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Ethanol-related death of a child: An unusual case report
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Forensic Science International 179 (2008) e1–e4 www.elsevier.com/locate/forsciint
Case report
Ethanol-related death of a child: An unusual case report Małgorzata Kłys *, Krzysztof Woz´niak, Sebastian Rojek, Ewa Rzepecka-Woz´niak, Piotr Kowalski Institute of Forensic Medicine, Department of Toxicology, Jagiellonian University Collegium Medicum, 16 Grzego´rzecka Street, 31-531 Krakow, Poland Received 10 December 2007; received in revised form 20 March 2008; accepted 24 March 2008 Available online 2 May 2008
Abstract The report describes a fatal outcome in a 5-year-old male who died after drinking a fatal dose of ethanol at the party held by his parents. Urine and blood alcohol level of the deceased was 0.4 and 0.5 g/dL, what might explain the sudden death of the child. In addition, the analysis of the boy’s hair demonstrated the presence of ethyl glucuronide (EtG), a marker of alcohol consumption; hair EtG concentration levels indicated that the boy might have occasionally imbibed alcohol prior to death. Pathological lesions of the liver observed in histopathology did not contradict such a hypothesis. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethanol; Ethyl glucuronide; Fatal ethanol poisoning; Child death; Hair analysis
1. Introduction As it has been proven by medico-legal practice, deaths of children and adolescents always evoke special interest and well-founded concerns [1–4]. In the archives of the Krakow Department of Forensic Medicine, hundreds of violent deaths of children aged from infancy to 18 years of life were registered each year [2]. These cases predominantly resulted from accidents, but the material also includes suicidal deaths. Although for many years, alcohol has been in Poland the leading poisonous substance involved in a considerable number of fatalities [5–7], it has to be admitted that the problem affects mainly adults. In the archival materials of the Institute of Forensic Medicine, Collegium Medicum, Jagiellonian University, Krakow, only isolated cases of fatal ethanol poisoning in older adolescents were registered. As it follows from data originating from pediatric sources [8–12], fatal poisonings in young children, including cases of ethanol poisoning, are relatively rare. This is why an accidental death of a 5-year-old boy in consequence of having ingested a known dose of alcohol, which is the subject of the present report, should be regarded as an
* Corresponding author: Tel.: +48 12 421 11 13; fax: +48 12 421 11 13. E-mail address: [email protected] (M. Kłys). 0379-0738/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2008.03.017
unusual case. The entire picture is supplemented with the analysis of the boy’s hair for detection of ethyl glucuronide (EtG), a marker of alcohol consumption, as a factor indicating intravital contacts of the child with alcohol. 2. Case report A 5-year-old boy participated in a party thrown by his parents. According to the supplied information, the child have drunk 120–150 g of vodka containing 40% of alcohol (50– 60 g of pure ethanol) in the evening. At that time, clear signs indicating state of inebriation were observed. These symptoms did not, however, alarm the boy’s parents, since, as it followed from the testimony of witnesses, such episodes had occurred in the past, ‘‘without any serious consequences’’. This time, however, the situation developed differently. The child was found dead in his bed in the morning of the following day. 3. Materials and methods 3.1. Biological materials Postmortem specimens were collected at autopsy of the victim, which was performed at the Institute of Forensic Medicine, Collegium Medicum, Jagiellonian University in Krakow, within 24 h following death.
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The samples were as follows: femoral vein blood, urine and a 4-cm long hair strand. Hair samples (without bulbs) were collected from the area at the back of the head with the use of a razor. Control autopsy blood, urine and hair samples (‘‘blank’’ samples) – free from any xenobiotics – were taken from non-poisoned subjects for analytical purposes.
3.2. Chemicals and reagents All chemicals and solvents were of the analytical grade. Standards of ethanol in water were obtained from Restek (Bellefonte, USA), tert-butanol was provided by Merck (Darmstadt, Germany), ethyl glucuronide and ethyl glucuronide-D5 (ethyl-D5-b-D-6-glucuronic acid) were purchased from Medichem (Stuttgart, Germany). Acetonitrile, acetone, ethyl acetate, ethanol, methanol and hexane were from Merck (Dramstadt, Germany). Formic acid, ammonium acetate and ammonium carbonate were from Riedel-de Hae¨n (Seelze, Germany), tris(hydroxymethyl)-aminomethane from SERVA (Heidelberg, Germany). Solid-phase extraction (SPE) cartridges, LiChrolut RP-18 (200 mg) and LiChrolut SPE-NH2 (200 mg), were supplied by Merck (Darmstadt, Germany).
