developments for monitoring alcohol consumption habits.
Worldwide the harmful use of alcohol results in about 2.5 million deaths every year and is also very costly to the society. Alcohol abuse is a major global contributing factor to death, disease and injury: to the drinker itself through the negative consequences of alcohol dependence, including liver diseases, cancer and injuries; to others through drunken driving and violence [1] . As in most western countries, health problems created by excessive alcohol consumption are still growing, it is important to develop effective methods for reducing alcohol consumption and to develop potent techniques for the early detection of hazardous drinking [2] . The examination of suspected alcohol dependence or abuse comprises symptoms, medical history, self-report forms, special questionnaires (e.g., AUDIT) and alcohol consumption markers [3] . These markers are formed endogenously and are a proof of ethanol intake. Due to the high and widespread ethanol consumption in the population, these biomarkers are gaining more and more importance in clinical and forensic questions, and also in traffic medicine [4] . About 95% of the consumed ethanol is oxidized to acetaldehyde and acetic acid (phase I metabolism), the
10.4155/BIO.14.195 © 2014 Future Science Ltd
rest experiences nonoxidative transformation (phase II metabolism). Among the formed direct, nonoxidative metabolites are ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanol (PEth) and fatty acid ethyl esters (FAEE). Besides the classical indirect markers carbohydrate-deficient transferrin (CDT), γ-glutamyl transpeptidase (GGT) or mean corpuscular volume (MCV), these direct markers have proven to be promising biomarkers for the detection of alcohol consumption [5,6] . In comparison to the clinically established indirect markers GGT, MCV and CDT, which are widely used to detect alcohol abuse, direct markers are less influenced by nonethanol-related factors, such as diseases, physiological abnormalities or genetic defects, since they are only formed when ethanol is consumed [2,7] . Sensitivity is strongly depending on thresholds and cut-offs, material for analysis (use of blood, urine, hair – which have different detection windows for the direct marker EtG), and utilization in different fields of application, such as abstinence monitoring, detection of social use of alcohol, differentiation between social consumption habits and alcohol abuse according to WHO definitions [8] .
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Alexandra Schröck*,1, Annette Thierauf2, Friedrich Martin Wurst3, Natasha Thon3 & Wolfgang Weinmann1 Institute of Forensic Medicine, University of Bern, Bühlstrasse 20, 3012 Bern, Switzerland 2 Institute of Forensic Medicine, University Medical Center Freiburg, Albertstrasse 9, 79104 Freiburg, Germany 3 Department of Psychiatry & Psychotherapy II, Christian-DopplerHospital, Paracelsus Medical University Salzburg, Ignaz-Harrer-Strasse 79, 5020 Salzburg, Austria *Author for correspondence: Tel.: +41 31 631 3050 Fax: +41 31 631 8580 [email protected] 1
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Review Schröck, Thierauf, Wurst, Thon & Weinmann With the alcohol biomarker PEth, it is possible to differentiate between social drinking and risky drinking habits. For high amounts of alcohol CDT and GGT are as suitable as PEth, but for medium amounts of alcohol PEth is more suitable than CDT [3] . Until 2012, analytical methods for the quantitation of PEth were not sensitive enough to detect single drinking. Recently, PEth was already detected after a single alcohol consumption using LC–MS/MS [9] . By lowering the detection limit of PEth by optimizing the analytical method, it is possible to get a higher sensitivity for PEth even when low amounts of alcohol were consumed. An overview of different kinds of alcohol markers in comparison to PEth is given. Indirect alcohol biomarkers The clinically established traditional markers of alcohol abuse CDT, GGT and MCV, which appear after a heavy long-term consumption of alcohol due to changes from the normal physiologic values, are still widely used [7] . However, these markers have only a moderate clinical sensitivity and specificity, which