[advances in clinical chemistry] volume 67 || ethyl glucuronide and ethyl sulfate

CHAPTER TWO

Ethyl Glucuronide and EthylSulfateNatalie E. Walsham*, Roy A. Sherwood†,1*Department of Clinical Biochemistry, University Hospital Lewisham, London, United Kingdom†Department of Clinical Biochemistry, King’s College Hospital NHS Foundation Trust, London,United Kingdom1Corresponding author: e-mail address: [email protected]

Contents

1. Introduction 482. Metabolism of Alcohol 493. Stability 514. Cutoff Values 515. Detection Times 526. Methods for Measurement of EtG and EtS 52

6.1 Hair EtG 547. Applications 55

7.1 Detoxification programs 567.2 Liver transplantation and liver disease 577.3 Fetal alcohol spectrum disorder 587.4 Postmortem 597.5 Sexual assault victims 607.6 Drink driving 60

8. Confounders Causing False-Positive or False-Negative Results 608.1 Urinary tract infection 608.2 Mouthwash 618.3 Hand sanitizers 618.4 Beverages and food 628.5 Drugs 628.6 Hair products 63

9. Conclusions 63Declarations 64References 64

Abstract

Alcohol misuse is associated with significant morbidity and mortality. Although clinicalhistory, examination, and the use of self-report questionnaires may identify subjectswith harmful patterns of alcohol use, denial or under-reporting of alcohol intake is com-mon. Existing biomarkers for detecting alcohol misuse include measurement of blood

Advances in Clinical Chemistry, Volume 67 # 2014 Elsevier Inc.ISSN 0065-2423 All rights reserved.http://dx.doi.org/10.1016/bs.acc.2014.09.006

47

http://dx.doi.org/10.1016/bs.acc.2014.09.006

or urine ethanol for acute alcohol consumption, and carbohydrate-deficient transferrinand gamma-glutamyl transferase for chronic alcohol misuse. There is a need for a bio-marker that can detect excessive alcohol consumption in the timeframe between 1 dayand several weeks. Ethyl glucuronide (EtG) is a direct metabolite of ethanol detectable inurine for up to 90 h and longer in hair. Because EtG has high specificity for excess alcoholintake, it has great potential for use in detecting “binge” drinking. Using urine or hair, thisnoninvasive marker has a role in a variety of clinical and forensic settings.

1. INTRODUCTION

Alcohol misuse is associated with significant morbidity and mortality

and is widely distributed throughout all socioeconomic groups worldwide.

The 2011 Global Status Report on Alcohol from the World Health Orga-

nization estimated that an excess of 70 million people worldwide had rec-

ognizable alcohol misuse [1]. Alcohol abuse was responsible for 2.25 million

deaths in the world each year (3.8% of the total). One-third of these were

associated with accidents. In its publication “Statistics on Alcohol: England

2013,” the Health and Social Care Information Centre estimated that alco-

hol misuse costs the National Health Service £3.5 billion each year [2].

Althoughmany subjects misusing alcohol can be identified by the clinical

history and examination or by self-report questionnaires such as the Alcohol

Use Disorders Identification Test (AUDIT) questionnaire, there are signif-

icant problems with deliberate under-reporting being common. A range of

biomarkers for the detection of harmful alcohol consumption has been

described [3]. These can be divided into direct and indirect markers. Direct

markers include ethanol itself or its metabolites. Indirect markers are depen-

dent on the action of alcohol at the molecular level or compounds released

from organ damage associated with ethanol or metabolites. Ethanol mea-

surements in breath or body fluids have high specificity for excessive alcohol

intake, but relatively narrow timeframes for positivity after alcohol con-

sumption (breath 4–6 h, blood 10–12 h, and urine 18–24 h). Other direct

biomarkers of alcohol intake rely on alternative pathways of alcohol metab-

olism and include ethyl glucuronide (EtG), ethyl sulfate (EtS), and

5-hydroxytryptophol (5-HTOL). The most commonly used indirect bio-

markers measured in blood are gamma-glutamyl transpeptidase (GGT),

carbohydrate-deficient transferrin (CDT), and erythrocyte mean corpuscu-

lar volume (MCV) [4]. Both GGT and MCV require significant alcohol

intake over a prolonged period of time (>1000 g over at least 2 weeks)

48 Natalie E. Walsham and Roy A. Sherwood

to become abnormal. GGT is both induced by alcohol itself and released by

hepatocytes damaged by alcohol or its metabolites, but it has poor specificity

due to its increase in liver disease not associated with alcohol misuse. This

particular problem is growing due to increased obesity in the developed

world. For example, hepatic steatosis associated with obesity and diabetes

mellitus causes increased GGT. In addition, MCV is increased in nutritional

deficiencies, particularly folate and/or vitamin B12 deficiency, which may

be present in those misusing alcohol with a chaotic lifestyle thus reducing

specificity. Although CDT has good specificity for alcohol misuse, it is best

used as a marker of chronic excessive alcohol consumption over 7–14 days.