3.3. Analytical procedure
3.3.1. Alcohol determination in blood and urine samples Blood and urine were diluted with 2 g/dL solution of tert-butanol (v/v, 1:3) and subjected to ‘‘head-space’’ analysis. The analysis was carried out with the use of a GC/FID Trace GC Ultra (ThermoElectron, USA) equipped with a Rtx1-BAC1 column (30 m, 0.53 mm ID, 3.0 mm film thickness) from Restek and initially using the temperature program starting at 40 8C, held for 4 min, then increasing temperature to 200 8C at 25 8C/1 min, and maintaining the process for 1 min. Flow carrier gas (helium) was 2.0 mL/min. Quantitative evaluation was carried out against alcohol standards at concentrations in the range of 0.015–0.5 g/dL.
3.4. Analysis of hair samples
3.4.1. Extraction from hair Decontamination of hair was carried out with the use of water, afterwards with acetone and methanol on an ultrasonic bath for 10 min; finally, the hair was dried. The hair samples (4 cm) were divided into two approximately 2-cm long segments. Two 2-cm long hair segments were pulverized in a mill-ball (Retsch MM 200, Germany) and 50 mg of homogenous aliquots were analyzed. To the samples, 2 mg/g ethyl glucuronide-D5 (internal standard), 1.5 mL of water and 3.5 mL acetonitrile were added and the specimens were incubated at 45 8C for 12 h, then put on an ultrasonic bath for 1 h. Afterwards, 100 mL of 0.1 M HCL was added to the samples, and the material was centrifuged at 2000 g for 15 min. The clear supernatant was applied to an aminopropyl column SPE-NH2 conditioned with methanol (3 mL), water (3 mL) and acetonitrile (3 mL). The cartridge was then washed with 3 mL of hexane and dried for 10 min. Elution was carried out with the use of a mixture of methanol/25% ammonia (98:2, v/v). The eluent was evaporated to dryness using a stream of nitrogen. The dried residue was reconstituted in 50 mL of 0.1% formic acid, 20 mL of the reconstituted extract was injected into the chromatographic system.
3.5. Calibration curves and quantitation Calibration curves were constructed after the analysis of 50 mg samples of drug-free hair (blank samples) containing known amounts of ethyl glucuronide. To prepare these standards, hair samples were spiked with the studied compound to the following concentrations: 25, 50, 100, 200, 500, 1000, 2000 and 5000 pg/mg. Ethyl glucuronide-D5 at the concentration of 2000 pg/mg was used. The samples were extracted according to the procedure described above. Calibration curves were constructed by plotting the peak area ratios of ethyl glucuronide/internal standard. Results of the validation procedure for hair determinations are presented in Table 1.
3.6. Instrumentation 3.6.1. Liquid chromatography A Finnigan MAT liquid chromatograph (San Jose, CA, USA) equipped with a pump Model TSP 4000 and an autosampler Model TSP AS 3000 with a 20 mL injection loop were used in gradient mode. The chromatographic separation was performed with a LiChroCART column (125 mm 3 mm i.d., 5 mm particle size) filled with Purospher RP 18 and a LiChroCART precolumn 4 mm 4 mm i.d., 5 mm particle size) filled with LiChrospher 60 RP-select B (Merck, Darmstadt, Germany). The mobile phases were [A], which was 0.1% formic acid in water, and [B], which was 95% acetonitrile + 5% of the phase [A]. The flow rate was 0.4 mL/min. The gradient for samples concerning ethyl glucuronide was programmed as follows: 95% [A] and 5% [B] for 1 min, followed by a linear change to 5% [A] and 95% [B] in 9 min, then 5% [A] and 95%[ B] was held for 3 min, then by a linear change to 95% [A] and 5% [B] for 2 min, then 95% [A] and 5% [B] was held for 5 min.
3.7. Detection by mass spectrometry A LCQ mass detector, ion trap (Finnigan MAT) equipped with an APCI source was used. The APCI inlet conditions were as follows: Sheath gas (nitrogen) pressure 0.9 L/min, auxiliary gas (helium) 0.15 L/min, heated vaporizer temperature 400 8C, heated capillary temperature 150 oC, discharge current 5 mA. Tandem mass spectrometric analysis was performed for EtG and EtG-D5. The surviving parent ions (EtG, m/z 221 ! m/z 221; EtG-D5, m/z 226 ! m/z 226; relative collision energy 3%) were monitored in negative-ion mode. Detailed analytical procedures were published by Kłys et al. [13].