means that a large portion of high consumers may have normal values and that an elevated value in a significant proportion of cases has a reason other than alcohol [3] . GGT is a membrane-bound glycoprotein enzyme located on the cell surface membrane of most cell types [2,7] . GGT has its highest activity in liver, kidney and bile. An increase of GGT values in serum is a sign of liver or bile damages. GGT is responsible for the extracellular catabolism of gluthatione, and catalyzes the transfer of the γ-glutamyl moiety of glutathione to various peptide acceptors. An increase in serum GGT activity may be due to increased synthesis of GGT as a result of enzyme induction by alcohol or drugs. Serum GGT has been widely used as an index of liver dysfunction and as a marker of alcohol misuse. An increase of GGT activity can be a sign for hepatobiliary diseases. Chronic alcohol intake also increases serum GGT activity. The specificities and sensitivities of GGT vary in different studies, but are usually slightly better than those of other commonly used long-term markers of alcohol intake. Serum GGT is also increased by age, smoking and various other diseases [2,7] . However, analysis of GGT from serum is a standard procedure in clinical chemistry. Key terms Social drinking: A daily alcohol intake ≤60 g of ethanol is classified as moderate, social drinking behavior. Risky drinking: A daily alcohol intake ≥60 g of ethanol is classified as heavy and risky drinking behavior.
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MCV (red blood cell volume) is an often used marker for the detection of excessive alcohol intake. In persons with high alcohol consumption, MCV increases and responds slowly (due to cell renewal within 2 to 4 months) to abstinence. The mechanisms of alcohol-induced increase of MCV are still unknown, but it seems that ethanol and its metabolites, especially acetaldehyde, have hematotoxic effects and may affect the red cell structure and its metabolism. Usually, the sensitivities and specificities of MCV are lower than those of GGT, and even moderate alcohol intake may increase MCV. Furthermore, MCV could also be elevated in patients with liver diseases, vitamin B12 or folic acid deficiency, hematological diseases, reticulocytosis or hypothyroidism, which limits the reliability of MCV for the detection of alcohol consumption [2,7] . CDT is a glycoprotein, which is important for the iron transport in plasma. Human transferrin is composed of 679 amino acids with two potential glycosylation sites, which usually bind two bi- and/or triantennary carbohydrate chains of variable composition, containing four different carbohydrates. Sialic acid is the only charged residue in these saccharide chains, and, when present, bears a negative charge [10] . CDT has a half-life of 7–10 days and is synthesized in the liver. As a result of heavy alcohol intake, the concentrations of desialylated isoforms of CDT in serum are increased. The exact mechanisms remain unknown, but both protein transport and enzyme activities seem to be influenced by ethanol. It is possible to analyze CDT by electrophoretic, chromatographic and immunometric methods. Currently, CDT is considered as the most useful marker for long-term alcohol misuse. A disadvantage of CDT is that its sensitivity varies greatly in different experimental conditions, clinical settings and populations. While alcohol intake above 60–80 g/day correlates well with increase of CDT, the results concerning the effects of moderate drinking are inconsistent. A high level of CDT is not always a sign of alcohol abuse, though. There are other factors such as gender, age, body mass index, smoking, liver disease, anorexia, pregnancy and hyperferremia, influencing the CDT level to obtain ‘false-positive’ results [2,7] . Analytical methods for CDT
For the identification and quantitation of CDT isoforms, the separation from other blood components is required [11] . While in former times the concentration of total CDT was determined, nowadays new analytical methods render a differentiation of CDT subtypes possible. Isoelectric focussing (IEF), capillary electrophoresis (CE) and liquid chromatography (LC) provide a distribution pattern of isoforms. IEF is highly selective and is used as a reference method for
