Measurement of Ethyl Glucuronide by Microgenics DRI

1
Measurement of Ethyl Glucuronide by Microgenics
DRI Enzyme Immunoassay
Analytical Unit1 Chemical Pathology2, St
Georges - University of London, UK1 Microgenics
GmbH , Hertfordshire, UK3
Jennifer Button1, Claire Mathers1, Rosemary Lee2,
Jas Dhillon3 and David W Holt1
RESULTS AND DISCUSSION
INTRODUCTION
DFSA There are a number of barriers to successful
prosecution of sexual assaults. Most notable is
the late presentation of victims and the
associated loss of evidence. Many cases hinge on
consent, but as the law stands, an individual is
not legally capable of providing consent when
incapacitated with alcohol or drugs. Many of the
drugs implicated in sexual crimes have a narrow
detection window, and alcohol is no exception. In
those individuals presenting to the police some
hours after the incident, EtG could be used to
establish alcohol consumption even after the
complete elimination of ethanol, and may
potentially be used to provide an indicator of an
individuals capability to have provided consent.
Ethyl Glucuronide (EtG) is a water soluble,
stable, non-volatile, direct metabolite of
ethanol. Metabolism occurs as soon as alcohol
enters the blood stream. More than 90 of alcohol
consumed is metabolised in the liver, via the
action of the enzyme alcohol dehydrogenase
(ADH).1,2 ADH catalyses the reaction in which
alcohol is oxidised to acetaldehyde.1 This is
then metabolised to acetate by aldehyde
dehydrogenase and then to carbon dioxide.1 Other
enzymes capable of metabolising alcohol include
catalase, the cytochrome P450 enzyme, CYP2E1 and
microsomal oxidising systems (MEOS).1,2 Between
5-8 of unchanged alcohol is excreted in the
urine, sweat and breath.2 The elimination of
ethanol by enzymatic conjugation with glucuronic
acid represents approximately 0.5 to 1.5 of the
total ethanol elimination.3 Whilst the
detection period for alcohol is relatively short,
EtG can be detected for up to 80 hours and peaks
at approximately 4 hours after alcohol
consumption.4-5 EtG offers several advantages
over traditional markers of alcohol abuse such as
gamma glutamyl transferase (GGT) mean corpuscular
volume (MCV) and carbohydrate deficient
transferrin (CDT). It is a direct, specific and
sensitive marker of alcohol consumption, being
present only if ethanol is consumed. It is not
influenced by age, gender, medication or
non-alcohol related disease and is not dependant
on chronic alcohol consumption.6 Unlike urinary
excretion of ethanol, EtG concentrations are
highly influenced by water intake.7 Normalisation
of EtG to creatinine is recommended. The
Microgenics DRI EtG assay is primarily targeted
at alcohol abstinence, where zero tolerance
policies are in existence. Such examples include
liver transplant recipients, recovering
alcoholics in withdrawal treatment programmes and
airline pilots. We investigated its application
in forensic settings such as in post-mortem cases
from Coroners and in the investigation of drug
facilitated sexual assault (DFSA). Coroners
Cases Several investigators have reported low or
absent ethanol concentrations in forensic
toxicology cases of known alcoholics, or those
with reliably witnessed alcohol consumption prior
to death. In many there is even evidence of
recent alcohol consumption in the form of cans or
bottles at the scene. In some, this phenomenon
can be explained by the presence of
?-hydroxybutyrate (BHB), a marker of alcoholic
ketoacidosis. However, there still remain a
number for which, no explanation can be found.
The detection of EtG in the absence of ethanol is
indicative of recent alcohol consumption even
after ethanol has been completely eliminated from
the body. Perhaps more problematic in Coroners
cases is the in-vitro, post-mortem production of
ethanol due to fermentation of sugars by bacteria
migrating from the gastro-intestinal tract as
cellular barriers break down. This process, also
common in samples obtained from diabetics or
those with urinary tract infections, is
accentuated by warm temperatures. Whilst this can
be inhibited by preservation with fluoride,
significant concentrations of alcohol may already
have been formed prior to sampling.