It will not test positive after a single session of heavy drinking. Excessive

drinking in one session, “binge” drinking, appears to be an increasing prob-

lem in many areas of the world. Therefore, there is a need for a biomarker of

alcohol misuse that can detect excessive consumption in the timeframe

between those tests that are positive in the first 24 h only and CDT (which

could be considered the HbA1c of alcohol intake).

2. METABOLISM OF ALCOHOL

The main metabolic pathway of ingested alcohol takes place in the

liver in a two-stage enzymatically catalyzed oxidation process. Alcohol is first

converted to acetaldehyde by alcohol dehydrogenase and then further

metabolized to acetate by aldehyde dehydrogenase. A small amount is

excreted unchanged in urine, sweat, and expired air.

EtG (ethyl β-D-6-glucuronide) is a direct metabolite of ethanol formed

by the enzymatic conjugation of ethanol with glucuronic acid in the liver

[5]. This phase II reaction is catalyzed by mitochondrial membrane-bound

UDP-glucuronosyltransferase. Ethanol is also conjugated to sulfate by sul-

fotransferase to form EtS (Fig. 1). These are minor pathways with less than

1% of ethanol ingested entering these pathways and, being water-soluble,

EtG and EtS are excreted in urine [6]. EtG and EtS are most commonlymea-

sured in urine as markers for alcohol intake, but can also be measured in

whole blood, serum/plasma, and a range of other body fluids or tissues.

Studies have shown that these minor metabolites are mainly distributed in

the plasma compartment of blood rather than the cellular compartment with

a median serum/plasma to whole blood ratio of 1.69 for EtG and 1.30 for

EtS [7].

As these metabolites are formed in the liver, maximal plasma metabolite

concentration occurs later than blood ethanol itself: approximately 2–3 h

49Ethyl Glucuronide and Ethyl Sulfate

later for EtG and 1–2 h later for EtS [8,9]. In a study conducted with vol-

unteers (n¼18), the maximal concentration of EtG and EtS in serum was

4000 and 2000 μg/L, respectively, following consumption of 32 g alcohol,

and 13,000 and 6000 μg/L, respectively, following consumption of 64 g

alcohol. Peak concentrations were reached 1–3 h after alcohol inges-

tion [10]. There appear, however, to be wide interindividual variations in

the maximum serum/plasma EtG and EtS concentration and there is a poor

correlation between the metabolites and blood ethanol concentration [8].

Studies have found that metabolite elimination occurs exponentially with

a median half-life of 2–4 h [9,11,12]. EtG can usually be detected in urine

for 72–90 h.

The elimination rate of EtG and EtS appears similar in healthy subjects

and heavy drinkers during alcohol detoxification [11]. This study found

decreased elimination rate and increased blood concentration in patients

with renal disease which would delay excretion of these metabolites.

Two small studies from one group have provided further evidence that renal

impairment may cause increased EtG and EtS in urine and increased EtG in

hair. In 14 subjects who each collected 10 urine samples after consuming

0.1–1.4 g of ethanol/kg body weight, detection times were found to be sig-

nificantly longer in patients with decreased renal function versus healthy

subjects (p<0.01). Significantly increased hair EtG was found in 12 patients

with renal disease versus 21 healthy volunteers (p¼0.009) [13,14]. A study

CH3CH2OHEthanol

Alcohol dehydrogenase

Aldehyde dehydrogenase

C2H5O-SO3HEthyl sulfate EtS

Sulfotransferase

CH3CHOAcetaldehyde

CH3COOHAcetic acid

HOOCC2H5O

O

OH

OH OH

Ethyl glucuronide EtG

UDP-glucuronosyltransferase

Figure 1 Metabolism of alcohol and formation of ethyl glucuronide and ethyl sulfate.


by Wurst et al.[15] found that EtG concentration was influenced by age,

gender, cannabis use, kidney disease, and the amount of ethanol ingested

in the previous month. Race, smoking, body mass index, liver cirrhosis,

the age at which subjects began drinking regularly, and total body water

had no significant influence on EtG concentration in urine [15].

In a way similar to some drug addicts, alcohol misusers sometimes

attempt to lower the EtG and EtS urine concentration by drinking large vol-

umes of water. Expressing EtG and EtS relative to urine creatinine can partly

overcome this dilutional effect [6,16]. However, Helander et al.[17]

reported that wide interindividual variation in EtG detection time was com-

mon despite normalization with creatinine.

The interaction of alcohol with other metabolic pathways has resulted

in several potential markers that have been compared to EtG. Fatty acid ethyl

esters (FAEEs) are esterification products of ethanol and fatty acids that

can be measured in blood and tissues as markers of alcohol intake [18]. Acute

alcohol intake alters the normal metabolism of serotonin (5-hydroxy-

tryptamine) to 5-hydroxyindole acetic acid (5-HIAA) resulting in the

formation of 5-HTOL, albeit at 1% of the 5-HIAA concentration. The ratio

of 5-HTOL to 5-HIAA in urine has been shown to be a more sensitive and

specific marker of alcohol ingestion than urine or blood ethanol, remaining

positive 6–15 h after the blood alcohol concentration (BAC) had returned to

baseline [19].

