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Disappearance of ethyl glucuronide during heavy putrefaction
Forensic Science International 176 (2008) 147–151 www.elsevier.com/locate/forsciint
Disappearance of ethyl glucuronide during heavy putrefaction Gudrun Høiseth *, Ritva Karinen, Lene Johnsen, Per Trygve Normann, Asbjørg S. Christophersen, Jørg Mørland Norwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse, Pb 4404 Nydalen, N-0403 Oslo, Norway Received 9 February 2007; received in revised form 19 July 2007; accepted 9 August 2007 Available online 19 September 2007
Abstract Introduction: There are previous publications showing the use of ethyl glucuronide (EtG), a non-oxidative metabolite of ethanol, as a marker of ante-mortem ingestion of alcohol in forensic autopsy cases. The problem of possible degradation or formation of EtG during putrefaction is however not well studied and the aim of this study was to investigate the possibility of false negative and false positive EtG results by an in vitro study. Further, we used the information from the in vitro study on real cases, to get an impression of the practical problem of degradation or formation of EtG. Methods: An in vitro study was carried out to study the concentrations of EtG in blood samples under controlled conditions during putrefaction. In addition, to illustrate the practical problem of degradation or formation of EtG, we used routine samples analysed for EtG in blood. Blood samples from forensic autopsies with ethanol detected but EtG not detected in blood, and therefore suspected post-mortem ethanol formation, were identified. Fifteen such cases had urine samples available, and these were analysed for EtG. We hypothesised that since concentrations are often higher in urine, there would still be traces of EtG left in this medium if post-mortem degradation was the reason for the negative result in blood. Results: In this in vitro experiment, EtG was very unstable in blood samples at 30/40 8C without preservatives. On the other hand, EtG was stable with potassium fluoride at room temperature, and there was no formation of EtG either at 30 8C without preservatives, or at room temperature with potassium fluoride. Of the 15 routine cases where EtG in blood was negative, and the ethanol detected was assumed endogenous, six were positive for EtG in urine. In these cases, ethanol was probably ingested, and the negative EtG in blood may be a false negative result due to degradation during putrefaction. Conclusion: Analysis of EtG in blood is a helpful tool to determine in vivo ingestion of ethanol in post-mortem cases. A negative result, however, especially in heavily putrefied cases, must be interpreted with caution. Analysis of an additional medium would be valuable in these cases. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethanol; Post-mortem; Ethyl glucuronide; Stability
1. Introduction Ethyl glucuronide (EtG) is a minor non-oxidative metabolite of ethanol, formed by conjugation with glucuronic acid catalysed by UDP-glucuronyl transferase [1,2]. The most frequent use of EtG has been its measurement in urine as a relapse marker with high specificity [3–7], but also analyses in serum or blood [1,8,9], as well as hair [10,11] have been published. EtG has also been found interesting in post-mortem cases as a marker of ante-mortem ingestion of alcohol. Previous studies have shown that EtG in blood is a helpful tool to
* Corresponding author. Tel.: +47 23 40 78 99; fax: +47 23 40 78 78. E-mail address: [email protected] (G. Høiseth). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.08.002
determine in vivo intake of ethanol in such cases [8,12,13] and the studies so far, although limited, indicate both a high sensitivity and specificity [8]. Heavily putrefied cases however, have not been specifically studied, and there are concerns about whether instability during putrefaction may lead to complete loss of EtG and hence false negative results. Also, it has not been excluded that EtG may be formed during putrefaction and if so lead to false positive results. Two recent papers have tried to answer this question. One study of EtG concentrations in post-mortem liver and muscle (n = 3) reported an average decrease of 30% over 4 weeks storage at room temperature [14]. These samples did not contain any preservatives (Weinmann, personal information). Also, there was no formation of EtG during 4 weeks of storage at room temperature (n = 5). Another study examined the
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stability in urine without preservatives and found a considerable decrease in EtG in urine samples stored at room temperature for a few days when the samples contained various bacteria, e.g. Escherichia coli [15]. These bacteria caused deglucuronidation of EtG as they contain beta-glucuronidase. When refrigerated, or when a preservative was added, the urine concentrations of EtG were stable [15]. In forensic toxicological analysis of post-mortem cases, there is possibility of degradation or formation of EtG both during putrefaction in the corpse post-mortem and during storage of samples after autopsy and addition of preservatives, before toxicological analysis. The aim of this study was to investigate the possibility of false negative and false positive EtG results by an in vitro study. Further, we used the information from the in vitro study on real cases, to get an impression of the practical problem of degradation or formation of EtG.
2.2. Real cases To get an impression of the practical problem of degradation of EtG, we also studied real cases. During a 3-month period, post-mortem cases sent to toxicological analysis at the Norwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse were routinely analysed for EtG in blood if post-mortem formation of ethanol was suspected or if concentrations of ethanol in blood or urine were between 0.1 and 0.5 g/L. A total of 39 blood samples containing potassium fluoride as a preservative, were satisfactory analysed for EtG during the 3-month period report. In 19 of these cases, EtG was positive. In the other 20, EtG was negative, and post-mortem formation of ethanol was therefore suspected. We report case histories, as well as findings of ethanol and EtG in these cases as we wanted to use this information to investigate the chance of false negative results. In 12 of these cases, a urine sample, which contained potassium fluoride as a preservative, was available. These urine samples were subject to analysis of EtG and we hypothesised that since EtG concentrations are much higher in urine [5,16], there could still be traces of EtG left in this medium if post-mortem degradation of EtG was the reason for the negative result in blood.