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Progress in monitoring alcohol consumption & alcohol abuse by phosphatidylethanol
transferrin isoforms. Separation proceeds in a gel containing a pH gradient according to the characteristic isoelectric points (pI) of the transferrin isoforms [11] . The disadvantage of IEF is its inaccuracy and imprecision in quantitative evaluation, which can be overcome by CE or by LC [10] . CE in fused-silica capillaries is an effective tool for the separation of the major transferrin isoforms [12] . A widespread method for CDT determination is microcolumn ion-exchange chromatography followed by immunometric quantitation based on the different charge characteristics of desialyzed transferrins. These recent methods indicate the measurement results as percentage of the total transferrin and allow for the detection of natural variability. Direct alcohol biomarkers In contrast to indirect markers, the direct markers are straightly related to the ethanol metabolism. They are formed in a nonoxidative phase II reaction. Some of them even occur after the uptake of trace amounts of ethanol. Aside from the detection of ethanol in blood, urine or exhaled air, there are the already abovementioned markers EtG, EtS, FAEE and PEth, which can be measured in blood and/or urine and also in hair (EtG) [3] . PEth represents a group of abnormal phospholipid homologs. The structure of PEth is a glycerol molecule with two fatty acid chains in sn-1 and sn-2 position, typically containing 14–22 carbon atoms with 0–6 double bonds, and with phosphoethanol as the head group. So far 48 different homologs were identified [13] . PEth is localized in cell membranes and is formed there from phosphatidylcholine only if ethanol is present. Ethanol acts as a co-substrate in the transphosphatidylation reaction catalyzed by the enzyme phospholipase D (PLD) (Figure 1) [3,14] . Compared with its formation, the elimination rate of PEth is slow. The elimination half-life time of PEth in human blood is approximately 4 days [7] . The concentration of PEth is highly correlated with the amount of ingested ethanol [15] . It is detectable in blood of alcohol abusers even up to 3 weeks after alcohol withdrawal; therefore it is a promising new biomarker for the detection of alcohol abuse [16,17] and for monitoring of abstinence. Analytical methods for PEth
When PEth was first measured in blood samples obtained from alcoholics, thin layer chromatography (TLC) with quantification by image analysis was used [18] . TLC separation was time consuming and the coefficient of variation (CV) was relatively high, however this study showed PEth as a possible marker of alcohol abuse with a high specificity and a long detection
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window. Other authors used mainly high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) [19–21] and nonaqueous capillary electrophoresis (NACE) combined with UV detection [22] . With an LOD of about 0.8 μmol/l (∼560 ng/ml PEth) [21] HPLC-ELSD has an essentially higher sensitivity than TLC, but is also time consuming with run times of about 60 min and ELSD has only been used for the detection of the total sum of PEth homologs. Compared with HPLC methods, NACE has shorter run times and needs less sample volume. NACE with UV detection has a slightly lower LOD of 0.4 μmol/l (∼280 ng/ml PEth) than the HPLC-ELSD methods, but it is also not possible to separate the different PEth homologs present in human blood. Using NACE, the detection times of PEth varied between the measured blood samples fortified with PEth standards and the clinical samples. This seemed to be due to the fact that the fortified samples only contained one PEth homolog (PEth 16:0/18:1). Also an internal standard (IS) was not included in the NACE method [22] . Coupling NACE with ESI–MS enhanced sensitivity (LOD of 0.1 μmol/l, ∼70 ng/ml PEth) and selectivity of this method, and increasing accuracy and precision was obtained by the use of an IS [23] . A more selective and sensitive detection method for PEth is MS with triple quadrupole or TOF analysis [19,24,25] . The mass spectrometric detection methods are advantageous compared with previous