3. STABILITY

EtG and EtS have been shown to be stable markers in vitro. Urine

samples stored at 4 �C for 5 weeks were found to have no change in EtG

concentration [20]. When stored at room temperature in ventilated vials,

the concentration of EtG was found to increase due to water evaporation.

During this study, there was no evidence of analyte decomposition. EtG-

positive tissue material allowed to slowly decompose at room temperature

exhibited decreased EtG concentration over time. No postmortem forma-

tion was found.

4. CUTOFF VALUES

Studies in healthy volunteers who ingested alcohol (0.1–0.8 g/kg

body weight) have consistently shown that the best cutoff value in urine

is 100–200 μg/L for EtG and 100–110 μg/L for EtS [17]. For clinical use,


cutoffs as high as 500 μg/L have been used to reduce the risk of false-positiveresults [17]. In a volunteer study, a maximal plasma EtG concentration

of 360 μg/L (range 280–410 μg/L) was found in samples taken 1.5–24 h

after a single alcohol dose (0.5 g/kg) [21]. Cutoffs of 500 μg/L for EtG

and 50–100 μg/L for EtS in urine were supported in a preliminary study

in healthy volunteers [22]. Meta-analysis of 15 studies found that mean

hair EtG concentration in social drinkers, heavy drinkers, and deceased

subjects with a known history of chronic alcohol misuse was 7.5, 142.7,

and 586.1 ng/g, respectively. A cutoff of 30 ng/g for EtG in hair was

proposed to limit false negatives and better distinguish social and heavy

drinkers [23].

5. DETECTION TIMES

There have been a number of studies characterizing the timeframe

during which EtG and EtS remain detectable in urine following alcohol

intake in healthy volunteers [6,8,9,24–27]. Although these studies involved

a range of alcohol doses (0.1–0.85 g/kg body weight), the detection time-

frame was relatively consistent (24–48 h) for both EtG and EtS. One study,

using a larger dose of alcohol (>1 g/kg), found that EtG remained above

the limit of detection (100 μg/L) for 39–102 h [28]. This longer timeframe

was in agreement with two studies conducted in alcohol-intoxicated sub-

jects (40–130 h) [17,29].

6. METHODS FOR MEASUREMENT OF EtG AND EtS

Various methods for measuring EtG and EtS have been published over

the past 10 years. The most commonly used methods are based on liquid

chromatography–mass spectrometry (LC-MS) because it is highly sensitive,

specific, and is able to simultaneously measure EtG and EtS [30–33]. This

technology has been used to determine EtG in urine, whole blood, serum,

meconium [34], saliva [35], hair [36], nails [37], and dried blood spots

[38,39].

LC-MS detection of EtG can be carried out using selected ion monitor-

ing of the precursor ion (m/z 221) and the principal daughter ion (m/z 75)

with penta-deuterated EtG (ETG-D5,m/z 226) as the internal standard [30].


The alternative transition m/z 221!85 has also been used [31,35].

Corresponding transitions for EtS are m/z 125!97 and m/z

125!80 [40]. Most LC-MS methods have a limit of quantitation (LOC)

of 50–100 μg/L for EtG and EtS. In some applications, urine can be injected

without extraction following centrifugation and dilution with water and

supplementation with an internal standard. Serum/plasma samples can be

analyzed after deproteinization with methanol or acetonitrile, centrifuga-

tion, and addition to an aliquot of the mobile phase. A comparison of five

LC-MS methods for measurement of urinary EtG and EtS recommended

that solid-phase extraction followed by LC-MS-MS should be adopted as

the reference method because of its high selectivity and sensitivity [41].

Other methods include reversed-phase liquid chromatography with

pulsed electrochemical detection [42], microwave-assisted extraction

followed by gas chromatography–mass spectrometry (GC-MS) [43,44],

GC-MS with solid-phase extraction for sweat samples [45] and GC-MS

of silylated derivatives [46]; capillary electrophoresis [47], capillary zone

electrophoresis–mass spectrometry [48], capillary isotachophoresis, and zone

electrophoresis [49]; and an ELISA based on polyclonal antibodies [50]

(Table 1). An LC-MS/MS method for urine EtG/EtS has been validated

using forensic guidelines [51].

A monoclonal antibody-based enzyme immunoassay (EIA) is commer-

cially available for EtG analysis in urine (DRI Ethyl Glucuronide Enzyme

Immunoassay, Thermo Fisher Scientific Diagnostics, Hemel Hempstead,

UK). Comparison with an established LC-MS method showed good agree-

ment (r2¼0.931), indicating a low cross-reactivity of the EtG antibody to

other urinary constituents [52]. The method evaluation showed the EIA

is sensitive, specific, and offers a low but clinically relevant measuring range

(0–500 μg/L). Higher results may be obtained by dilution (detection limit of

100 μg/L). Although correlation to LC-MS was good, the EIA method is

considered a screening test. EtG-positive samples should always be con-

firmed by LC-MS/MS with EtS measurement to rule out false positives

(see Section 8).