2. Materials and methods 2.1. In vitro study (summarised in Table 1)
2.3. Analytical methods 2.1.1. Stability without preservatives To simulate the stability of EtG during putrefaction in a corpse before sampling, blood samples were collected from 10 different corpses at forensic autopsies, five without EtG and five containing EtG. No preservatives were added. In the five EtG-negative cases, the absence of ethanol and EtG in blood was verified by analysis before start of the study. To a 5 mL plastic tube, 0.1 mL of a solution containing 44.4 mg EtG/L in water (MEDICHEM1, Steinenbronn, Germany) was added to 2.9 mL blood to a final theoretical concentration of 1.5 mg/L. The concentrations were immediately controlled by analysis and the samples were incubated at 40 8C. In the five EtG-positive cases, the initial ethanol and EtG concentrations were determined and 3 mL blood was transferred to a 5 mL plastic tube. The samples were incubated at 30 8C. Analyses were made at days 0, 3, 6, 11, 15 and 21.
Ethanol was analysed in blood and urine by using headspace gas chromatography equipped with a flame ionisation detector [17]. EtG in blood and urine was analysed using previously published fully validated methods [8,18]. The limit of detection (LOD) for EtG was 0.02 mg/L in blood and 0.09 mg/L in urine.
3. Results 3.1. In vitro study The results of the in vitro studies are summarised in Table 1. In the five samples without preservatives at 40 8C, spiked with EtG, the initial concentrations of EtG ranged between 1.2 and 1.6 mg/ L. This deviation from the theoretical spiked concentration of 1.5 mg/L is, in addition to analytical variations, caused by the character of post-mortem blood, which makes pipetting of exact volumes difficult. The concentrations fell from the initial concentration after 3 days, and by day 21, all samples were negative for EtG (Fig. 1a). In the five samples without preservatives at 30 8C, containing real EtG, the initial concentrations of ethanol were in the range 1.5–4.3 g/L, and of EtG 1.0–6.5 mg/L. The concentrations of EtG started declining from these initial values after 3–6 days. Only one sample was still positive for EtG after 21 days and this showed a decline in concentration from 6.5 to 2.9 mg/L (Fig. 1b). In two of these cases, one single data point (day 3) had to be excluded because of addition of an insufficient amount of internal standard during analysis.
2.1.2. Stability with preservatives To investigate the stability of EtG when stored in the laboratory, real samples arriving for routine toxicological analysis were used. These samples contained 300 mL of an 11.5 M potassium fluoride solution as preservative. Blood samples from four different corpses were selected shortly after arrival at the laboratory and analysed for ethanol and EtG. Three milliliters of blood was transferred to a 5 mL plastic tube and the samples were stored at room temperature (20 8C). Analyses were made at days 0, 2, 7, 10 and 18. 2.1.3. Formation of EtG To investigate the possible formation of EtG, blood from 10 corpses negative for ethanol and EtG were collected, five containing potassium fluoride and five without preservatives. To a 5 mL plastic tube, 60 mL of a solution containing 50 g/L ethanol in water (Merck, Darmstadt, Germany) was added to 3.0 mL blood to a final theoretical concentration of 1.0 g/L. The concentration of ethanol and absence of EtG were controlled by analysis. The five samples without preservatives were incubated at 30 8C and the five samples with Table 1 Summary of in vitro study of the stability and formation of EtG Conditions
Simulation of
N
Origin of EtG/ethanol
Results
Without preservatives, 40 8C Without preservatives, 30 8C With fluoride, room temperature Without preservatives, 30 8C With fluoride, room temperature
Stability during putrefaction (Fig. 1a) Stability during putrefaction (Fig. 1b) Stability during storage (Fig. 2) Formation during putrefaction Formation during storage
5 5 4 5 5
Spiked with EtG Real EtG Real EtG Spiked with ethanol Spiked with ethanol
All samples negative for EtG after 21 days All but one sample negative for EtG after 21 days Concentrations of EtG unchanged during 18 days No formation of EtG during 21 days No formation of EtG during 21 days
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4. Discussion
Fig. 1. (a) Concentrations of EtG in blood in five samples without preservatives spiked with EtG and incubated at 40 8C. (b) Concentrations of EtG in blood in five samples without preservatives containing real EtG incubated at 30 8C.
In the four samples with potassium fluoride at room temperature, containing real EtG, the ethanol concentrations were in the range 2.1–3.0 g/L. The initial concentrations of EtG were in the range 0.8–2.7 mg/L, and there was no change in concentrations from these initial values (Fig. 2). No formation of EtG was observed during 21 days, in samples spiked with ethanol, neither in the five samples without preservatives at 30 8C, nor in the five samples containing potassium fluoride at room temperature. 3.2. Real cases A total of 39 cases were routinely analysed for EtG in blood during a period of 3 months. In 19 of these, EtG was positive, with a median concentration of 1.6 mg/L (range 0.2–34.9). In the other 20 cases, EtG was negative and the ethanol detected was therefore suspected to be of post-mortem origin. These are shown in Table 2 as case 1–20. In 15 of these, a urine sample was available, and the results of analysis of EtG in urine demonstrated that 6 of the 15 cases were positive for EtG in urine.
Fig. 2. . Concentrations of EtG in blood in four samples with potassium fluoride stored at room temperature.