4. Results Autopsy of the corpse with a body length of 118 cm and body mass of approximately 20 kg demonstrated the presence of the following pathologies: pulmonary edema and congestion of internal organs, a single subepicardial petechia and disseminated, small foci of increased transparency in the parenchyma of the right hepatic lobe, as well as macrovesicular dispersed steatosis of the liver. Histopathological examinations of sections collected from the brain, cardiac muscle, lungs, kidneys and liver showed pulmonary edema, massive congestion of all the internal
Table 1 Validation parameters for ethyl glucuronide in hair Analyte n = 5
Ethyl glucuronide
LOD (pg/mg)
15
LLOQ (pg/mg)
25
LOL (pg/mg)
25–5000
r2
0.9992
Recovery (% S.D.)
Intraday precision (R.S.D., %)
Interday precision (R.S.D., %)
200 pg/ mg
2000 pg/ mg
200 pg/ mg
2000 pg/ mg
200 pg/ mg
2000 pg/ mg
86.4 3.7
91.9 2.8
10.3
4.9
13.7
8.6
M. Kłys et al. / Forensic Science International 179 (2008) e1–e4
e3
Table 2 Toxicological findings in the child Xenobiotic
Ethanol Ethyl glucuronide
Blood (g/dL)
0.4 –
Urine (g/dL)
Hair (pg/mg)
0.5 –
organs, as well as macrovesicular steatosis of the liver confirmed by Sudan III staining. Blood and urine toxicology demonstrated the presence of alcohol, 0.4 and 0.5 g/dL, respectively. Segmental analysis of a hair strand with a total length of 4 cm showed ethyl glucuronide being present in both analyzed 2-cm long segments; its concentration levels indicated probable consumption of alcohol. The method of determination of EtG in hair was validated and some data are presented in Table 1. The results of toxicological findings are presented in Table 2. 5. Discussion The toxicological results revealed a high alcohol level in the child’s blood and urine, indicating that death had occurred during the phase of alcohol elimination. In view of the generally accepted principles of medico-legal investigation [14,15], the blood alcohol level determined in the deceased child (0.4 g/dL) justifies the verdict of a sudden death in consequence of alcohol inebriation. The analysis of data from the case history, which reports the child as having drunk approximately 50–60 g of alcohol over a short period suggested that alcohol consumed in such an amount must have constituted a lethal dose for the boy in question. Ethyl alcohol, which constitutes approximately 0.5–1.5% of the total alcohol consumed, is coupled with glucuronic acid in a phase II reaction, thus forming ethyl glucuronide [14,16,17]. The determination of EtG levels represents a useful forensic tool, as well as a possible method for monitoring alcohol consumption of patients being treated for alcoholism [18–21]. Hair levels of ethyl glucuronide in alcohol-consuming individuals demonstrate a broad range of values, depending on the frequency of alcohol intake. Following the suggestion of Appenzeller et al. [22], a social drinker is defined as an individual consuming 11–40 g of alcohol daily, while a heavy drinker exceeds a daily dose of 60 g of alcohol. As it was proven by the studies carried out by these investigators, the concentration of EtG measured in hair and the daily alcohol intake were highly significantly correlated. Using the equation of the curve fitting EtG concentration in hair vs. alcohol consumption, the authors calculated the following theoretical cut-off value: 4–15 pg/mg EtG for social drinkers and a concentration above 23 pg/mg for heavy drinkers. In turn, Pragst and Balikova [23], as well as Yegles et al. [17] suggested that detection of EtG in hair in the amount above 30 pg/mg could be a strong evidence for alcohol abuse; in this group of subjects, the researchers obtained very high values, in the range of EtG concentrations between 30 and 13,300 pg/mg. At the same time, these authors pointed to the fact that a
I segment proximal
II segment distal
– 46
– 54
