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Caveats against an improper use of hair testing to support the diagnosis of chronic excessive alcohol consumption, following the “Consensus” of the Society of Hair Testing 2009 [Forensic Science International 196 (2010) 2]
Forensic Science International 207 (2011) e69–e70
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Letter to the Editor Caveats against an improper use of hair testing to support the diagnosis of chronic excessive alcohol consumption, following the ‘‘Consensus’’ of the Society of Hair Testing 2009 [Forensic Science International 196 (2010) 2]
Sir, The reason for the present letter to the Editor lies in the recent publication [Forensic Science International 196 (2010) 2] of a ‘‘Consensus’’ of the Society of Hair Testing (SoHT), adopted on June 16, 2009 in Rome on the use of hair testing for ‘‘objectively’’ inferring chronic excessive consumption of alcohol. The effort made by the SoHT is certainly appreciable, since it aims to face a problem of great importance to clinical and forensic toxicologists, i.e. the diagnosis of excessive ethyl alcohol consumption, nosographically classifiable as abuse or dependence. This is an interdisciplinary, difficult and delicate issue, about which a lot has already been written and, this notwithstanding, is still debated. This being so, in the light of our personal clinical and laboratory experience, we reviewed the case series of our institutes and the literature on the topic to analyse the experimental data and scientific evidence in the light of the 17 points detailed and shared by the SoHT (evidence that was not cited in the references of the above-mentioned publication). Fifty-three scientific works listed in the MedLine database – search criteria: ‘‘(EtG OR Ethyl glucuronide) AND (hair OR hair testing) – discuss the measurement of EtG in a keratin matrix (10 of them are reviews), while the studies on the measurement of FAEE in the hair – search criteria: ‘‘(FAEE OR fatty acid ethyl esters) AND (hair OR hair testing) – amount to 38 (8 of which are reviews). Among these studies, to the best of our knowledge, only 14 present data on EtG (10 studies) and/or FAEE (five studies) concentrations in the keratin matrix of ‘‘normal subjects’’ (i.e. socially acceptable alcohol users) [1–14]. It is also worth emphasising that the total number of ‘‘normal subjects’’ analysed in these studies amount to only 144 individuals. In the light of the theoretical assumptions and experimental findings discussed below, the inter-individual variation can be expected to be very considerable, and therefore should be well ascertained preliminarily to any effort to indentify cut-off concentrations capable of ensuring a high sensitivity and specificity of diagnosis of chronic alcohol abuse. It is therefore particularly important to emphasise the following points: 1. The enzymatic transformation of ethanol into EtG and FAEE occurs in a very small fraction, accounting for less than 1% of the total intake. This means that inter-individual variability and even intra-individual conditions (enzyme induction) may lead to important differences in the formation of the two metabolites 0379-0738/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2010.10.005
and consequently in their concentrations in the hair matrix of individuals ingesting the same amounts of alcohol. 2. The proposed mechanism of an acidic, water-soluble EtG compound becoming embedded in the hair matrix, mainly via perspiration [15], has been shown to be extremely inefficient, given the pg/mg amounts of EtG that are determined in the hair matrix by comparison with the concentrations measured in sweat, which range from 1.7 to 103 ng/mL [16]. This leads to the consideration that the well-known inter-individual differences in sweating may give rise to a different incorporation of EtG in the hair. On the other hand, the high water solubility of EtG may lead to an easy removal of this metabolite from the hair matrix with normal hair washing, representing another cause of variability; this consideration is also supported by the easy extraction of EtG from hair samples (with incubation and sonication in water) applied in most analytical procedures. 3. In the case of FAEE, which are very lipophilic compounds, the proposed mechanism of its embedding in the hair matrix is mainly via sebum [15]. Here again, the well-known considerable inter-individual differences in sebum production from the scalp may lead to a different incorporation of FAEE in the hair. 4. The non-homogeneous and non-synchronous growth of scalp hair may also reasonably be responsible for a marked intraindividual variability in the concentrations of both EtG and FAEE in the hair. It should be also pointed out that, only after many years of debate, hair analysis for controlled/illicit drugs has been accepted in the last decade as a marker of past chronic use or ‘‘exposure’’ to these compounds. This acceptance came after extensive studies, which led, however, to the identification of potential interferences (e.g. external contamination), artefacts and limitations of the test, including the lack of any precise quantitative and chronological correlation between drug intake and drug concentrations in the hair. All these data contributed to the development of a clear frame for the practical application of toxicological hair analysis. It is important to add that any consumption of illegal drugs and, in some settings, exposure to them, is illicit per se, so the test does not need to guarantee a close quantitative correlation with a drug’s consumption in order to be of diagnostic value (particularly in the forensic environment). In the light of the above evidence and considerations, multicentre studies need to be performed on adequate case series (promoted by international scientific societies such as the Society of Hair Testing or the International Association of Forensic Toxicologists, or independent, multicentre, cooperative studies) with a view to characterising the real distribution of EtG and FAEE concentrations in the hair of social drinkers of the population, including different ethnic and geographical origins, in order to define and validate cut-off concentrations with an adequate diagnostic efficiency for use in the forensic practice.