To ascertain if EtG crosses the human placenta to the fetus, a method for

the measurement of EtG in placental perfusate and tissue was developed

using headspace solid-phase microextraction coupled with GC-MS. This

was used in an ex vivo placental perfusion model to show that EtG could

be detected in the fetal circulation within 20 min [53]. EtG has also been

measured in dental tissue by LC-MS/MS and correlated well with theMich-

igan Alcohol Screening Test (r¼0.914) [54].


6.1. Hair EtGAnalysis of drugs of abuse in hair samples has long been used to identify

chronic use over extended timeframes (weeks to months). As such, there

has been considerable interest in testing hair EtG to extend the detection

period beyond traditional markers such as CDT. Hair EtG, as a marker

for detection of alcohol intake, has been recently reviewed [55].

Table 1 Characteristics of selected methods for the measurement of EtG

Method ExtractionSamplematrix LoD Ref.

Electrospray LC-MS Direct injection Urine 100 μg/L [30]

Electrospray LC-MS Direct injection Urine – [31]

Anion exchange LC-MS/MS Direct injection Urine 100 μg/L [32]

Electrospray LC-MS/MS Protein

precipitation

Serum 0.2 μmol/L [33]

LC-MS/MS Solid-phase Meconium 5 ng/g [34]

UPLC-MS/MS Solid-phase Oral fluid 4.4 μg/L [35]

GC-MS/MS Solid-phase Hair 8.4 ng/g [36]

LC-MS/MS Water Nails 10 ng/g [37]

LC-MS/MS Methanol Blood spots 0.1 mg/L [38]

Electrospray LC-MS Direct injection Urine 50 μg/L [40]

LC-pulsed ECD Liquid–liquid Urine 10 μg/L [42]

GC-MS Microwave assisted Urine 100 μg/L [43]

GC-MS Microwave assisted Hair 0.3 ng/mg [44]

GC-MS Solid phase Sweat 1 μg/L [45]

GC-MS Silylated

derivatives

Hair – [46]

Capillary electrophoresis Direct injection Serum 100 μg/L [47]

Capillary isotachophoresis

+CZE

Water dilution Serum 0.01 μmol/

L

[49]

ELISA Direct sample Serum 300 μg/L [50]

EIA Direct sample Urine 100 μg/L [51]

LC, liquid chromatography; MS, mass spectrometry; GC, gas chromatography; LoD, limit of detection; UPLC,ultra-performance liquid chromatography; ECD, electrochemical detection; CZE, capillary zone electropho-resis; ELISA, enzyme linked immunosorbent assay; EIA, enzyme immunoassay.


Initial methods using GC-MS for hair EtG were hampered by LOCs

(0.5–2.0 μg/g) [46,56,57]. Over the past decade, LC-MS methods devel-

oped for urine EtG have contributed to substantially improve the LOC

in hair so that it is now typically in the range of 2-10 ng/g. [58–65]. An

EtG cutoff value of 4–30 ng/g in hair has been proposed to distinguish

social (<20 g ethanol/day) and heavy drinkers (>40 g ethanol/day). This

approach has yielded good sensitivity and specificity (90–95%). In subjects

with low to moderate alcohol intake, i.e., daily consumption of 16–32 g

alcohol over a 3-month period, the maximum hair EtG concentration

was 11 ng/g [66]. This study proposed an abstinence threshold of

<7 ng/g and an excessive consumption threshold of >30 ng/g. The latter

cutoff produced a higher positivity rate versus CDT in a fitness-to-drive fol-

lowing previous alcohol problems program [67]. Because of the false pos-

itivity concerns in cases with legal implications (fitness-to-drive, workplace

testing, child custody, etc.), several groups have recommended a combina-

tion of hair EtG and FAEE measurement [68,69].

Hair analysis for EtG requires extraction prior to analysis by any of the

methods detailed earlier. Washing the hair sample with dichloromethane

and methanol followed by sonication (30 min) extracts more than 50% of

the EtG [70]. An alternative approach is to usemicropulverization [71]. Body

site is independent, i.e., chest, arm, and leg hair samples provide equivalent

EtG values when compared to scalp hair [72]. Recent methods for hair

EtG analysis include hydrophilic interaction liquid chromatography–tandem

mass spectrometry (HILIC-MS/MS) with liquid–liquid extraction that has a

lower LOC of 0.18 ng/g [73] and UHPLC-MS/MS with an LOC of

1.0 ng/g [74,75].

Hair and nail EtG was measured in 606 undergraduate students by

LC-MS/MS [76]. Nail EtG demonstrated better sensitivity versus hair

EtG for detecting any weekly alcohol use (p¼0.02).