negative hair EtG result did not completely exclude alcohol abuse, because also in many cases no EtG was detected in some hair samples obtained from alcoholics. Hair EtG analysis in alcohol abusers was subject to research carried out in previous years also by other investigators [13,16,18,24]. Their results do not show drastic differences in hair EtG concentration values, although classification of particular individuals as heavy drinkers or social drinkers based on their hair EtG content may – especially in the case of borderline values – suggest some, albeit slight, differences. Based on the criteria proposed by Appenzeller et al. [22], Pragst and Balikova [23], and Yegles et al. [17], the level of ethyl glucuronide ranging between 44 and 54 pg/mg found at autopsy in the hair of the deceased child allows for classifying the boy as a heavy drinker. Some indications on the possible antemortem alcohol consumption by the child have been obtained from histopathology of the liver. Although the observed lesions are not markedly characteristic, they do not contradict the hypothesis on alcohol abuse by the child. Steatosis demonstrated in the postmortem examination of liver sections indicates the presence of a toxic factor, which might have triggered such lesions. As it follows from reports of various investigators [25,26], fatty degeneration of the liver observed in cases associated with alcohol consumption and abuse could not have developed following a single instance of alcohol intake, even in a lethal dose. The unique character of the case analyzed in the present report lies in a comprehensive determination – based on postmortem toxicology and histopathology, as well as on detection of ethyl glucuronide in hair – that the child consumed alcohol on numerous occasions. In view of the presented material, it seems likely that such a comprehensive analysis of an alcohol-associated death in a young child has not been reported to date. Thus, the case may constitute a contribution to the casuistry of unusual cases of fatal ethanol poisonings. Acknowledgements The authors gratefully acknowledge M. Sc, W. Klementowicz for their assistance and contribution to this work. References [1] M. Kotwica, A. Jarosz, Z. Kołacin´ski, Sources of poisoning exposures in children during 1990–1995: an analysis of the National Poison Information Centre files, Int. J. Occup. Med. Environ. Health 10 (1997) 177–180. [2] M. Kłys, Fatal poisonings in the medico-legal aspect with special attention of children and young people in the material of the Institute of Forensic Medicine in Cracow in the years 1998–2002, Przeg. Lek. 60 (11) (2003) 762–767.
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[3] H. Wiseman, K. Guest, V.S.G. Murray, G.N. Volans, Accidental poisoning in childhood: a multicentre survey. 1. General epidemiology, Hum. Toxicol. 6 (1987) 293–301. [4] A.G. Marchi, G. Messi, S. Renier et all, The risk associated with poisonings in children, Vet. Hum. Toxicol. 36 (1994) 112–116. [5] M. Kłys, E. Baran, Fatal poisonings in the material of the Institute of Forensic Medicine in Krako´w in the years 1946–1995, Arch. Med Sa˛d i Krym. 46 (1996) 297–307. [6] K. Sancewicz-Pach, A. Kamen´czak, E. Klag, M. Kłys, Acute poisonings with chemical compounds among adolescent and adult inhabitants of Krako´w in the year 1997, Przeg. Lek. 56 (6) (1999) 409–414. [7] D. Targosz, K. Sancewicz-Pach, B. Szkolnicka, M. Kłys, Chemical poisonings among adult and adolescent Krako´w inhabitants in 1972 and 2002, Przeg. Lek. 61 (4) (2004) 251–255. [8] M. Kotwica, A. Rogaczewska, Causes of poisoning in children during the period 1990–1993, Przeg. Lek. 53 (1996) 329–333. [9] D. Targosz, K. Sancewicz-Pach, B. Szkolnicka, M. Mikrus, M. Kłys, Frequency and pattern of poisoning in adult and adolescent Krako´w population in 2003 and 1983, Przeg. Lek. 62 (2005) 446–452. [10] U. Engqvist, P.A. Rydeluis, Death and suicide among former child and adolescent psychiatric patients, BMC Psychiatr. 2 (2006) 40–51. [11] C. Ulmeanu, V.G. Nitescu Girnita, Mortality rate in acute poisoning in a pediatric toxicology department, Przeg Lek. 62 (6) (2005) 453–455. [12] F. Sharif, R.A. Khan, P. Keenan, Poisoning in a pediatric hospital, J. Med. Sci. 172 (2) (2003) 78–80. [13] M. Kłys, M. S´cislowski, S. Rojek, J. Kołodziej, A fatal clomipramine intoxication case of a chronic alcoholic patient: application of postmortem hair analysis method of clomipramine and ethyl glucuronide using LC/ APCI/MS, Legal Med. 7 (2005) 319–325. [14] R.C. Baselt, Disposition of Toxic Drugs and Chemicals in Man, 5th ed., Chemical Toxicology Institute, Foster City, CA, 2000, pp. 323–326.
