Transcript Herbal Medicines?: A Case Study

Measurement of Ethyl Glucuronide by Microgenics
Analytical
1
Unit
®
DRI
Enzyme Immunoassay
2
Pathology ,
& Chemical
St Georges - University of London,
3
Microgenics GmbH , Hertfordshire, UK
1
UK
Jennifer Button1, Claire Mathers1, Rosemary Lee2, Jas Dhillon3 and David W Holt1
INTRODUCTION
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.
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
individual’s capability to have provided consent.
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
EtG
Creatinine Etg:Creatinine
Cause of Death
(mg/dL) (ng/mL) (mmol/L)
(ng/mmol)
27
14140
4.4
3213.6
Unknown
284
61400
3.5
17542.9
Head injury
18
<100
2.2
N/A
Hanging
188
166800
9.3
17935.5
Multiple injuries
279
<100
1.3
N/A
Overdose
<10
<100
10.0
N/A
Unknown
<10
25600
16.5
1551.5
Unknown
252
1303000
8.4
155119.0
Overdose
RESULTS AND DISCUSSION
Ethanol
(mg/dL)
EtG
(ng/mL)
Creatinine
(mmol/L)
Etg:Creatinine
(ng/mmol)
Time post incident
(hrs)
266
143800
2.7
53259.3
5.5
174
171700
2.3
74652.2
4.5
126
1301000
12.9
100852.7
8
<10
113100
17.5
6462.9
30
<10
72
6.6
10.9
3.5
<10
30
13.3
2.3
55
<10
187
10.9
17.2
18
159
196600
4.4
44681.8
N/A
<10
1766
16.5
107.0
8.5
<10
524
7.6
68.9
38
<10
525
18.1
29.0
12
186
272700
4.9
55653.1
12
<10
232800
16.0
14550.0
N/A
100
>2000000
8.7
N/A
5
<10
149
6.0
24.8
N/A
<10
153
6.1
25.1
N/A
<10
6100
5.2
1173.1
1
<10
2400
3.2
750.0
3
<10
72700
11.8
6161.0
1
<10
425
7.2
59.0
42
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 (EtG:Creatinine, 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.
Table 1. EtG concentrations in a random selection of Coronial cases.
CONCLUSION
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
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.
References:
(1) Drummer, O.H. The Forensic Pharmacology of Drugs of Abuse, London, Arnold, 2001.
(2) Shepherd, R. Simpson’s 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, 172;119-124.
(6) Kapur, B. IATDMCT Compass 2007 6(3);7-11.
(7) Goll, M. J Anal Toxicol 2002, 26;262-265.
(8) Schmitt, G. Forensic Sci 1999, 42;1099.