Forensic Science International 243 (2014) 44–46
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Testing for ethanol markers in hair: Discrepancies after simultaneous quantification of ethyl glucuronide and fatty acid ethyl esters P. Kintz a,*, D. Nicholson b a b
X-Pertise Consulting, 84 route de Saverne, F-67205 Oberhausbergen, France DNA Worldwide Group, K10 The Courtyard, Jenson Avenue, Commerce Park, Frome BA11 2FG, UK
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 13 April 2014
The hair of 97 cases were analysed for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE, including ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) according to the Society of Hair Testing guidelines to examine the role of both tests in documenting chronic excessive alcohol drinking, particularly when the results are in contradiction. 27 (27.8%) results were EtG negative and FAEE positive, when applying the SoHT cut-offs, probably due to the use of alcohol-containing hair products. Four cases (4.1%) were EtG positive and FAEE negative that were attributed to the use of herbal lotions containing EtG. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ethyl glucuronide Fatty acid ethyl esters Hair analysis Alcohol abuse
1. Introduction In human, ethanol is mainly metabolized by oxidation in the liver (about 90–95%) and in lesser quantities by the kidneys, lungs and skin. Phase II of ethanol metabolism involves biotransformation to ethyl glucuronide (EtG) that represents about 0.5% of total ethanol elimination [1]. Following ethanol intake, this nonvolatile, water soluble conjugate is reported to be detectable for an extended period of time after the complete elimination of the parent compound from the body: up to 18 h in blood and up to 80 h in urine. EtG can accumulate in hair and its first identification was reported by Sachs [2] in 1993. Due to its high volatility, ethanol itself cannot be used as an analyte, because it does not incorporate into the hair shaft. Detection of EtG in hair is considered to be associated with alcohol consumption, whereas a negative result does not unambiguously exclude the alcohol abuse [3]. Investigations on fatty acid ethyl esters (FAEE) has been proposed by Pragst and colleagues [4] to monitor alcohol consumption. FAEE are a group of more than 20 substances and are formed in presence of ethanol and free fatty acids, triglycerides, lipoproteins or phospholipids by FAEE synthases as well as by nonspecific enzymes such as carboxylesterase, lipoprotein lipase, carboxylester lipase or cholesterol esterase found in the liver but also in hair roots. FAEE determination is of interest as they appear
* Corresponding author. Tel.: +33 388266431; fax: +33 388272403. E-mail addresses: [email protected], [email protected] (P. Kintz). http://dx.doi.org/10.1016/j.forsciint.2014.03.012 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
responsive to alcohol-induced organ damage. Four FAEE (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are suitable markers for the detection of heavy alcohol consumption and show differential concentrations in hair of children, adult teetotalers and social drinkers in comparison with FAEE concentrations found in hair of alcoholics. Hydrophilic EtG is mainly formed in the liver cells and incorporated into the hair matrix by sweat. In contrast, lipophilic FAEE are built by esterification in blood and almost all other tissues. They are incorporated into the hair matrix by sebum [5]. Comparison with traditional indirect alcohol markers in blood such as CDT or gGT showed for FAEE and for EtG advantages in sensitivity and in specificity [6–8]. The Society of Hair Testing [9] provides guidelines for hair testing for chronic excessive alcohol consumption. The cut-off for EtG in hair to strongly suggest chronic excessive alcohol consumption is proposed at 30 pg/mg scalp hair measured in the 0–3 up to 0–6 cm proximal segment. The cut-off for the sum of the four FAEE esters in hair to strongly suggest chronic excessive alcohol consumption is proposed at 0.5 ng/mg scalp hair measured in the 0–3 cm proximal segment. If the proximal 0–6 cm segment is used the proposed cut-off is 1.0 ng/mg scalp hair. In both cases, if samples less than 3 cm are used the results should be interpreted with caution. It has been proposed to use, for mutual confirmation, both EtG and FAEE analyses at the same time [10–12]. Unfortunately, only 69–75% of the results (n = 1657) were in agreement in the first study [10]. There were more positive results (18.5%) with FAEE than with EtG (positive FAEE and negative EtG), probably due to
P. Kintz, D. Nicholson / Forensic Science International 243 (2014) 44–46