e70
Letter to the Editor / Forensic Science International 207 (2011) e69–e70
To avoid raising medicolegal litigation issues, it would be appropriate – for the numerous above-mentioned reasons – to omit the use of hair testing for EtG and FAEE in the forensic practice (i.e. to ascertain an individual’s ability to drive and/or to work, or mental capacity at the time of committing a crime). Results from hair testing for alcohol abuse should not be used in the forensic context until further studies will be concluded providing that epidemiological evidence necessary to support an objective diagnosis with the degree of ‘‘high probability or near certainty’’ needed to satisfy the ‘‘conditionalist theory’’ in any legal debate.
[11] P. Kintz, M. Villain, E. Vallet, M. Etter, G. Salquebre, V. Cirimele, Ethylglucuronide: unusual distribution between head hair and pubic hair, Forensic Sci. Int. 176 (2008) 87–90. [12] L. Morini, L. Politi, A. Polettini, Ethylglucuronide in hair. A sensitive and specific marker of chronic heavy drinking, Addiction 104 (2009) 915–920. [13] Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography–tandem mass spectrometry following solid-phase extraction, Anal. Bional. Chem. 394 (2009) 1895–1901. [14] L. Morini, A. Zucchella, A. Polettini, L. Politi, A. Groppi, Effect of bleaching on ethyl glucuronide in hair: a in vitro experiment, Forensic Sci. Int. 198 (2010) 23–27. [15] F. Pragst, M. Rothe, B. Moench, M. Hastedt, S. Herre, D. Simmert, Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: interpretation and advantages, Forensic Sci. Int. 196 (2010) 101–110. [16] C. Schummer, B.M. Appenzeller, R. Wennig, Quantitative determination of ethyl glucuronide in sweat, Ther. Drug Monit. 30 (2008) 536–539.