[15] A.C. Moffat, M.D. Osselton, B. Widdop, Clarke’s Analysis of Drugs and Poisons in Pharmaceuticals, Body Fluids and Postmortem Material, 3rd ed., Pharmaceutical Press, London, Chicago, 2004, pp. 991–992. [16] I. Janda, W. Weinmann, T. Kuehnle, M. Lahode, A. Alt, Determination of ethyl glucuronide in human hair by SPE and LC–MS/MS, Forensic Sci. Int. 128 (2002) 59–65. [17] 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 teetotalers, Forensic Sci. Int. 145 (2004) 167–173. [18] G. Skopp, G. Schmitt, L. Potsch, P. Drooner, R. Aderjan, R. Mattern, Ethyl glucuronide in human hair, Alcohol 35 (2000) 283–285. [19] F. Pragst, K. Spiegel, F. Sporkert, M. Bohnenkamp, Are the possibilities for the detection of chronically elevated alcohol consumption by hair analysis ? A report about the state of investigation, Forensic Sci. Int. 107 (2000) 201–223. [20] S. Seidl, A. Wurst, A. Alt, Ethyl glucuronide—a biological marker for recent alcohol consumption, Addict. Biol. 6 (2001) 205–212. [21] F. Musshoff, T. Daldrup, Determination of biological markers for alcohol abuse, J. Chromatogr. B 713 (1998) 245–264. [22] B.M. Appenzeller, R. Aqirman, P. Neuberg, M. Yegles, R. Wennig, Segmental determination of ethyl glucuronide in hair: a pilot study, Forensic Sci. Int. 173 (2007) 87–89. [23] F. Pragst, M. Balikova, State of the art in hair analysis for detection of drug and alcohol abuse, Clin. Chim. Acta 370 (2006) 17–49. [24] A. Alt, I. Janda, S. Seidl, F.M. Wurst, Determination of ethyl glucuronide in hair samples, Alcohol 35 (2000) 313–314. [25] C.S. Lieber, Ethanol metabolism, cirrhosis and alcoholism, Clin. Chim. Acta 257 (1997) 59–84. [26] M.B. Gatch, H. Lal, Pharmacological treatment of alcoholism, Prog. Neuro-Psychopharmacol. Biol. Psychiatry 22 (1998) 917–944.
Forensic Science International 179 (2008) e1–e4 www.elsevier.com/locate/forsciint
Case report
Ethanol-related death of a child: An unusual case report Małgorzata Kłys *, Krzysztof Woz´niak, Sebastian Rojek, Ewa Rzepecka-Woz´niak, Piotr Kowalski Institute of Forensic Medicine, Department of Toxicology, Jagiellonian University Collegium Medicum, 16 Grzego´rzecka Street, 31-531 Krakow, Poland Received 10 December 2007; received in revised form 20 March 2008; accepted 24 March 2008 Available online 2 May 2008
Abstract The report describes a fatal outcome in a 5-year-old male who died after drinking a fatal dose of ethanol at the party held by his parents. Urine and blood alcohol level of the deceased was 0.4 and 0.5 g/dL, what might explain the sudden death of the child. In addition, the analysis of the boy’s hair demonstrated the presence of ethyl glucuronide (EtG), a marker of alcohol consumption; hair EtG concentration levels indicated that the boy might have occasionally imbibed alcohol prior to death. Pathological lesions of the liver observed in histopathology did not contradict such a hypothesis. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Ethanol; Ethyl glucuronide; Fatal ethanol poisoning; Child death; Hair analysis
1. Introduction As it has been proven by medico-legal practice, deaths of children and adolescents always evoke special interest and well-founded concerns [1–4]. In the archives of the Krakow Department of Forensic Medicine, hundreds of violent deaths of children aged from infancy to 18 years of life were registered each year [2]. These cases predominantly resulted from accidents, but the material also includes suicidal deaths. Although for many years, alcohol has been in Poland the leading poisonous substance involved in a considerable number of fatalities [5–7], it has to be admitted that the problem affects mainly adults. In the archival materials of the Institute of Forensic Medicine, Collegium Medicum, Jagiellonian University, Krakow, only isolated cases of fatal ethanol poisoning in older adolescents were registered. As it follows from data originating from pediatric sources [8–12], fatal poisonings in young children, including cases of ethanol poisoning, are relatively rare. This is why an accidental death of a 5-year-old boy in consequence of having ingested a known dose of alcohol, which is the subject of the present report, should be regarded as an
* Corresponding author: Tel.: +48 12 421 11 13; fax: +48 12 421 11 13. E-mail address: [email protected] (M. Kłys). 0379-0738/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2008.03.017