HPLC-ELSD or NACE-UV methods, because they have lower LODs and single PEth homologs can be differentiated, whereas with ELSD or UV-detection, it was only possible to detect the sum of PEth homologs with less sensitivity. For our research on PEth, an LC–MS/MS method was developed for quantitation of the PEth homologs 16:0/16:0, 18:1/18:1, 16:0/18:2 and 16:0/18:1 with an LOD of 100 ng/ml. The most abundant homologs in human blood are PEth 16:0/18:1 and PEth 16:0/18:2 [26,27] , with a nearly 10 times higher area ratio of PEth 16:0/18:1 than the area ratio of PEth 16:0/16:0 and 18:1/18:1. For each above-mentioned PEth homolog, the corresponding deuterated IS was synthesized in-house by transphosphatidylation reaction of the corresponding phosphatidylcholines (PC) with D6 -ethanol enzyme-catalyzed by phospholipase D according to a published method with minor modifications [26,28] . To assess the time window of detectability of PEth after single alcohol consumption, the combination of liquid–liquid extraction and LC–MS/ MS was switched to protein precipitation followed by online-SPE-LC–MS/MS. This improved the LOD from 100 ng/ml to 10 ng/ml. Table 1 gives an overview
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O R1
phosphatidic acid
O
O R2
H2O
O O
H
O
P
O
O–
R1 O CH3 +
H3C
O O
O
O
R2
N
H3C
O
O O
R1
phospholipase D
P
O
O–
phosphatidylcholine
R2
EtOH
O O
H3C
phosphatidylethanol
O
O
O
P O–
Figure 1. Enzymatic formation of phosphatidic acid from phosphatidylcholine, in the presence of ethanol phosphatidylethanol is formed.
about the different analysis methods for PEth and their LODs and LOQs. EtG and EtS are direct metabolites of ethanol. They are widely used for clinical and forensic applications. EtG and EtS are detectable in blood for up to 8 h; in urine the detection window is up to 80 h after heavy alcohol consumption. EtG and EtS are formed by a nonoxidative elimination of ethanol, catalyzed by the enzymes uridine diphosphateglucuronosyltransferase or sulfotransferase, respectively, being less than 0.1% of the ethanol intake excreted through this phase II metabolism [6] . Already after consumption of trace amounts of alcohol, EtG and EtS are detectable in Key terms Online SPE: The instrumental setup for online SPE uses a modified HPLC system. The sample is loaded onto a trapping column where the sample is concentrated and washed. Then a switching valve is switched and the mixture is eluted through an analytical column, which separates the sample chromatographically. Society of Hair Testing (SoHT): The Society of Hair Testing (SoHT) was founded in 1995 and has the goal to promote research in hair testing technologies in forensic and clinical sciences.
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urine [31] , and are still present after ethanol has been eliminated. Therefore, the analysis of EtG and EtS is a well-suited method for the detection of recent alcohol exposure and for abstinence monitoring [5,7,32] . However, there is the possibility that EtG can be produced in urine after sampling, if the specimen contains bacteria such as Escherichia coli and ethanol, which might be produced from sugar and yeast, thus causing positive EtG results. Furthermore, EtG, is sensitive to bacterial degradation if the samples are contaminated with bacteria and transported without cooling or if stored improperly, causing ‘false negative’ results [33–35] . For EtS neither postsampling formation nor degradation has been found in urine samples. With the collection of dried urine spots (DUS), it is possible to inhibit bacterial degradation [36] , but for better clinical specificity, a combination of both, EtG and EtS has been recommended. EtG is as well detectable in hair. The detection window of EtG can be several months, depending on the hair length. In Switzerland and Germany, the analysis of EtG in hair is meanwhile used for abstinence monitoring in driving aptitude testing before re-issuing a driver’s license after ‘drunken driving’. The Society of Hair Testing (SoHT) suggested a cut-off of 30 pg/mg to differentiate excessive alcohol
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Progress in monitoring alcohol consumption & alcohol abuse by phosphatidylethanol