References [1] G. Skopp, G. Schmitt, L. Potsch, P. Dronner, R. Aderjan, R. Mattern, Ethylglucuronide in human hair, Alcohol Alcohol. 35 (2000) 283–285. [2] A. Alt, I. Janda, S. Seidl, F.M. Wurst, Determination of ethyl glucuronide in hair samples, Alcohol Alcohol. 35 (2000) 313–314. [3] V. Auwarter, F. Sporkert, S. Hartwig, F. Pragst, H. Vater, A. Diefenbacher, Fatty acid ethyo`l esters in hair as markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers and teetotalers, Clin. Chem. 47 (2001) 2114–2123. [4] F. Pragst, V. Auwarter, F. Sporkert, K. Spiegel, Analysis of fatty acid ethyl esters in hair as possible markers of chronically elevated alcohol consumption by7 headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC–MS), Forensic Sci. Int. 121 (2001) 76–88. [5] 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. [6] S. Hartwig, V. Auwarter, F. Pragst, Fatty acid ethyl esters in scalp, pubic, axillary, beard and body hair as markers for alcohol misuse, Alcohol Alcohol. 38 (2003) 163–167. [7] M Yegles, A. Labarthe, V. Auwarter, S. Hartwig, H. Vater, R. wennig, F. Pragsts, Comparison of ethylglucuronide and fatty acid ethil esters concentrations in hair of alcoholics, social drinkers and teetotalers, Forensic Sci. Int. 145 (2004) 167–173. [8] V. Auwarter, B. Kiessling, F. Pragst, Squalene in hair – a natural reference substance for the improved interpretation of fatty acid ethil esters concentrations with respect to alcohol misuse, Forensic Sci. Int. 145 (2004) 149–159. [9] L. Politi, L. Morini, F. Leone, A. Polettini, Ethyl glucuronide in hair: i sit a reliable marker of chronic high levels of alcohol consumption? Addiction 101 (2006) 1408–1412. [10] F.M. Wurst, K.M. Dursteler–Mac Farland, V. Auwarter, S. ergovic, N. Thon, M. Yegle, C. Halter, W. Weinmann, G. Wiesbeck, Assessment of alcohol use among methadone maintenance patients by direct ethanol metabolites and self reports, Alcohol. Clin. Exp. Res. 32 (2008) 1552–1557.
Franco Tagliaro* Department of Public Health and Community Medicine, Unit of Forensic Medicine, University of Verona, Verona, Italy Federica Bortolotti Department of Public Health and Community Medicine, Unit of Forensic Medicine, University of Verona, Verona, Italy Guido Viel Forensic Toxicology and Antidoping, University Hospital of Padova, Padova, Italy Santo Davide Ferrara Forensic Toxicology and Antidoping, University Hospital of Padova, Padova, Italy *Corresponding author at: Department of Public Health and Community Medicine, Unit of Forensic Medicine, Policlinico ‘‘G.B. Rossi’’, Piazzale L.A. Scuro 10, 37134 Verona, Italy E-mail address: [email protected] (F. Tagliaro) 13 August 2010 Available online 28 October 2010
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Letter to the Editor Caveats against an improper use of hair testing to support the diagnosis of chronic excessive alcohol consumption, following the ‘‘Consensus’’ of the Society of Hair Testing 2009 [Forensic Science International 196 (2010) 2]
Sir, The reason for the present letter to the Editor lies in the recent publication [Forensic Science International 196 (2010) 2] of a ‘‘Consensus’’ of the Society of Hair Testing (SoHT), adopted on June 16, 2009 in Rome on the use of hair testing for ‘‘objectively’’ inferring chronic excessive consumption of alcohol. The effort made by the SoHT is certainly appreciable, since it aims to face a problem of great importance to clinical and forensic toxicologists, i.e. the diagnosis of excessive ethyl alcohol consumption, nosographically classifiable as abuse or dependence. This is an interdisciplinary, difficult and delicate issue, about which a lot has already been written and, this notwithstanding, is still debated. This being so, in the light of our personal clinical and laboratory experience, we reviewed the case series of our institutes and the literature on the topic to analyse the experimental data and scientific evidence in the light of the 17 points detailed and shared by the SoHT (evidence that was not cited in the references of the above-mentioned publication). Fifty-three scientific works listed in the MedLine database – search criteria: ‘‘(EtG OR Ethyl glucuronide) AND (hair OR hair testing) – discuss the measurement of EtG in a keratin matrix (10 of them are reviews), while the studies on the measurement of FAEE in the hair – search criteria: ‘‘(FAEE OR fatty acid ethyl esters) AND (hair OR hair testing) – amount to 38 (8 of which are reviews). Among these studies, to the best of our knowledge, only 14 present data on EtG (10 studies) and/or FAEE (five studies) concentrations in the keratin matrix of ‘‘normal subjects’’ (i.e. socially acceptable alcohol users) [1–14]. It is also worth