7. APPLICATIONS

The measurement of EtG has been carried out in a variety of clinical

and forensic settings. Alcohol misuse can be implicated in a significant pro-

portion of subjects admitted to a hospital emergency department with gas-

trointestinal symptoms or following minor injury. Self-report of alcohol

intake using the AUDIT questionnaire tends to be unreliable due to under-

estimation of alcohol consumption by the subjects. Two studies on the use of


EtG measurements in the emergency room setting have been carried out

using urine [18] and plasma [77]. Most subjects either tested negative for

blood ethanol or had low BAC in the range 0.01–0.07 g/L. Interestingly,

a substantial percentage (25–38%) tested positive for EtG irrespective of pos-

itive (�8 points) or negative AUDIT score.

7.1. Detoxification programsMonitoring abstinence in subjects undergoing alcohol detoxification pro-

grams is important. Blood or urine ethanol measurement is problematic

due to the relatively short timeframe for these markers following alcohol

ingestion. In a group of 139 detoxified alcohol-dependent patients followed

up for 12 weeks after discharge from in-patient treatment, 28% of subjects

denying relapse tested positive for EtG and EtS by LC-MS/MS [78]. Sim-

ilarly 4 out of 30 patients, in whom neither clinical assessment nor routine

laboratory testing suggested relapse, tested positive for urine EtG at concen-

trations from 4200 to 196,600 μg/L [19]. It should be noted, however, that

the subject with the highest urine EtG concentration had detectable serum

EtG. A double-blind placebo-controlled oral acamprosate study was con-

ducted in 56 alcohol-dependent subjects (30males) [79]. Urine was obtained

at baseline and weekly for EtG and EtS. On the first day, 72% of subjects

tested positive. This number decreased to 31% after 3 weeks with no differ-

ence between the acamprosate and placebo groups. Significantly, 28% of

samples from subjects who denied alcohol consumption in the day prior

to testing were positive for EtG and EtS. In a similar study of 24 out-patients

undergoing treatment for alcohol or drug dependency, urine EtG and EtS

were compared to self-reporting [80]. This study found high concordance

(87%) for self-report and EtG/EtS results. A single patient specimenwas pos-

itive for EtS only.

Subjects undergoing opioid maintenance therapy often abuse alcohol,

but often deny it with negative AUDIT scores. Urine and hair EtGmeasure-

ment identified cases of excess alcohol intake in subjects on a methadone

maintenance program [81–83].Many of these would have beenmissed using

self-report alone. In health-care professionals recovering from substance-

related disorders, complete abstinence from drugs, including alcohol, is

required before they can return to work. Random urine testing is usually

incorporated into such programs. In one study, 100 urines were collected

and tested for alcohol use [84]. Although none tested positive for alcohol,

seven tested positive for EtG (0.5–196 mg/L).


Oral fluid EtG has been measured in a Norwegian employee recruitment

exercise [85]. In this nondetoxification study, about 2.1% tested positive

for EtG (>2.2 μg/L). Hair testing for EtG and FAEE has potential applica-

tion in workplace testing for employees in high-risk occupations [86].

7.2. Liver transplantation and liver diseaseOrthotopic liver transplantation (OLT) for treatment of end-stage liver dis-

ease resulting from alcohol misuse remains controversial because a substan-

tial percentage of subjects (20–25%) return to harmful drinking. As such,

most transplant programs require a period of abstinence to remain on

the waiting list. Detection of alcohol misuse in these patients represents

a challenge because GGT is typically increased due to hepatic fibrosis

and CDT may be increased secondary to reduced clearance from the cir-

culation into bile [87]. In addition, patients refrain from drinking in the

24–36-h period prior to breath, blood, or urine alcohol testing. Another

study was conducted in 18 OLT candidates who denied alcohol consump-

tion [88]. This report found that almost half (49%) of urine specimens were

positive for EtG, whereas only 1 of 127 breath alcohol tests was positive.

A cross-sectional anonymous study of adult OLT candidates (n¼109)

found that 20% of subjects were positive for urine EtG and EtS versus

4% by self-reported questionnaire [89]. A large study of OLT candidates

(n¼141) reported a positive predictive value (PPV) of 89.3% and negative

predictive value of 98.9% for urine EtG in detecting alcohol misuse [90].

This German report demonstrated that EtG was clearly superior to CDT,

MCV, or GGT.

These findings have been confirmed in two recent studies including one

that used hair EtG. In a study of 121 OLT candidates/recipients, urine EtG

was compared to serum and urine ethanol, CDT, and the AUDIT-c ques-

tionnaire [91]. Alcohol consumption was defined as a positive AUDIT-c or

by patient confirmation when challenged with the test results. Receiver

Operator Characteristics analysis found that urine EtGwas the best predictor

of alcohol consumption (AUC 0.94) versus CDT (AUC 0.63). Urine EtG

combined with the AUDIT-c increased the AUC to 0.98. In 63 OLT can-

didates, hair EtG was compared to urine EtG, blood ethanol, and CDT [92].