consumption from social drinking. For abstinence monitoring, a cut-off of 7 pg/mg EtG in hair was recommended [37,38] . Positive results are always associated with alcohol consumption, but in cases where EtG is not detected alcohol consumption cannot be excluded definitely [39–45] . FAEE are nonoxidative products of ethanol metabolism [46] . They are formed by esterification of fatty acids with ethanol, catalyzed by FAEE synthases; and also carboxylesterases and glutathione transferase have FAEE synthase activity [46,47] . As FAEE are bound to albumin or lipoproteins, they can be transferred between lipid compartments. Elevated levels of FAEE are detectable in blood shortly after alcohol intake and remain elevated in serum for approximately 24 h [7] . In hair, FAEE are suitable long-term markers of high and risky alcohol consumption [48] ; and at least 15 different FAEE have been identified. Thereof ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate have been chosen as ethanol consumption markers [49,50] . For quantitation of FAEE by GC–MS, the sum of these four FAEE has been used [48] . In hair the sum of the
Review
concentrations of the four FAEE with a cut-off value of 0.5 ng/mg reveals a sensitivity of 90% and a specificity of 90% [38] . For detecting prenatal exposure to alcohol, FAEE in meconium have been proposed as biomarkers [7] . Figure 2 gives an overview of the different kinds of biomarkers described in this paper and their detection windows with respect to consumption habits. Recent developments in PEth analysis & applications The clinically established indirect markers GGT, MCV and CDT are widely used to detect alcohol abuse, but these markers are not sensitive and specific enough to identify the extent of alcohol abuse [2,7] , as they can be influenced by other factors such as gender, age, body mass index, smoking and liver diseases. Currently, the most useful indirect marker for long-term alcohol misuse is CDT. It has a wide window of detection, shows a significantly higher clinical specificity than GGT or MCV, but also lacks sensitivity, as CDT is only detectable after high and long-term alcohol abuse. Therefore, slowly eliminated direct markers with improved
Table 1. Overview of LOD and LOQ of PEth with different methods. Procedure
LOD
LOQ
Analytes and IS
Ref.
‐
0.22 μmol/l (150 ng/ml)
Total sum of PEth homologs, IS: phosphatidylbutanol
[20]†
HPLC–ELSD
0.8 μmol/l (560 ng/ml)
‐
Total sum of PEth homologs, no IS
‐
0.7 μmol/l (500 ng/ml)
Total sum of PEth homologs, IS: phosphatidylbutanol
NACE–UV
0.4 μmol/l (280 ng/ml)
‐
Total sum of PEth homologs, no IS
NACE–MS (QQQ)
0.1 μmol/l (70 ng/ml)
0.4 μmol/l (280 ng/ml)
Single PEth homologs, IS: phosphatidylbutanol
[23]†
0.001 μmol/l (0.7 ng/ml)
Single PEth homologs, IS: phosphatidylbutanol
[25]† [24]†
LC–HRMS/MS (Orbitrap) 0.0005 μmol/l (0.35 ng/ml)
[21] [3] [22]
LC/Q–ToF–MS/MS
0.001 μmol/l (1 ng/ml)
0.001 μmol/l (1 ng/ml)
Single PEth homologs, IS: phosphatidylpropanol
LC–MS (Q)
0.07 μmol/l (5 ng/ml)
‐
Total sum of PEth homologs, no IS
0.02 μmol/l (14 ng/ml)
0.1 μmol/l (70 ng/ml)
Single PEth homologs, IS: phosphatidylpropanol
[27]†
LC–MS/MS (QQQ)
0.02 μmol/l (14 ng/ml) for PEth 16:0/16:0; 0.024 μmol/l (17 ng/ml) for PEth 18:1/18:1
0.064 μmol/l Single PEth homologs, (45 ng/ml) for PEth IS: phosphatidylpropanol 16:0/16:0; 0.077 μmol/l (54 ng/ml) for PEth 18:1/18:1
[29]†
0.01 μmol/l (7 ng/ml)
0.03 μmol/l (21 ng/ml)
Single PEth homologs, IS: corresponding penta-deuterated IS
[26]†
Online-SPE LC–MS/MS (QQQ)
0.014 μmol/l (10 ng/ml)
0.028 μmol/l (20 ng/ml)
Single PEth homologs, IS: corresponding penta-deuterated IS
[30]
[19]
‐: No data available. Mass-spectrometric analyzers: PEth: Phosphatidylethanol; Q: Quadrupole, QQQ: Triple quadrupole or hybride triple quadrupole ion trap used in triple quadrupole mode; QToF: Quadrupole-time-of-flight. † Method was validated
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BAC Single/regular consume Short-term markers
EtG/EtS (blood) EtG/EtS (urine) 6
4
2
Days PEth (blood)
High-risk consume Intermediary long-term markers
CDT (serum) GGT (serum) MCV (blood)
10
6
8
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Weeks
Abstinence/social consume/high-risk consume Long-term markers EtG/FAEE (hair) 8
6
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Months