unusual case. The entire picture is supplemented with the analysis of the boy’s hair for detection of ethyl glucuronide (EtG), a marker of alcohol consumption, as a factor indicating intravital contacts of the child with alcohol. 2. Case report A 5-year-old boy participated in a party thrown by his parents. According to the supplied information, the child have drunk 120–150 g of vodka containing 40% of alcohol (50– 60 g of pure ethanol) in the evening. At that time, clear signs indicating state of inebriation were observed. These symptoms did not, however, alarm the boy’s parents, since, as it followed from the testimony of witnesses, such episodes had occurred in the past, ‘‘without any serious consequences’’. This time, however, the situation developed differently. The child was found dead in his bed in the morning of the following day. 3. Materials and methods 3.1. Biological materials Postmortem specimens were collected at autopsy of the victim, which was performed at the Institute of Forensic Medicine, Collegium Medicum, Jagiellonian University in Krakow, within 24 h following death.
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The samples were as follows: femoral vein blood, urine and a 4-cm long hair strand. Hair samples (without bulbs) were collected from the area at the back of the head with the use of a razor. Control autopsy blood, urine and hair samples (‘‘blank’’ samples) – free from any xenobiotics – were taken from non-poisoned subjects for analytical purposes.
3.2. Chemicals and reagents All chemicals and solvents were of the analytical grade. Standards of ethanol in water were obtained from Restek (Bellefonte, USA), tert-butanol was provided by Merck (Darmstadt, Germany), ethyl glucuronide and ethyl glucuronide-D5 (ethyl-D5-b-D-6-glucuronic acid) were purchased from Medichem (Stuttgart, Germany). Acetonitrile, acetone, ethyl acetate, ethanol, methanol and hexane were from Merck (Dramstadt, Germany). Formic acid, ammonium acetate and ammonium carbonate were from Riedel-de Hae¨n (Seelze, Germany), tris(hydroxymethyl)-aminomethane from SERVA (Heidelberg, Germany). Solid-phase extraction (SPE) cartridges, LiChrolut RP-18 (200 mg) and LiChrolut SPE-NH2 (200 mg), were supplied by Merck (Darmstadt, Germany).
3.3. Analytical procedure
3.3.1. Alcohol determination in blood and urine samples Blood and urine were diluted with 2 g/dL solution of tert-butanol (v/v, 1:3) and subjected to ‘‘head-space’’ analysis. The analysis was carried out with the use of a GC/FID Trace GC Ultra (ThermoElectron, USA) equipped with a Rtx1-BAC1 column (30 m, 0.53 mm ID, 3.0 mm film thickness) from Restek and initially using the temperature program starting at 40 8C, held for 4 min, then increasing temperature to 200 8C at 25 8C/1 min, and maintaining the process for 1 min. Flow carrier gas (helium) was 2.0 mL/min. Quantitative evaluation was carried out against alcohol standards at concentrations in the range of 0.015–0.5 g/dL.
3.4. Analysis of hair samples
3.4.1. Extraction from hair Decontamination of hair was carried out with the use of water, afterwards with acetone and methanol on an ultrasonic bath for 10 min; finally, the hair was dried. The hair samples (4 cm) were divided into two approximately 2-cm long segments. Two 2-cm long hair segments were pulverized in a mill-ball (Retsch MM 200, Germany) and 50 mg of homogenous aliquots were analyzed. To the samples, 2 mg/g ethyl glucuronide-D5 (internal standard), 1.5 mL of water and 3.5 mL acetonitrile were added and the specimens were incubated at 45 8C for 12 h, then put on an ultrasonic bath for 1 h. Afterwards, 100 mL of 0.1 M HCL was added to the samples, and the material was centrifuged at 2000 g for 15 min. The clear supernatant was applied to an aminopropyl column SPE-NH2 conditioned with methanol (3 mL), water (3 mL) and acetonitrile (3 mL). The cartridge was then washed with 3 mL of hexane and dried for 10 min. Elution was carried out with the use of a mixture of methanol/25% ammonia (98:2, v/v). The eluent was evaporated to dryness using a stream of nitrogen. The dried residue was reconstituted in 50 mL of 0.1% formic acid, 20 mL of the reconstituted extract was injected into the chromatographic system.