Although 19 patients (30%) admitted alcohol consumption in the previous

6 months, 39 patients (62%) tested positive for at least one marker. In the

44 patients claiming abstinence, 52% had one positive marker with hair

EtG above the cutoff (30 ng/g) in 83% of cases providing a specificity of


98% and a PPV of 92%. Interestingly, the authors claimed that renal and liver

function had no effect on hair EtG concentration.

Others evaluated urine EtG and EtS in patients with liver disease

(n¼120) and hair EtG in patients with liver disease (n¼200) [93,94]. Urine

EtG (cutoff 100 μg/L) had a sensitivity of 76% and specificity of 93%. Urine

EtS (cutoff 25 μg/L) had a sensitivity of 82% and specificity of 86%. Hair

EtG (cutoff 8 ng/g) demonstrated an AUC of 0.93 for detecting ethanol

ingestion (average 28 g of ethanol a day over a 3-month period).

7.3. Fetal alcohol spectrum disorderFetal alcohol syndrome (FAS) and fetal alcohol spectrum disorder (FASD)

are recognized as a cause of congenital abnormalities, cognitive dysfunction,

and developmental delay. It is estimated that FAS affects 2/1000 and FASD

9/1000 live births in the developed world. Diagnosis after birth, however, is

difficult. EtG and EtS have been measured by LC-MS/MS in meconium

samples from the infants of 177 randomly selected women from Italy and

Spain [95]. EtG was detectable in over 80% of samples while EtS was only

found in 50%. A cutoff of 2 nmol/g was found to have 100% sensitivity and

specificity to distinguish heavy maternal ethanol consumption during preg-

nancy from occasional or no use (defined by questionnaire and meconium

FAEE measurement). This cutoff was validated in a study from the same

group using a subset of mothers who self-reported alcohol consumption

during pregnancy [96]. This study showed that neonatal hair EtG was a poor

predictor of maternal alcohol intake. A similar study of 602 meconium sam-

ples from a maternal health evaluation in Germany found only 97 (16.3%) of

cases had detectable EtG [97]. In none of the 602 cases did the mothers

report serious alcohol consumption and no evidence of FAS or FASD were

found in the newborn infants. When EtG was compared to FAEE, a cutoff

of 274 ng/g provided the best agreement between the two markers. Two

outliers (EtG 10,200 and 82,000 ng/g) suggested heavy alcohol consump-

tion that was not reported. The authors concluded that combined EtG

and FAEE in meconium minimized both false-positive and false-negative

results. An LC-MS/MS method has been developed for the simultaneous

measurement of FAEE, EtG, and EtS [98]. An ELISA has been developed

and validated for the measurement of EtG in meconium [99]. An EtG cutoff

of 0.9 nmol/g provided excellent sensitivity (100%) and good specificity

(78%) when compared to LC-MS/MS confirmation.


Urine and hair EtG and EtS measurement during pregnancy has been

reported [100]. In this Swedish study, women (n¼103) provided urine

and hair for EtG, EtS, and FAEE measurement and completed the AUDIT

questionnaire. Although 26 women (25.2%) were identified as possible

alcohol consumers and 7 women had hair EtG or FAEE concentrations

highly suspicious of heavy drinking, only 1 was positive by self-reported

AUDIT questionnaire. An Italian group compared the performance of

FAEE and EtG in meconium with maternal hair and nail EtG in predicting

fetal exposure to alcohol [101]. Similar results to other groups were

obtained for FAEE and EtG in meconium. None, however, tested positive

for hair or nail EtG despite confirmed alcohol consumption in 18 of

151 cases.

7.4. PostmortemConfirming a role for alcohol as a contributor to cause of death has been

difficult due to the inherent instability of peptide markers such as CDT

postmortem. EtG was compared to CDT in serum, urine, cerebrospinal

fluid (CSF), and vitreous humor in postmortem forensic cases with a pos-

itive (n¼38) and negative (n¼22) history of alcohol misuse [102]. EtG

(mean�SD) in urine (339,000�389,000 μg/L; p<0.001), vitreous

humor (4200�4800 μg/L; p<0.001), serum (6900�8900 μg/L;p<0.01), and CSF (1700�2.7 μg/L; p<0.01) were significantly higher

in the alcohol-positive group, whereas CDT was only increased in CSF.

The same group demonstrated that the commercially available immunoas-

say (Thermo Scientific) could also be applied to vitreous humor samples

and correlated well with LC-MS/MS (r¼0.94) [103]. An immunoassay

cutoff of 300 μg/L for vitreous humor EtG provided high sensitivity

(92%). In contrast, blood alcohol (cutoff 100 mg/L) was positive in only

68% of cases. Postmortem urine (n¼800) was tested for EtG by immuno-

assay and LC-MS/MS [104]. The LC-MS/MS method had a statistically

significant proportional bias (p<0.0001). An LC-MS/MS cutoff of

100 μg/L provided the best sensitivity and specificity that equated to a

92 μg/L immunoassay cutoff.