Figure 2. Overview of the different kinds of biomarkers described in this paper and their detection windows with respect to consumption habits. BAC: Blood alcohol concentration; CDT: Carbohydrate-deficient transferrin; EtG: Ethyl glucuronide; FAEE: Fatty acid ethyl esters; GGT: γ-glutamyl transpeptidase; MCV: Mean corpuscular volume; PEth: Phosphatidylethanol.
specificity and sensitivity seem to be highly desirable in clinical and forensic issues [3] . One such biomarker that has gained increasing interest over the last few years is PEth, which is only formed after ethanol intake. Thus, the specificity of PEth is close to 100% whereas that for GGT and CDT is only about 40%. The two most
abundant PEth homologs in human blood are PEth 16:0/18:1 and PEth 16:0/18:2 [26,27] . Studies [3,15] showed that the sensitivity of PEth is much better for the detection of alcohol consumption than CDT. Up to now PEth was not sensitive enough for the detection of small amounts of alcohol, but by optimizing
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0 0
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Figure 3. Phosphatidylethanol concentrations after repeated drinking on five subsequent days. PEth: Phosphatidylethanol. Data taken from [51] (two examples shown).
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the analysis method the LOD could be lowered. With the new LOD of 10 ng/ml (instead of 100 ng/ml), first drinking studies were performed to define the detection window of the PEth homolog PEth 16:0/18:1 after ingestion of a single amount of alcohol, which led to a blood alcohol concentration (BAC) of 1‰. In a first study with cumulative drinking up to a BAC of 1‰ on five subsequent days, Gnann et al. [9] showed that PEth values of alcoholics (PEth values > 700 ng/ ml) cannot be reached (Figure 3) . Thus, it is possible to differentiate with the alcohol biomarker PEth between social drinking and risky drinking. In the most recent pilot study, two volunteers (male and female) took an individual calculated single dose of alcohol, which led to a BAC of 1‰ after an abstinence period of 2 weeks. In the week after the experiment, one blood and one urine sample was taken every day, in the second week samples were taken every 2 days. The test persons stayed abstinent throughout these weeks. At the start of the study, blood samples were negative for PEth. After drinking alcohol, which led to a BAC of 1‰, formation of PEth was observed over several hours, as long as ethanol was present in blood. After elimination of ethanol, PEth decreased with a half-life time of approximately 4 days, and was detectable in blood for up to 10 days [30] . Conclusion The study of Gnann et al. [9] and the described pilot study with a single drinking event show the potential of PEth for abstinence monitoring, since PEth could be detected even after a single drinking event for several days. Until now, positive PEth levels have only been found when ethanol was consumed, demonstrating the specificity of PEth. In general, the risk of sample manipulation or dilution after urine collection or the instability of the analyte due to bacterial contamination and degradation are of major concern when using urine as test specimen for alcohol markers or drugs of abuse. In hair analysis, hair type, color, length and treatment (by hair cosmetics or heat) may have a strong influence on alcohol markers in hair. In contrast, blood samples do not have these disadvantages and are often taken in clinical settings also for other markers. Sampling of venous blood is a welldefined process. For high-throughput analysis blood can be used, while hair need laborious sample preparation and a high degree of automation has not yet been achieved for hair sample work-up. Due to the high specificity of PEth as marker for alcohol consumption and due to its rather slow elimination compared with EtG or EtS, PEth is predesti-
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Key term Dried blood spots (DBS): A simple and cost-effective sampling method is the collection of venous or capillary blood on filter cards. This method is used since years for neonatal metabolic screening, and started to be of interest for therapeutic drug monitoring.