This study showed that EtG in blood samples may be very unstable at high temperatures without preservatives, but stable at room temperature in containers with added potassium fluoride. The present study also strengthened the assumption that no EtG is formed post-mortem. Incubating samples at 30/40 8C without preservatives aimed to simulate putrefaction in a corpse before sample collection. This method had obvious weaknesses, and this study only gave an indication of the problem. The best model would have been to follow the concentrations of EtG in a corpse during putrefaction, but such a procedure was considered unethical. Thus, we cannot exclude that EtG levels would change differently over time in a corpse than in a blood sample. Further, many aspects concerning the blood samples studied were subject to uncertainty, e.g. presence of bacteria. Neither did we know the degree of putrefaction of the blood before sampling. This problem is partly evaded in the part of the study of stability where the samples were spiked with EtG, but since there are previous reports that stability of substances in spiked and real samples may be different [19], we also studied samples containing real EtG. However, it seemed like the stability was equal in both situations. We chose to use two different temperatures in the experiment. This was done to get an impression of the importance of the temperature of the corpse’s environment, and showed that the instability was present at both temperatures. The intervals between the analyses were also shorter in the start of the study period. This design was chosen to discover an eventually very fast decline in concentrations. The instability of EtG in blood without preservatives was more pronounced than previously published [14]. This might be explained by the high temperatures in this study compared to the previous study (30/40 8C versus room temperature). The results in the present study were more comparable with the findings of a relatively high instability of EtG in urine containing bacteria when stored at room temperature without preservatives [15]. For putrefaction, 30 and 40 8C must be considered extreme conditions, above what is usually the case for most corpses in Norway, but possible in more southern countries. This study thus probably illustrated a worst-case scenario, and its aim was not to elucidate the instability of EtG under all possible conditions. Only one sample was still positive for EtG after end of the study at 21 days, and this sample also showed a considerable decline in concentration of EtG. Two weeks after end of the study, this sample was reanalysed, and it was negative for EtG at that time. The stability during storage in containers with preservative was satisfactory. The small variations observed were within analytical variation. This was consistent with the previous study showing stable concentrations of EtG in urines at room temperature when preservative was added [15]. Studies of other analytes, as benzodiazepines, also show better stability when preservative is added [20]. The present work, like a previous study [14], failed to show any formation of EtG from ethanol, neither in a situation without preservatives simulating putrefaction in a corpse, nor
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Table 2 Concentrations of ethanol and EtG in blood and urine plus case history, in 20 cases where a negative EtG in blood led to suspicion of post-mortem formation of ethanol Case no.
Ethanol in blood (g/L)
Ethanol in urine (g/L)
Case history
Time between death and autopsy (days)
EtG in blood (mg/L)
EtG in urine (mg/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
0.3 0.4 0.6 0.3 0.4 0.1 0a 0 0.1 0.2 0.4 0.4b 0.4a 0 0.2a 0.1 1.0c 0.3 0.7c 0.6
0 0.4 0.5 0.5 0.3 0.2 0.1 0.2 0 0.3 0.6 0.2 0.1 0.3 0 N.A 0.8 0.4 N.A N.A
Found dead at home Found dead in apartment Found dead Found dead at home Found dead in bathroom Found dead at home Traffic accident Intoxication Found dead at home Death from dissection of the aorta Dead from tuberculosis Found dead on bathroom floor with heater cables Found dead at home Found dead in apartment, cardiac death Found dead in apartment Drug-abuser found dead at home Found dead under a bridge, larvae-eaten Found dead in the sea Drug-abuser found dead Cardiac death, found with alcohol bottles
18 5 8 14 14 10 2 7 18 6 40 N.A 12 3 18 N.A 30 30 14 18
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0.3 0.3 0.7 2.8 7.5 244 N.A N.A N.A N.A N.A
Time between death and autopsy is based on assumed time of death from the forensic pathologist. Peripheral blood if not otherwise noted. N.A, not available. a Heart blood. b Blood from pericardium. c Pleural fluid.
with potassium fluoride, simulating storage in the laboratory. It therefore seemed that positive EtG results verify ingestion of ethanol. EtG was found in 19 out of 39 of the real cases analysed for EtG in the present study, and was accordingly interpreted as evidence for ante-mortem ethanol intake. A negative result, however, as seen in 20 out of 39 of the real cases (Table 2), might be a false negative due to EtG degradation. Post-mortem urine was available in 15 of these, and in six, we found EtG in urine (Table 2). A possible explanation of this might be that EtG instability during putrefaction had resulted in complete loss of EtG from blood, but still with its presence in urine, where concentrations are higher. Another, less likely explanation is that EtG was totally eliminated from blood, but detectable in urine due to the longer detection time [18,21]. Ethanol was ingested, but might have been totally eliminated and the ethanol detected might have been formed post-mortem. If so, the negative EtG in blood correctly indicated post-mortem formation of ethanol. The remaining nine cases where urine was available had no trace of EtG neither in blood nor urine and the assumption of post-mortem formation of ethanol was therefore more reliable. The analysis of two different media must be considered valuable. However, these urines were not investigated for bacterial contamination. We can therefore not totally exclude false negative results also in these samples. Recently, it has become clear that ethanol also undergoes sulphate conjugation to form ethyl sulphate (EtS) [22]. In a previous study, none of the bacteria that caused deglucuronidation of EtG contained beta-sulphatase, and as expected, the concentrations of EtS did not decrease during storage at room temperature without preservative [15]. There is reason to