3.5. Calibration curves and quantitation Calibration curves were constructed after the analysis of 50 mg samples of drug-free hair (blank samples) containing known amounts of ethyl glucuronide. To prepare these standards, hair samples were spiked with the studied compound to the following concentrations: 25, 50, 100, 200, 500, 1000, 2000 and 5000 pg/mg. Ethyl glucuronide-D5 at the concentration of 2000 pg/mg was used. The samples were extracted according to the procedure described above. Calibration curves were constructed by plotting the peak area ratios of ethyl glucuronide/internal standard. Results of the validation procedure for hair determinations are presented in Table 1.
3.6. Instrumentation 3.6.1. Liquid chromatography A Finnigan MAT liquid chromatograph (San Jose, CA, USA) equipped with a pump Model TSP 4000 and an autosampler Model TSP AS 3000 with a 20 mL injection loop were used in gradient mode. The chromatographic separation was performed with a LiChroCART column (125 mm 3 mm i.d., 5 mm particle size) filled with Purospher RP 18 and a LiChroCART precolumn 4 mm 4 mm i.d., 5 mm particle size) filled with LiChrospher 60 RP-select B (Merck, Darmstadt, Germany). The mobile phases were [A], which was 0.1% formic acid in water, and [B], which was 95% acetonitrile + 5% of the phase [A]. The flow rate was 0.4 mL/min. The gradient for samples concerning ethyl glucuronide was programmed as follows: 95% [A] and 5% [B] for 1 min, followed by a linear change to 5% [A] and 95% [B] in 9 min, then 5% [A] and 95%[ B] was held for 3 min, then by a linear change to 95% [A] and 5% [B] for 2 min, then 95% [A] and 5% [B] was held for 5 min.
3.7. Detection by mass spectrometry A LCQ mass detector, ion trap (Finnigan MAT) equipped with an APCI source was used. The APCI inlet conditions were as follows: Sheath gas (nitrogen) pressure 0.9 L/min, auxiliary gas (helium) 0.15 L/min, heated vaporizer temperature 400 8C, heated capillary temperature 150 oC, discharge current 5 mA. Tandem mass spectrometric analysis was performed for EtG and EtG-D5. The surviving parent ions (EtG, m/z 221 ! m/z 221; EtG-D5, m/z 226 ! m/z 226; relative collision energy 3%) were monitored in negative-ion mode. Detailed analytical procedures were published by Kłys et al. [13].
4. Results Autopsy of the corpse with a body length of 118 cm and body mass of approximately 20 kg demonstrated the presence of the following pathologies: pulmonary edema and congestion of internal organs, a single subepicardial petechia and disseminated, small foci of increased transparency in the parenchyma of the right hepatic lobe, as well as macrovesicular dispersed steatosis of the liver. Histopathological examinations of sections collected from the brain, cardiac muscle, lungs, kidneys and liver showed pulmonary edema, massive congestion of all the internal
Table 1 Validation parameters for ethyl glucuronide in hair Analyte n = 5
Ethyl glucuronide
LOD (pg/mg)
15
LLOQ (pg/mg)
25
LOL (pg/mg)
25–5000
r2
0.9992
Recovery (% S.D.)