emphasising that the total number of ‘‘normal subjects’’ analysed in these studies amount to only 144 individuals. In the light of the theoretical assumptions and experimental findings discussed below, the inter-individual variation can be expected to be very considerable, and therefore should be well ascertained preliminarily to any effort to indentify cut-off concentrations capable of ensuring a high sensitivity and specificity of diagnosis of chronic alcohol abuse. It is therefore particularly important to emphasise the following points: 1. The enzymatic transformation of ethanol into EtG and FAEE occurs in a very small fraction, accounting for less than 1% of the total intake. This means that inter-individual variability and even intra-individual conditions (enzyme induction) may lead to important differences in the formation of the two metabolites 0379-0738/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2010.10.005
and consequently in their concentrations in the hair matrix of individuals ingesting the same amounts of alcohol. 2. The proposed mechanism of an acidic, water-soluble EtG compound becoming embedded in the hair matrix, mainly via perspiration [15], has been shown to be extremely inefficient, given the pg/mg amounts of EtG that are determined in the hair matrix by comparison with the concentrations measured in sweat, which range from 1.7 to 103 ng/mL [16]. This leads to the consideration that the well-known inter-individual differences in sweating may give rise to a different incorporation of EtG in the hair. On the other hand, the high water solubility of EtG may lead to an easy removal of this metabolite from the hair matrix with normal hair washing, representing another cause of variability; this consideration is also supported by the easy extraction of EtG from hair samples (with incubation and sonication in water) applied in most analytical procedures. 3. In the case of FAEE, which are very lipophilic compounds, the proposed mechanism of its embedding in the hair matrix is mainly via sebum [15]. Here again, the well-known considerable inter-individual differences in sebum production from the scalp may lead to a different incorporation of FAEE in the hair. 4. The non-homogeneous and non-synchronous growth of scalp hair may also reasonably be responsible for a marked intraindividual variability in the concentrations of both EtG and FAEE in the hair. It should be also pointed out that, only after many years of debate, hair analysis for controlled/illicit drugs has been accepted in the last decade as a marker of past chronic use or ‘‘exposure’’ to these compounds. This acceptance came after extensive studies, which led, however, to the identification of potential interferences (e.g. external contamination), artefacts and limitations of the test, including the lack of any precise quantitative and chronological correlation between drug intake and drug concentrations in the hair. All these data contributed to the development of a clear frame for the practical application of toxicological hair analysis. It is important to add that any consumption of illegal drugs and, in some settings, exposure to them, is illicit per se, so the test does not need to guarantee a close quantitative correlation with a drug’s consumption in order to be of diagnostic value (particularly in the forensic environment). In the light of the above evidence and considerations, multicentre studies need to be performed on adequate case series (promoted by international scientific societies such as the Society of Hair Testing or the International Association of Forensic Toxicologists, or independent, multicentre, cooperative studies) with a view to characterising the real distribution of EtG and FAEE concentrations in the hair of social drinkers of the population, including different ethnic and geographical origins, in order to define and validate cut-off concentrations with an adequate diagnostic efficiency for use in the forensic practice.
e70
Letter to the Editor / Forensic Science International 207 (2011) e69–e70
To avoid raising medicolegal litigation issues, it would be appropriate – for the numerous above-mentioned reasons – to omit the use of hair testing for EtG and FAEE in the forensic practice (i.e. to ascertain an individual’s ability to drive and/or to work, or mental capacity at the time of committing a crime). Results from hair testing for alcohol abuse should not be used in the forensic context until further studies will be concluded providing that epidemiological evidence necessary to support an objective diagnosis with the degree of ‘‘high probability or near certainty’’ needed to satisfy the ‘‘conditionalist theory’’ in any legal debate.
