1. Measurement of Ethyl Glucuronide by Microgenics DRI ® Enzyme Immunoassay Jennifer Button 1, Claire Mathers 1, Rosemary Lee 2, Jas Dhillon 3 and David W Holt 1 Analytical Unit 1 & Chemical Pathology 2, St Georges - University of London, UK 1 Microgenics GmbH, Hertfordshire, UK 3 References: 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. INTRODUCTION EXPERIMENTAL RESULTS AND DISCUSSION CONCLUSION (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. 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. Ethanol (mg/dL) EtG (ng/mL) Creatinine (mmol/L) Etg:Creatinine (ng/mmol) Cause of Death 27141404.43213.6Unknown 284614003.517542.9Head injury 18<1002.2N/AHanging 1881668009.317935.5Multiple injuries 279<1001.3N/AOverdose <10<10010.0N/AUnknown <102560016.51551.5Unknown 25213030008.4155119.0Overdose RESULTS 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. 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. Table 1. EtG concentrations in a random selection of Coronial cases. Table 2. EtG concentrations in DFSA cases. 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