EtG has also been detected in postmortem hair samples together with

tissue samples (gluteal and abdominal fat, liver, and brain) from intoxicated

subjects [105]. It is unclear, however, why EtG was not detected in the liver

or gluteal fat of one subject who died intoxicated.


7.5. Sexual assault victimsDelay in testing for alcohol intake is common in cases of sexual assault due to the

late presentation ofmany victims.Urine from59 female victims of sexual assault

in Norway was tested for EtG and EtS by UPLC-MS/MS [106]. EtG and EtS

were positive in 45 of 48 cases with self-reported alcohol intake, whereas

ethanol was only detected in 20 cases (sensitivity: EtG 94%; ethanol 42%).

7.6. Drink drivingIn a number of European countries, drink-driving offenders have to prove

abstinence for a period of time to regain their driving licenses. Recently, the

United Kingdom replaced GGT and MCV with CDT. In Germany, a pro-

gram that encompasses both urine alcohol and drug tests is in place. In a

Canadian study, drivers regaining their licenses were required to install igni-

tion interlock devices that prevented the vehicle being driven if BAC limit

was exceeded. Urine EtG/EtS and hair EtG were compared with conven-

tional biomarkers and the ignition interlock BAC [107,108]. The authors

concluded that testing for EtG in either urine or hair improved the detection

rate for problem drinkers. Similar conclusions for hair EtG were reached in

Switzerland [67] and Germany [109].

8. CONFOUNDERS CAUSING FALSE-POSITIVE ORFALSE-NEGATIVE RESULTS

8.1. Urinary tract infectionAs EtG measurement becomes commonplace in clinical and medico-legal

settings, i.e., the use of urine EtG in Germany for return of driving licenses

after conviction of driving under the influence of alcohol, it has become

increasingly important to understand the potential causes of false-positive

or false-negative results.

False-positive and false-negative results for urine EtG have been reported

when bacteria are present. Glucuronide and sulfate conjugates are cleaved

by β-glucuronidase and sulfatase enzymes, respectively. Studies have shown

that EtG, but not EtS, was sensitive to bacterial hydrolysis when exposed

to Escherichia coli and Clostridium sordellii [110,111]. As E. coli is the most

common pathogen in urinary tract infections (UTIs), falsely decreased

EtG may occur in its presence. Under these conditions, EtG should be

combined with EtS LC-MS analysis because EtS appears unaffected by


bacterial contamination. Preservatives such as fluoride and immediately

freezing specimens may prevent or mitigate bacterial growth [110]. Other

urine preservatives such as boric acid have not been investigated with respect

to EtG.

Interestingly, EtGmay be formed postcollection in samples infected with

E. coli in the presence of ethanol via fermentative processes [112]. This risk is

increased in diabetic subjects if glycosuria is also present. Formation of EtG

postcollection may not always be prevented by fluoride preservatives or by

storage at 4 �C [112], and therefore, caution is advised when interpreting

results. Formation of EtS in these bacterially contaminated samples did

not occur, supporting the recommendation that EtS should accompany

or be used to verify EtG results.

8.2. MouthwashIt is important to determine if sources of ethanol other than overt consump-

tion can be responsible for the presence of EtG or EtS. Although ethanol

absorbed into the body from alcohol-based mouthwash may result in the

presence of EtG in the urine, normal routine use did not generate high urine

values [113]. Routine alcohol-based mouthwash use, i.e., three times a day

after meals, resulted in 29% of subjects having urine EtG >50 μg/L. In two

smaller studies (n¼14 subjects), only one person was positive for urine EtG,

whereas seven had detectable EtS (maximum concentration 104 μg/L)[114,115].

8.3. Hand sanitizersIt has also been demonstrated that EtG was detected in urine when alcohol-

containing hand sanitizer gels are used frequently [116]. These products typ-

ically contain 60–65% ethanol by weight. When used eight times over an

8-h period, urine EtG and EtS up to 103 and 51 μg/L respectively, were

reported. A study on intensive use of hand sanitizers was conducted in vol-

unteers (n¼11) who cleansed their hands with an alcohol-based sanitizer

(62% ethanol) every 5 min for 10 h on three consecutive days [117]. Urine

EtG and EtS could be detected (maximum concentration 2001 and 84 μg/L,respectively) at the end of the study day. Only two specimens had detectable

EtG the next morning (96 and 139 μg/L) and only one had detectable EtS

(64 μg/L). A more recent study suggested that inhalation of ethanol vapor

not transdermal absorption caused the increase in EtG [118]. Using LC-MS/

MS, 2-propyl glucuronide, a metabolite of 2-propanol (a compound


frequently used in disinfectants), was found in urine. As such, the presence of

this metabolite could potentially be used to identify false-positive EtG

results. The methodology employed for urine EtG measurement is highly

important with respect to false positivity. Positive results after the use of a

hand sanitizer in one case could not be confirmed by LC-MS/MS [119].