believe that the lack of sulphatases in bacteria makes EtS more stable during putrefaction also in post-mortem blood. This remains to be studied, but EtS could for this reason be a valuable supplement to EtG. In conclusion, analysis of EtG in blood is a helpful tool to determine in vivo ingestion of ethanol in post-mortem cases. This study showed that it has a high specificity, but a lower sensitivity in post-mortem cases. Therefore, a positive result probably verifies ingestion of ethanol, while a negative result, especially in heavy putrefied cases, must be interpreted with caution. Analysis of an additional medium, or also of EtS, would be valuable in these cases. Acknowledgement To Professor Sidsel Rogde at the Institute of Forensic Medicine, University if Oslo, for providing samples. Reference [1] G. Schmitt, R. Aderjan, T. Keller, M. Wu, Ethyl glucuronide: an unusual ethanol metabolite in humans. Synthesis, analytical data, and determination in serum and urine, J Anal. Toxicol. 19 (1995) 91–94. [2] R.S. Foti, M.B. Fisher, Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs, Forensic Sci. Int. 153 (2005) 109–116. [3] F.M. Wurst, R. Vogel, K. Jachau, A. Varga, C. Alling, A. Alt, G.E. Skipper, Ethyl glucuronide discloses recent covert alcohol use not detected by standard testing in forensic psychiatric inpatients, Alcohol.-Clin. Exp. Res. 27 (2003) 471–476. [4] K. Borucki, R. Schreiner, J. Dierkes, K. Jachau, D. Krause, S. Westphal, F.M. Wurst, C. Luley, H. Schmidt-Gayk, Detection of recent ethanol intake with new markers: comparison of fatty acid ethyl esters in serum
G. Høiseth et al. / Forensic Science International 176 (2008) 147–151
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
and of ethyl glucuronide and the ratio of 5-hydroxytryptophol to 5hydroxyindole acetic acid in urine, Alcohol.-Clin. Exp. Res. 29 (2005) 781–787. J. Bergstrom, A. Helander, A.W. Jones, Ethyl glucuronide concentrations in two successive urinary voids from drinking drivers: relationship to creatinine content and blood and urine ethanol concentrations, Forensic Sci. Int. 133 (2003) 86–94. H. Dahl, N. Stephanson, O. Beck, A. Helander, Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide, J Anal. Toxicol. 26 (2002) 201–204. T. Sarkola, H. Dahl, C.J. Eriksson, A. Helander, Urinary ethyl glucuronide and 5-hydroxytryptophol levels during repeated ethanol ingestion in healthy human subjects, Alcohol Alcohol. 38 (2003) 347–351. G. Hoiseth, R. Karinen, A.S. Christophersen, L. Olsen, P.T. Normann, J. Morland, A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol, Forensic Sci. Int. 165 (2007) 41–45. L. Morini, L. Politi, A. Zucchella, A. Polettini, Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography–electrospray tandem mass spectrometry, Clin. Chim. Acta 376 (2007) 213–219. L. Politi, L. Morini, F. Leone, A. Polettini, Ethyl glucuronide in hair: is it a reliable marker of chronic high levels of alcohol consumption? Addiction 101 (2006) 1408–1412. M. Yegles, A. Labarthe, V. Auwarter, S. Hartwig, H. Vater, R. Wennig, F. Pragst, Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers, Forensic Sci. Int. 145 (2004) 167–173. F.M. Wurst, R. Schuttler, C. Kempter, S. Seidl, T. Gilg, K. Jachau, A. Alt, Can ethyl glucuronide be determined in post-mortem body fluids and tissues? Alcohol Alcohol. 34 (1999) 262–263. H. Schloegl, T. Rost, W. Schmidt, F.M. Wurst, W. Weinmann, Distribution of ethyl glucuronide in rib bone marrow, other tissues and body liquids as
[14]
[15]
[16]
[17]
[18]
[19] [20] [21]
[22]
151
proof of alcohol consumption before death, Forensic Sci. Int. 156 (2006) 213–218. H. Schloegl, S. Dresen, K. Spaczynski, M. Stoertzel, F.M. Wurst, W. Weinmann, Stability of ethyl glucuronide in urine, post-mortem tissue and blood samples, Int. J. Legal Med. 120 (2006) 83–88. A. Helander, H. Dahl, Urinary tract infection: a risk factor for false-negative urinary ethyl glucuronide but not ethyl sulfate in the detection of recent alcohol consumption, Clin. Chem. 51 (2005) 1728–1730. N. Stephanson, H. Dahl, A. Helander, O. Beck, Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography– mass spectrometry, Ther. Drug Monit. 24 (2002) 645–651. L. Kristoffersen, L.E. Stormyhr, A. Smith-Kielland, Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma, Forensic Sci. Int. 161 (2006) 151–157. G. Hoiseth, J.P. Bernard, R. Karinen, L. Johnsen, A. Helander, A.S. Christophersen, J. Morland, A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology, Forensic Sci. Int. 172 (2007) 119–124. A.S. Christophersen, Tetrahydrocannabinol stability in whole blood: plastic versus glass containers, J Anal. Toxicol. 10 (1986) 129–131. M.D. Robertson, O.H. Drummer, Stability of nitrobenzodiazepines in postmortem blood, J. Forensic Sci. 43 (1998) 5–8. G. Schmitt, P. Droenner, G. Skopp, R. Aderjan, Ethyl glucuronide concentration in serum of human volunteers, teetotalers, and suspected drinking drivers, J. Forensic Sci. 42 (1997) 1099–1102. F.M. Wurst, S. Dresen, J.P. Allen, G. Wiesbeck, M. Graf, W. Weinmann, Ethyl sulphate: a direct ethanol metabolite reflecting recent alcohol consumption, Addiction 101 (2006) 204–211.