nated for abstinence monitoring and also for detection of problematic alcohol consumption habits. For the detection of problematic drinking, a threshold of 1 μmol/l (∼700 ng/ml) PEth in blood (for PEth 16:0/18:1) has been proposed. Still there are further experiments needed, because up to now, ‘normal’ ranges of PEth, in other words, concentrations typical for social drinking over a long time period have not been established for large collectives. With lowered detection limits, it is now the starting point to establish a cut-off for abstinence monitoring, not including PEth formation from ‘hidden alcohol in nutrition’ – and for further evaluation of the threshold between social drinking and risky drinking habits and alcohol abuse. Future perspective The newest developments in PEth analysis and controlled drinking studies show the potential of PEth in abstinence monitoring. Until now, positive PEth levels were only found when ethanol had been consumed prior to blood sampling. However, further investigations with larger numbers of persons are necessary, to determine whether physiological abnormalities can be detected, and to determine cut-off levels for drinking habits according to WHO categories (abstinence, social drinking and risky alcohol consumption) [8] . For routine analysis of PEth, there is the possibility to speed up the method with UHPLC and by the use of well plate formats [52] . Another promising development might be the analysis of PEth in dried blood spots (DBS) [53,54] . Studies on DBS showed good robustness and the advantage that neoformation of PEth in the presence of BAC does not occur after drying blood on filter paper for DBS sampling [30,54] . Capillary blood sampling is used since many years for neonatal screening [55] and has additionally been applied for neonatal screening of prenatal alcohol exposure [56,57] . However for routine application, this method still needs to be evaluated. Capillary blood sampling would mean easier and faster sample collection, as sampling can also be done by nonmedical personnel. Acknowledgement We would like to thank S. Lanz (IRM Bern) for technical support in setting up the online SPE-LC–MS/MS system, and the team of FTC Bern for measuring blood alcohol concentrations.
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Review Schröck, Thierauf, Wurst, Thon & Weinmann Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employ-
ment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary • Worldwide the harmful use of alcohol results in about 2.5 million deaths every year, and health problems created by excessive alcohol consumption are still growing in most western countries. • Therefore it is important to develop potent techniques for the early detection of hazardous drinking. Besides self-report forms, special questionnaires (e.g., AUDIT), this can be done by alcohol consumption markers.
Different kinds of alcohol biomarkers • Indirect markers, such as CDT, GGT and MCV, appear after a heavy long-term consumption of alcohol due to changes from the normal physiologic values and are widely used, but have only a moderate clinical sensitivity and specificity. • Direct markers are only formed after ethanol consumption. Some of them even occur after the uptake of trace amounts of ethanol. Aside from the detection of ethanol in blood, urine or exhaled air, the markers EtG, EtS, FAEE and PEth can be measured in blood and/or urine and also in hair.
Phosphatidylethanol • The direct alcohol biomarker phosphatidylethanol (PEth) represents a group of abnormal phospholipid homologs. PEth can be used to differentiate social drinking from risky drinking.
Perspective • Drinking studies show the potential of PEth in abstinence monitoring. • Cut-off levels for abstinence, social drinking and risky alcohol consumption have to be determined. PEth might also be detected in dried blood spots, as this method showed good robustness and PEth is more stable in dried blood spots than in whole blood. http://whqlibdoc.who.int/hq/2000/who_msd_ msb_00.4.pdf, pp. 50–54 (2000).
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