Intraday precision (R.S.D., %)
Interday precision (R.S.D., %)
200 pg/ mg
2000 pg/ mg
200 pg/ mg
2000 pg/ mg
200 pg/ mg
2000 pg/ mg
86.4 3.7
91.9 2.8
10.3
4.9
13.7
8.6
M. Kłys et al. / Forensic Science International 179 (2008) e1–e4
e3
Table 2 Toxicological findings in the child Xenobiotic
Ethanol Ethyl glucuronide
Blood (g/dL)
0.4 –
Urine (g/dL)
Hair (pg/mg)
0.5 –
organs, as well as macrovesicular steatosis of the liver confirmed by Sudan III staining. Blood and urine toxicology demonstrated the presence of alcohol, 0.4 and 0.5 g/dL, respectively. Segmental analysis of a hair strand with a total length of 4 cm showed ethyl glucuronide being present in both analyzed 2-cm long segments; its concentration levels indicated probable consumption of alcohol. The method of determination of EtG in hair was validated and some data are presented in Table 1. The results of toxicological findings are presented in Table 2. 5. Discussion The toxicological results revealed a high alcohol level in the child’s blood and urine, indicating that death had occurred during the phase of alcohol elimination. In view of the generally accepted principles of medico-legal investigation [14,15], the blood alcohol level determined in the deceased child (0.4 g/dL) justifies the verdict of a sudden death in consequence of alcohol inebriation. The analysis of data from the case history, which reports the child as having drunk approximately 50–60 g of alcohol over a short period suggested that alcohol consumed in such an amount must have constituted a lethal dose for the boy in question. Ethyl alcohol, which constitutes approximately 0.5–1.5% of the total alcohol consumed, is coupled with glucuronic acid in a phase II reaction, thus forming ethyl glucuronide [14,16,17]. The determination of EtG levels represents a useful forensic tool, as well as a possible method for monitoring alcohol consumption of patients being treated for alcoholism [18–21]. Hair levels of ethyl glucuronide in alcohol-consuming individuals demonstrate a broad range of values, depending on the frequency of alcohol intake. Following the suggestion of Appenzeller et al. [22], a social drinker is defined as an individual consuming 11–40 g of alcohol daily, while a heavy drinker exceeds a daily dose of 60 g of alcohol. As it was proven by the studies carried out by these investigators, the concentration of EtG measured in hair and the daily alcohol intake were highly significantly correlated. Using the equation of the curve fitting EtG concentration in hair vs. alcohol consumption, the authors calculated the following theoretical cut-off value: 4–15 pg/mg EtG for social drinkers and a concentration above 23 pg/mg for heavy drinkers. In turn, Pragst and Balikova [23], as well as Yegles et al. [17] suggested that detection of EtG in hair in the amount above 30 pg/mg could be a strong evidence for alcohol abuse; in this group of subjects, the researchers obtained very high values, in the range of EtG concentrations between 30 and 13,300 pg/mg. At the same time, these authors pointed to the fact that a
I segment proximal
II segment distal
– 46
– 54
negative hair EtG result did not completely exclude alcohol abuse, because also in many cases no EtG was detected in some hair samples obtained from alcoholics. Hair EtG analysis in alcohol abusers was subject to research carried out in previous years also by other investigators [13,16,18,24]. Their results do not show drastic differences in hair EtG concentration values, although classification of particular individuals as heavy drinkers or social drinkers based on their hair EtG content may – especially in the case of borderline values – suggest some, albeit slight, differences. Based on the criteria proposed by Appenzeller et al. [22], Pragst and Balikova [23], and Yegles et al. [17], the level of ethyl glucuronide ranging between 44 and 54 pg/mg found at autopsy in the hair of the deceased child allows for classifying the boy as a heavy drinker. Some indications on the possible antemortem alcohol consumption by the child have been obtained from histopathology of the liver. Although the observed lesions are not markedly characteristic, they do not contradict the hypothesis on alcohol abuse by the child. Steatosis demonstrated in the postmortem examination of liver sections indicates the presence of a toxic factor, which might have triggered such lesions. As it follows from reports of various investigators [25,26], fatty degeneration of the liver observed in cases associated with alcohol consumption and abuse could not have developed following a single instance of alcohol intake, even in a lethal dose. The unique character of the case analyzed in the present report lies in a comprehensive determination – based on postmortem toxicology and histopathology, as well as on detection of ethyl glucuronide in hair – that the child consumed alcohol on numerous occasions. In view of the presented material, it seems likely that such a comprehensive analysis of an alcohol-associated death in a young child has not been reported to date. Thus, the case may constitute a contribution to the casuistry of unusual cases of fatal ethanol poisonings. Acknowledgements The authors gratefully acknowledge M. Sc, W. Klementowicz for their assistance and contribution to this work. References [1] M. Kotwica, A. Jarosz, Z. Kołacin´ski, Sources of poisoning exposures in children during 1990–1995: an analysis of the National Poison Information Centre files, Int. J. Occup. Med. Environ. Health 10 (1997) 177–180. [2] M. Kłys, Fatal poisonings in the medico-legal aspect with special attention of children and young people in the material of the Institute of Forensic Medicine in Cracow in the years 1998–2002, Przeg. Lek. 60 (11) (2003) 762–767.
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