Disappearance of ethyl glucuronide during heavy putrefaction Gudrun Høiseth *, Ritva Karinen, Lene Johnsen, Per Trygve Normann, Asbjørg S. Christophersen, Jørg Mørland Norwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse, Pb 4404 Nydalen, N-0403 Oslo, Norway Received 9 February 2007; received in revised form 19 July 2007; accepted 9 August 2007 Available online 19 September 2007
Abstract Introduction: There are previous publications showing the use of ethyl glucuronide (EtG), a non-oxidative metabolite of ethanol, as a marker of ante-mortem ingestion of alcohol in forensic autopsy cases. The problem of possible degradation or formation of EtG during putrefaction is however not well studied and the aim of this study was to investigate the possibility of false negative and false positive EtG results by an in vitro study. Further, we used the information from the in vitro study on real cases, to get an impression of the practical problem of degradation or formation of EtG. Methods: An in vitro study was carried out to study the concentrations of EtG in blood samples under controlled conditions during putrefaction. In addition, to illustrate the practical problem of degradation or formation of EtG, we used routine samples analysed for EtG in blood. Blood samples from forensic autopsies with ethanol detected but EtG not detected in blood, and therefore suspected post-mortem ethanol formation, were identified. Fifteen such cases had urine samples available, and these were analysed for EtG. We hypothesised that since concentrations are often higher in urine, there would still be traces of EtG left in this medium if post-mortem degradation was the reason for the negative result in blood. Results: In this in vitro experiment, EtG was very unstable in blood samples at 30/40 8C without preservatives. On the other hand, EtG was stable with potassium fluoride at room temperature, and there was no formation of EtG either at 30 8C without preservatives, or at room temperature with potassium fluoride. Of the 15 routine cases where EtG in blood was negative, and the ethanol detected was assumed endogenous, six were positive for EtG in urine. In these cases, ethanol was probably ingested, and the negative EtG in blood may be a false negative result due to degradation during putrefaction. Conclusion: Analysis of EtG in blood is a helpful tool to determine in vivo ingestion of ethanol in post-mortem cases. A negative result, however, especially in heavily putrefied cases, must be interpreted with caution. Analysis of an additional medium would be valuable in these cases. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethanol; Post-mortem; Ethyl glucuronide; Stability
1. Introduction Ethyl glucuronide (EtG) is a minor non-oxidative metabolite of ethanol, formed by conjugation with glucuronic acid catalysed by UDP-glucuronyl transferase [1,2]. The most frequent use of EtG has been its measurement in urine as a relapse marker with high specificity [3–7], but also analyses in serum or blood [1,8,9], as well as hair [10,11] have been published. EtG has also been found interesting in post-mortem cases as a marker of ante-mortem ingestion of alcohol. Previous studies have shown that EtG in blood is a helpful tool to
* Corresponding author. Tel.: +47 23 40 78 99; fax: +47 23 40 78 78. E-mail address: [email protected] (G. Høiseth). 0379-0738/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2007.08.002
determine in vivo intake of ethanol in such cases [8,12,13] and the studies so far, although limited, indicate both a high sensitivity and specificity [8]. Heavily putrefied cases however, have not been specifically studied, and there are concerns about whether instability during putrefaction may lead to complete loss of EtG and hence false negative results. Also, it has not been excluded that EtG may be formed during putrefaction and if so lead to false positive results. Two recent papers have tried to answer this question. One study of EtG concentrations in post-mortem liver and muscle (n = 3) reported an average decrease of 30% over 4 weeks storage at room temperature [14]. These samples did not contain any preservatives (Weinmann, personal information). Also, there was no formation of EtG during 4 weeks of storage at room temperature (n = 5). Another study examined the
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stability in urine without preservatives and found a considerable decrease in EtG in urine samples stored at room temperature for a few days when the samples contained various bacteria, e.g. Escherichia coli [15]. These bacteria caused deglucuronidation of EtG as they contain beta-glucuronidase. When refrigerated, or when a preservative was added, the urine concentrations of EtG were stable [15]. In forensic toxicological analysis of post-mortem cases, there is possibility of degradation or formation of EtG both during putrefaction in the corpse post-mortem and during storage of samples after autopsy and addition of preservatives, before toxicological analysis. The aim of this study was to investigate the possibility of false negative and false positive EtG results by an in vitro study. Further, we used the information from the in vitro study on real cases, to get an impression of the practical problem of degradation or formation of EtG.
2.2. Real cases To get an impression of the practical problem of degradation of EtG, we also studied real cases. During a 3-month period, post-mortem cases sent to toxicological analysis at the Norwegian Institute of Public Health, Division of Forensic Toxicology and Drug Abuse were routinely analysed for EtG in blood if post-mortem formation of ethanol was suspected or if concentrations of ethanol in blood or urine were between 0.1 and 0.5 g/L. A total of 39 blood samples containing potassium fluoride as a preservative, were satisfactory analysed for EtG during the 3-month period report. In 19 of these cases, EtG was positive. In the other 20, EtG was negative, and post-mortem formation of ethanol was therefore suspected. We report case histories, as well as findings of ethanol and EtG in these cases as we wanted to use this information to investigate the chance of false negative results. In 12 of these cases, a urine sample, which contained potassium fluoride as a preservative, was available. These urine samples were subject to analysis of EtG and we hypothesised that since EtG concentrations are much higher in urine [5,16], there could still be traces of EtG left in this medium if post-mortem degradation of EtG was the reason for the negative result in blood.