Distinguishing between ethanol present due to
alcohol consumption or that produced via in-vitro
fermentation is of particular importance in road
traffic collisions. EtG can be used to determine
this. It should, however, be noted that bacterial
glucoronidases can result in the breakdown of
EtG, so that ethanol may be detected in the
absence of this metabolite.
EXPERIMENTAL
EtG was measured in urine samples obtained from
20 DFSA cases and 8 randomly selected post-mortem
cases, using the Microgenics DRI EtG Enzyme
Immunoassay on the Olympus AU400 platform. Assays
were semi-quantitative (0, 100 (LLOQ), 500, 1000,
2000 (ULOQ) ng/mL) with 4 QC levels employed
(375, 625, 750, 1250ng/mL). Cut offs of 500ng/mL
or 1000ng/mL are typically recommended. Samples
above the top calibrator for EtG were diluted
using saline. EtG results were corrected for the
effect of internal dilution using creatinine,
measured using the Jaffe reaction on the Siemens
Advia 2400 and were compared to ethanol
concentrations measured by head Space GC-FID on
the Shimadzu GC 2014.
RESULTS
Ethanol (mg/dL) EtG (ng/mL) Creatinine (mmol/L) EtgCreatinine (ng/mmol) Cause of Death
27 14140 4.4 3213.6 Unknown
284 61400 3.5 17542.9 Head injury
18 lt100 2.2 N/A Hanging
188 166800 9.3 17935.5 Multiple injuries
279 lt100 1.3 N/A Overdose
lt10 lt100 10.0 N/A Unknown
lt10 25600 16.5 1551.5 Unknown
252 1303000 8.4 155119.0 Overdose
Table 2. EtG concentrations in DFSA cases.
In an in-house study looking at the effect of an
alcohol containing mouthwash (Sainsbury's total
care active performance mouthwash), used as
directed, resulted in an EtG of 375ng/mL
(EtGCreatinine, 65.8ng/mmol) 3 hours post use.
Further work is required to establish the effect
of false positives resulting from incidental
alcohol exposure, such as from medication,
hygiene products, cosmetics and foods. This is of
particular importance in countries such as
Germany, where 1 year alcohol abstinence is
required for drivers who have lost their licences
following a charge of driving impairment or where
liver transplantation is conditional on
abstinence.
CONCLUSION
Table 1. EtG concentrations in a random selection
of Coronial cases.
EtG is a direct and more specific and sensitive
indicator of alcohol consumption than traditional
markers such as GGT, MCV and CDT. The Microgenics
DRI EtG assay has application in not only the
clinical, but also forensic settings including
Coroners and DFSA cases. However, if employed
semi-quantitatively in both fields, a two tier
calibration needs to be employed to encompass the
range of concentrations observed.
The red circles on table 1 highlight, even in a
small sample population, examples of where EtG
can be used as a biomarker to indicate recent
alcohol consumption where there is low or absent
ethanol. In isolation an alcohol concentration of
27mg/dL may previously have been attributed to
post-mortem fermentation. The green circles
indicate a possible case of bacterial
glucoronidase degradation of EtG, as 279mg/dL is
too great a concentration to solely attribute to
fermentation and too high not to have seen some
EtG formation. The red circles in table 2
illustrate a late report (30hrs post incident) of
sexual assault, with absent alcohol but positive
EtG. This result may help to strengthen a case
where a defence of consent has been made in the
presence of demonstratable impairment. As the
data base of information builds EtG
concentrations may even be able to distinguish
between moderate and chronic alcohol abuse.8
References
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Drugs of Abuse, London, Arnold, 2001. (2)
Shepherd, R. Simpsons Forensic Medicine, 12th
Edition, London, Arnold, 2003. (3) Janda, I.
Alt, A. J Chromatog B 2001 758 229-234. (4)
Wurst, F.M. Addiction 2003, 98 (S2) 51-61. (5)
Hoiseth, G. Forensic Sci Int. 2007,
172119-124. (6) Kapur, B. IATDMCT Compass 2007
6(3)7-11. (7) Goll, M. J Anal Toxicol 2002,
26262-265. (8) Schmitt, G. Forensic Sci 1999,
421099.