[11] P. Kintz, M. Villain, E. Vallet, M. Etter, G. Salquebre, V. Cirimele, Ethylglucuronide: unusual distribution between head hair and pubic hair, Forensic Sci. Int. 176 (2008) 87–90. [12] L. Morini, L. Politi, A. Polettini, Ethylglucuronide in hair. A sensitive and specific marker of chronic heavy drinking, Addiction 104 (2009) 915–920. [13] Determination of ethyl-glucuronide in hair for heavy drinking detection using liquid chromatography–tandem mass spectrometry following solid-phase extraction, Anal. Bional. Chem. 394 (2009) 1895–1901. [14] L. Morini, A. Zucchella, A. Polettini, L. Politi, A. Groppi, Effect of bleaching on ethyl glucuronide in hair: a in vitro experiment, Forensic Sci. Int. 198 (2010) 23–27. [15] F. Pragst, M. Rothe, B. Moench, M. Hastedt, S. Herre, D. Simmert, Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: interpretation and advantages, Forensic Sci. Int. 196 (2010) 101–110. [16] C. Schummer, B.M. Appenzeller, R. Wennig, Quantitative determination of ethyl glucuronide in sweat, Ther. Drug Monit. 30 (2008) 536–539.
References [1] G. Skopp, G. Schmitt, L. Potsch, P. Dronner, R. Aderjan, R. Mattern, Ethylglucuronide in human hair, Alcohol Alcohol. 35 (2000) 283–285. [2] A. Alt, I. Janda, S. Seidl, F.M. Wurst, Determination of ethyl glucuronide in hair samples, Alcohol Alcohol. 35 (2000) 313–314. [3] V. Auwarter, F. Sporkert, S. Hartwig, F. Pragst, H. Vater, A. Diefenbacher, Fatty acid ethyo`l esters in hair as markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers and teetotalers, Clin. Chem. 47 (2001) 2114–2123. [4] F. Pragst, V. Auwarter, F. Sporkert, K. Spiegel, Analysis of fatty acid ethyl esters in hair as possible markers of chronically elevated alcohol consumption by7 headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC–MS), Forensic Sci. Int. 121 (2001) 76–88. [5] 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. [6] S. Hartwig, V. Auwarter, F. Pragst, Fatty acid ethyl esters in scalp, pubic, axillary, beard and body hair as markers for alcohol misuse, Alcohol Alcohol. 38 (2003) 163–167. [7] M Yegles, A. Labarthe, V. Auwarter, S. Hartwig, H. Vater, R. wennig, F. Pragsts, Comparison of ethylglucuronide and fatty acid ethil esters concentrations in hair of alcoholics, social drinkers and teetotalers, Forensic Sci. Int. 145 (2004) 167–173. [8] V. Auwarter, B. Kiessling, F. Pragst, Squalene in hair – a natural reference substance for the improved interpretation of fatty acid ethil esters concentrations with respect to alcohol misuse, Forensic Sci. Int. 145 (2004) 149–159. [9] L. Politi, L. Morini, F. Leone, A. Polettini, Ethyl glucuronide in hair: i sit a reliable marker of chronic high levels of alcohol consumption? Addiction 101 (2006) 1408–1412. [10] F.M. Wurst, K.M. Dursteler–Mac Farland, V. Auwarter, S. ergovic, N. Thon, M. Yegle, C. Halter, W. Weinmann, G. Wiesbeck, Assessment of alcohol use among methadone maintenance patients by direct ethanol metabolites and self reports, Alcohol. Clin. Exp. Res. 32 (2008) 1552–1557.
Franco Tagliaro* Department of Public Health and Community Medicine, Unit of Forensic Medicine, University of Verona, Verona, Italy Federica Bortolotti Department of Public Health and Community Medicine, Unit of Forensic Medicine, University of Verona, Verona, Italy Guido Viel Forensic Toxicology and Antidoping, University Hospital of Padova, Padova, Italy Santo Davide Ferrara Forensic Toxicology and Antidoping, University Hospital of Padova, Padova, Italy *Corresponding author at: Department of Public Health and Community Medicine, Unit of Forensic Medicine, Policlinico ‘‘G.B. Rossi’’, Piazzale L.A. Scuro 10, 37134 Verona, Italy E-mail address: [email protected] (F. Tagliaro) 13 August 2010 Available online 28 October 2010