2. Materials and methods 2.1. In vitro study (summarised in Table 1)
2.3. Analytical methods 2.1.1. Stability without preservatives To simulate the stability of EtG during putrefaction in a corpse before sampling, blood samples were collected from 10 different corpses at forensic autopsies, five without EtG and five containing EtG. No preservatives were added. In the five EtG-negative cases, the absence of ethanol and EtG in blood was verified by analysis before start of the study. To a 5 mL plastic tube, 0.1 mL of a solution containing 44.4 mg EtG/L in water (MEDICHEM1, Steinenbronn, Germany) was added to 2.9 mL blood to a final theoretical concentration of 1.5 mg/L. The concentrations were immediately controlled by analysis and the samples were incubated at 40 8C. In the five EtG-positive cases, the initial ethanol and EtG concentrations were determined and 3 mL blood was transferred to a 5 mL plastic tube. The samples were incubated at 30 8C. Analyses were made at days 0, 3, 6, 11, 15 and 21.
Ethanol was analysed in blood and urine by using headspace gas chromatography equipped with a flame ionisation detector [17]. EtG in blood and urine was analysed using previously published fully validated methods [8,18]. The limit of detection (LOD) for EtG was 0.02 mg/L in blood and 0.09 mg/L in urine.
3. Results 3.1. In vitro study The results of the in vitro studies are summarised in Table 1. In the five samples without preservatives at 40 8C, spiked with EtG, the initial concentrations of EtG ranged between 1.2 and 1.6 mg/ L. This deviation from the theoretical spiked concentration of 1.5 mg/L is, in addition to analytical variations, caused by the character of post-mortem blood, which makes pipetting of exact volumes difficult. The concentrations fell from the initial concentration after 3 days, and by day 21, all samples were negative for EtG (Fig. 1a). In the five samples without preservatives at 30 8C, containing real EtG, the initial concentrations of ethanol were in the range 1.5–4.3 g/L, and of EtG 1.0–6.5 mg/L. The concentrations of EtG started declining from these initial values after 3–6 days. Only one sample was still positive for EtG after 21 days and this showed a decline in concentration from 6.5 to 2.9 mg/L (Fig. 1b). In two of these cases, one single data point (day 3) had to be excluded because of addition of an insufficient amount of internal standard during analysis.
2.1.2. Stability with preservatives To investigate the stability of EtG when stored in the laboratory, real samples arriving for routine toxicological analysis were used. These samples contained 300 mL of an 11.5 M potassium fluoride solution as preservative. Blood samples from four different corpses were selected shortly after arrival at the laboratory and analysed for ethanol and EtG. Three milliliters of blood was transferred to a 5 mL plastic tube and the samples were stored at room temperature (20 8C). Analyses were made at days 0, 2, 7, 10 and 18. 2.1.3. Formation of EtG To investigate the possible formation of EtG, blood from 10 corpses negative for ethanol and EtG were collected, five containing potassium fluoride and five without preservatives. To a 5 mL plastic tube, 60 mL of a solution containing 50 g/L ethanol in water (Merck, Darmstadt, Germany) was added to 3.0 mL blood to a final theoretical concentration of 1.0 g/L. The concentration of ethanol and absence of EtG were controlled by analysis. The five samples without preservatives were incubated at 30 8C and the five samples with Table 1 Summary of in vitro study of the stability and formation of EtG Conditions
Simulation of
N
Origin of EtG/ethanol
Results
Without preservatives, 40 8C Without preservatives, 30 8C With fluoride, room temperature Without preservatives, 30 8C With fluoride, room temperature
Stability during putrefaction (Fig. 1a) Stability during putrefaction (Fig. 1b) Stability during storage (Fig. 2) Formation during putrefaction Formation during storage
5 5 4 5 5
Spiked with EtG Real EtG Real EtG Spiked with ethanol Spiked with ethanol
All samples negative for EtG after 21 days All but one sample negative for EtG after 21 days Concentrations of EtG unchanged during 18 days No formation of EtG during 21 days No formation of EtG during 21 days
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4. Discussion
Fig. 1. (a) Concentrations of EtG in blood in five samples without preservatives spiked with EtG and incubated at 40 8C. (b) Concentrations of EtG in blood in five samples without preservatives containing real EtG incubated at 30 8C.
In the four samples with potassium fluoride at room temperature, containing real EtG, the ethanol concentrations were in the range 2.1–3.0 g/L. The initial concentrations of EtG were in the range 0.8–2.7 mg/L, and there was no change in concentrations from these initial values (Fig. 2). No formation of EtG was observed during 21 days, in samples spiked with ethanol, neither in the five samples without preservatives at 30 8C, nor in the five samples containing potassium fluoride at room temperature. 3.2. Real cases A total of 39 cases were routinely analysed for EtG in blood during a period of 3 months. In 19 of these, EtG was positive, with a median concentration of 1.6 mg/L (range 0.2–34.9). In the other 20 cases, EtG was negative and the ethanol detected was therefore suspected to be of post-mortem origin. These are shown in Table 2 as case 1–20. In 15 of these, a urine sample was available, and the results of analysis of EtG in urine demonstrated that 6 of the 15 cases were positive for EtG in urine.
Fig. 2. . Concentrations of EtG in blood in four samples with potassium fluoride stored at room temperature.