1-Propylglucuronide and 2-propylglucuronide were detected, i.e., in vivo

metabolites of 1-propanol and 2-propanol, respectively. Interestingly, the

two parent compounds accounted for 75% by weight of the sanitizer

solution.

8.4. Beverages and foodLow alcohol or “alcohol-free” beers have become popular in many parts of

the world. Despite having up to 0.5% alcohol, these are still deemed non-

alcoholic. Four volunteers who consumed 2.5 L of these nonalcoholic beers

had urine EtG concentrations ranging from 300 to 14,100 μg/L the next

morning [120]. Positive EtG results were also found in another study

13 h after consumption of nonalcoholic beers [121]. In the same study,

the authors showed that consuming foodstuffs that contain alcohol caused

positive urine EtG results including samples taken 5 h after eating sauerkraut

and 3.5 h after consuming matured bananas [121]. Similarly, in vivo fermen-

tation of baker’s yeast to ethanol with subsequent formation of EtG and EtS

has been reported [122]. Paradoxically, ingestion of brewer’s yeast did not

result in any positive EtG or EtS results.

8.5. DrugsThe case below highlights the importance of method selection for measuring

urine EtG. In this report, the patient was taking a number of medications

including levetiracetam, gabapentin, clomethiazol, and chloral hydrate

[123]. Despite confirmed alcohol abstinence, the patient had urine EtG

(up to 8000 μg/L) as determined by commercial immunoassay. Further

investigation by LC-MS/MS revealed no urine EtG or EtS. To validate

these findings, urine was collected from a control subject who ingested

500 mg chloral hydrate. The control urine was found to have an EtG con-

centration of 280 μg/L. Trichloroethyl glucuronide was proposed as the

most likely cross-reacting compound. Unfortunately, this premise could

not be confirmed due to the lack of a pure standard.


8.6. Hair productsInterest in quantifying hair EtG has led a number of groups to investigate

the impact of various hair treatments on false-positive and false-negative

results. LC-MS/MS demonstrated the presence of EtG (64%) and EtS

(27%) in 11 herbal hair tonics [124]. EtG concentration ranged from

70 to 1060 μg/L. A case report of an individual with a hair EtG concen-

tration of 910 ng/g, but normal CDT and GGT, who regularly used a hair

tonic was investigated as a potential false-positive [125]. Overnight incuba-

tion of EtG-free hair in the lotion resulted in a hair EtG of 140 ng/g.

Another report, however, found no increase in hair EtG in seven volun-

teers using a hair tonic for up to 1 month despite the tonic containing 44%

(v/v) ethanol [126]. Although coloring does not to affect hair EtG content,

bleaching and perming caused decreased hair EtG (mean decrease 73.5%

and 95.7%, respectively) [127]. In vitro experiments using hydrogen perox-

ide to simulate bleaching and ammonium thioglycate to simulate perming

showed similar decreases in hair EtG suggesting chemical degradation

of EtG.

9. CONCLUSIONS

EtG was first described as a metabolite of ethanol in 1967 [128]; how-

ever, the increased use of mass spectrometry over the past decade has resulted

in the development of accurate and reliable methods for EtG and EtS in bio-

logic samples. Although most methods were initially developed for urine,

there has been renewed interest in testing hair to increase the timeframe

for detecting alcohol misuse. Published data suggest that EtG has potential

as a marker of high sensitivity and specificity for the detection of alcohol mis-

use in a variety of settings in both clinical and forensic medicine.

As a noninvasivemarker, EtG in urine or hair could have a role in screen-

ing, diagnosis, and monitoring treatment in selected groups of subjects or in

general population studies. Urine EtG remains positive for periods of up to

48–72 h following heavy alcohol consumption. As such, EtG has potential

use in the intermediate timeframes, i.e., between those times in which eth-

anol and GGT/CDTmeasurements are performed. This approach has been

successfully applied to establish abstinence in patients on liver transplant

waiting lists and in alcohol detoxification programs. Whether EtG will be

adopted in workplace monitoring or regranting driving licenses requires

further work.


The availability of an immunoassay for EtG that can be performed on

general clinical chemistry analyzers will make it easier to conduct larger stud-

ies. Because bacterial UTIs cause both false-positive and false-negative EtG

results, mass spectrometry-based methods that measure both EtG and EtS

may be preferable. Nearly all methods for hair measurement of EtG and

EtS use mass spectrometry which allows for identification of other alcohols

that could interfere with immunoassay-based methods.

Further work is clearly required before the full potential of these direct

ethanol biomarkers can be realized and incorporated into the armamentar-

ium of alcohol biomarkers in general.

DECLARATIONSConflicts of interest None.

Funding No funding applicable to this review.

Ethical approval Ethical approval not required with regard to the content of this review.

Guarantor R. A. S.

Contributorship The authors contributed equally to the work.

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[advances in clinical chemistry] volume 67 || ethyl glucuronide and ethyl sulfate

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