This study showed that EtG in blood samples may be very unstable at high temperatures without preservatives, but stable at room temperature in containers with added potassium fluoride. The present study also strengthened the assumption that no EtG is formed post-mortem. Incubating samples at 30/40 8C without preservatives aimed to simulate putrefaction in a corpse before sample collection. This method had obvious weaknesses, and this study only gave an indication of the problem. The best model would have been to follow the concentrations of EtG in a corpse during putrefaction, but such a procedure was considered unethical. Thus, we cannot exclude that EtG levels would change differently over time in a corpse than in a blood sample. Further, many aspects concerning the blood samples studied were subject to uncertainty, e.g. presence of bacteria. Neither did we know the degree of putrefaction of the blood before sampling. This problem is partly evaded in the part of the study of stability where the samples were spiked with EtG, but since there are previous reports that stability of substances in spiked and real samples may be different [19], we also studied samples containing real EtG. However, it seemed like the stability was equal in both situations. We chose to use two different temperatures in the experiment. This was done to get an impression of the importance of the temperature of the corpse’s environment, and showed that the instability was present at both temperatures. The intervals between the analyses were also shorter in the start of the study period. This design was chosen to discover an eventually very fast decline in concentrations. The instability of EtG in blood without preservatives was more pronounced than previously published [14]. This might be explained by the high temperatures in this study compared to the previous study (30/40 8C versus room temperature). The results in the present study were more comparable with the findings of a relatively high instability of EtG in urine containing bacteria when stored at room temperature without preservatives [15]. For putrefaction, 30 and 40 8C must be considered extreme conditions, above what is usually the case for most corpses in Norway, but possible in more southern countries. This study thus probably illustrated a worst-case scenario, and its aim was not to elucidate the instability of EtG under all possible conditions. Only one sample was still positive for EtG after end of the study at 21 days, and this sample also showed a considerable decline in concentration of EtG. Two weeks after end of the study, this sample was reanalysed, and it was negative for EtG at that time. The stability during storage in containers with preservative was satisfactory. The small variations observed were within analytical variation. This was consistent with the previous study showing stable concentrations of EtG in urines at room temperature when preservative was added [15]. Studies of other analytes, as benzodiazepines, also show better stability when preservative is added [20]. The present work, like a previous study [14], failed to show any formation of EtG from ethanol, neither in a situation without preservatives simulating putrefaction in a corpse, nor
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Table 2 Concentrations of ethanol and EtG in blood and urine plus case history, in 20 cases where a negative EtG in blood led to suspicion of post-mortem formation of ethanol Case no.
Ethanol in blood (g/L)
Ethanol in urine (g/L)
Case history
Time between death and autopsy (days)
EtG in blood (mg/L)
EtG in urine (mg/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
0.3 0.4 0.6 0.3 0.4 0.1 0a 0 0.1 0.2 0.4 0.4b 0.4a 0 0.2a 0.1 1.0c 0.3 0.7c 0.6
0 0.4 0.5 0.5 0.3 0.2 0.1 0.2 0 0.3 0.6 0.2 0.1 0.3 0 N.A 0.8 0.4 N.A N.A
Found dead at home Found dead in apartment Found dead Found dead at home Found dead in bathroom Found dead at home Traffic accident Intoxication Found dead at home Death from dissection of the aorta Dead from tuberculosis Found dead on bathroom floor with heater cables Found dead at home Found dead in apartment, cardiac death Found dead in apartment Drug-abuser found dead at home Found dead under a bridge, larvae-eaten Found dead in the sea Drug-abuser found dead Cardiac death, found with alcohol bottles
18 5 8 14 14 10 2 7 18 6 40 N.A 12 3 18 N.A 30 30 14 18
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0.3 0.3 0.7 2.8 7.5 244 N.A N.A N.A N.A N.A
Time between death and autopsy is based on assumed time of death from the forensic pathologist. Peripheral blood if not otherwise noted. N.A, not available. a Heart blood. b Blood from pericardium. c Pleural fluid.
with potassium fluoride, simulating storage in the laboratory. It therefore seemed that positive EtG results verify ingestion of ethanol. EtG was found in 19 out of 39 of the real cases analysed for EtG in the present study, and was accordingly interpreted as evidence for ante-mortem ethanol intake. A negative result, however, as seen in 20 out of 39 of the real cases (Table 2), might be a false negative due to EtG degradation. Post-mortem urine was available in 15 of these, and in six, we found EtG in urine (Table 2). A possible explanation of this might be that EtG instability during putrefaction had resulted in complete loss of EtG from blood, but still with its presence in urine, where concentrations are higher. Another, less likely explanation is that EtG was totally eliminated from blood, but detectable in urine due to the longer detection time [18,21]. Ethanol was ingested, but might have been totally eliminated and the ethanol detected might have been formed post-mortem. If so, the negative EtG in blood correctly indicated post-mortem formation of ethanol. The remaining nine cases where urine was available had no trace of EtG neither in blood nor urine and the assumption of post-mortem formation of ethanol was therefore more reliable. The analysis of two different media must be considered valuable. However, these urines were not investigated for bacterial contamination. We can therefore not totally exclude false negative results also in these samples. Recently, it has become clear that ethanol also undergoes sulphate conjugation to form ethyl sulphate (EtS) [22]. In a previous study, none of the bacteria that caused deglucuronidation of EtG contained beta-sulphatase, and as expected, the concentrations of EtS did not decrease during storage at room temperature without preservative [15]. There is reason to
believe that the lack of sulphatases in bacteria makes EtS more stable during putrefaction also in post-mortem blood. This remains to be studied, but EtS could for this reason be a valuable supplement to EtG. In conclusion, analysis of EtG in blood is a helpful tool to determine in vivo ingestion of ethanol in post-mortem cases. This study showed that it has a high specificity, but a lower sensitivity in post-mortem cases. Therefore, a positive result probably verifies ingestion of ethanol, while a negative result, especially in heavy putrefied cases, must be interpreted with caution. Analysis of an additional medium, or also of EtS, would be valuable in these cases. Acknowledgement To Professor Sidsel Rogde at the Institute of Forensic Medicine, University if Oslo, for providing samples. Reference [1] G. Schmitt, R. Aderjan, T. Keller, M. Wu, Ethyl glucuronide: an unusual ethanol metabolite in humans. Synthesis, analytical data, and determination in serum and urine, J Anal. Toxicol. 19 (1995) 91–94. [2] R.S. Foti, M.B. Fisher, Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs, Forensic Sci. Int. 153 (2005) 109–116. [3] F.M. Wurst, R. Vogel, K. Jachau, A. Varga, C. Alling, A. Alt, G.E. Skipper, Ethyl glucuronide discloses recent covert alcohol use not detected by standard testing in forensic psychiatric inpatients, Alcohol.-Clin. Exp. Res. 27 (2003) 471–476. [4] K. Borucki, R. Schreiner, J. Dierkes, K. Jachau, D. Krause, S. Westphal, F.M. Wurst, C. Luley, H. Schmidt-Gayk, Detection of recent ethanol intake with new markers: comparison of fatty acid ethyl esters in serum
G. Høiseth et al. / Forensic Science International 176 (2008) 147–151
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
and of ethyl glucuronide and the ratio of 5-hydroxytryptophol to 5hydroxyindole acetic acid in urine, Alcohol.-Clin. Exp. Res. 29 (2005) 781–787. J. Bergstrom, A. Helander, A.W. Jones, Ethyl glucuronide concentrations in two successive urinary voids from drinking drivers: relationship to creatinine content and blood and urine ethanol concentrations, Forensic Sci. Int. 133 (2003) 86–94. H. Dahl, N. Stephanson, O. Beck, A. Helander, Comparison of urinary excretion characteristics of ethanol and ethyl glucuronide, J Anal. Toxicol. 26 (2002) 201–204. T. Sarkola, H. Dahl, C.J. Eriksson, A. Helander, Urinary ethyl glucuronide and 5-hydroxytryptophol levels during repeated ethanol ingestion in healthy human subjects, Alcohol Alcohol. 38 (2003) 347–351. G. Hoiseth, R. Karinen, A.S. Christophersen, L. Olsen, P.T. Normann, J. Morland, A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol, Forensic Sci. Int. 165 (2007) 41–45. L. Morini, L. Politi, A. Zucchella, A. Polettini, Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography–electrospray tandem mass spectrometry, Clin. Chim. Acta 376 (2007) 213–219. L. Politi, L. Morini, F. Leone, A. Polettini, Ethyl glucuronide in hair: is it a reliable marker of chronic high levels of alcohol consumption? Addiction 101 (2006) 1408–1412. M. Yegles, A. Labarthe, V. Auwarter, S. Hartwig, H. Vater, R. Wennig, F. Pragst, Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers, Forensic Sci. Int. 145 (2004) 167–173. F.M. Wurst, R. Schuttler, C. Kempter, S. Seidl, T. Gilg, K. Jachau, A. Alt, Can ethyl glucuronide be determined in post-mortem body fluids and tissues? Alcohol Alcohol. 34 (1999) 262–263. H. Schloegl, T. Rost, W. Schmidt, F.M. Wurst, W. Weinmann, Distribution of ethyl glucuronide in rib bone marrow, other tissues and body liquids as
[14]
[15]
[16]
[17]
[18]
[19] [20] [21]
[22]
151
proof of alcohol consumption before death, Forensic Sci. Int. 156 (2006) 213–218. H. Schloegl, S. Dresen, K. Spaczynski, M. Stoertzel, F.M. Wurst, W. Weinmann, Stability of ethyl glucuronide in urine, post-mortem tissue and blood samples, Int. J. Legal Med. 120 (2006) 83–88. A. Helander, H. Dahl, Urinary tract infection: a risk factor for false-negative urinary ethyl glucuronide but not ethyl sulfate in the detection of recent alcohol consumption, Clin. Chem. 51 (2005) 1728–1730. N. Stephanson, H. Dahl, A. Helander, O. Beck, Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography– mass spectrometry, Ther. Drug Monit. 24 (2002) 645–651. L. Kristoffersen, L.E. Stormyhr, A. Smith-Kielland, Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma, Forensic Sci. Int. 161 (2006) 151–157. G. Hoiseth, J.P. Bernard, R. Karinen, L. Johnsen, A. Helander, A.S. Christophersen, J. Morland, A pharmacokinetic study of ethyl glucuronide in blood and urine: applications to forensic toxicology, Forensic Sci. Int. 172 (2007) 119–124. A.S. Christophersen, Tetrahydrocannabinol stability in whole blood: plastic versus glass containers, J Anal. Toxicol. 10 (1986) 129–131. M.D. Robertson, O.H. Drummer, Stability of nitrobenzodiazepines in postmortem blood, J. Forensic Sci. 43 (1998) 5–8. G. Schmitt, P. Droenner, G. Skopp, R. Aderjan, Ethyl glucuronide concentration in serum of human volunteers, teetotalers, and suspected drinking drivers, J. Forensic Sci. 42 (1997) 1099–1102. F.M. Wurst, S. Dresen, J.P. Allen, G. Wiesbeck, M. Graf, W. Weinmann, Ethyl sulphate: a direct ethanol metabolite reflecting recent alcohol consumption, Addiction 101 (2006) 204–211.