hair cosmetic treatments, since hair spray increases the portion of positive FAEE results, and bleaching and dying increase the portion of negative EtG results, mostly in women. The possibility of having positive EtG results associated to negative FAEE results, representing 10% of the cases, was not documented. However, given the legal consequences of a positive result, it was suggested by some teams not to anymore perform hair analyses for alcohol markers [13]. According to the SoHT on point 18 of its consensus [9], it is not advisable to use the results of hair testing for alcohol markers in isolation. The present paper deals with the interpretation of 97 cases where EtG and FAEE were simultaneously measured in human hair and the discrepancies that were observed. 2. Materials and methods Between January 2012 and August 2013, we received 97 files with a view to assessing if there is any evidence of historical alcohol abuse by the donor of the sample. The items were received sealed and the chain of custody was intact. The samples were logged onto the system and processed at the laboratory. At the request of the solicitor, the hair was examined, with the aid of scientific support staff for EtG and FAEE over a 0–3 or 0–6 month period. The hair length was always the same for both tests. All analyses were achieved under ISO17025 accreditation, using previously published methods. EtG was tested by liquid chromatography–tandem mass spectrometry after solid-phase extraction and separation on a hydrophilic interaction liquid chromatography column [14]. For the determination of FAEE a procedure based on headspace solid-phase micro extraction followed by gas chromatography–mass spectrometry was applied [15]. All the hair specimens met the SoHT criteria in terms of anatomical origin (head hair) and length of the submitted segment (3 or 6 cm). Self-reported alcohol consumption was not known. For some subjects, the current medical treatment was available, but this was not considered in the discussion, given the chemical profile of the involved pharmaceuticals. The use of cosmetics and hair sprays or lotions was sometimes indicated but cannot be considered as a general rule. 3. Results and discussion It is published in the literature [5,10–12] that FAEE and EtG are complementary to each other also with respect to their different sensitivity to hair cosmetics. False positive FAEE results due to ethanol containing sprays or lotions would not be ‘‘confirmed’’ because the same specimen would have a negative EtG result, and false negative results caused by bleaching or dyeing will be compensated by a positive FAEE finding. From the data pool of individuals analyzed over the 20 month period for alcohol markers in hair, only those in accordance with the SoHT consensus were included in this evaluation. In all the 97 cases, EtG and FAEE were measured at the same time. In Table 1, the percentages of the four possible EtG-FAEE result combinations are shown. The results were in agreement in 68% of the cases (positive EtG and positive FAEE, negative EtG and negative FAEE). This is close to the 69–75% agreement published by Suesse et al. [10]. There were 27.8% of cases where the results for EtG were negative while the results for FAEE were positive, when applying the SoHT cut-offs. This is presented Table 2. The influence of the Table 1 Distribution of the results after combined testing of EtG and FAEE. Pos EtG/pos FAEE
Neg EtG/neg FAEE
Pos EtG/neg FAEE
Neg EtG/pos FAEE
22.7%
45.4%
4.1%
27.8%
45
Table 2 Individual results in case of neg EtG and pos FAEE. Case
Gender
Age
Length of segment
EtG (pg/mg)
FAEE (ng/mg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
F Fa F F F Fb Mb F M M Fb M F Fa Ma M Fb M Fb F M Ma F Fb M F Fb
44 34 24 25 20 23 25 36 34 36 43 36 20 18 25 50 34 36 20 27 38 47 35 41 40 20 34
6 cm 3 cm 3 cm 6 cm 3 cm 3 cm 3 cm 3 cm 3 cm 3 cm 6 cm 3 cm 3 cm 3 cm 3 cm 3 cm 6 cm 3 cm 3 cm 3 cm 3 cm 3 cm 6 cm 6 cm 3 cm 6 cm 3 cm
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Testing for ethanol markers in hair: Discrepancies after simultaneous quantification of ethyl glucuronide and fatty acid ethyl esters P. Kintz a,*, D. Nicholson b a b
X-Pertise Consulting, 84 route de Saverne, F-67205 Oberhausbergen, France DNA Worldwide Group, K10 The Courtyard, Jenson Avenue, Commerce Park, Frome BA11 2FG, UK
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 13 April 2014
The hair of 97 cases were analysed for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE, including ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) according to the Society of Hair Testing guidelines to examine the role of both tests in documenting chronic excessive alcohol drinking, particularly when the results are in contradiction. 27 (27.8%) results were EtG negative and FAEE positive, when applying the SoHT cut-offs, probably due to the use of alcohol-containing hair products. Four cases (4.1%) were EtG positive and FAEE negative that were attributed to the use of herbal lotions containing EtG. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ethyl glucuronide Fatty acid ethyl esters Hair analysis Alcohol abuse
1. Introduction In human, ethanol is mainly metabolized by oxidation in the liver (about 90–95%) and in lesser quantities by the kidneys, lungs and skin. Phase II of ethanol metabolism involves biotransformation to ethyl glucuronide (EtG) that represents about 0.5% of total ethanol elimination [1]. Following ethanol intake, this nonvolatile, water soluble conjugate is reported to be detectable for an extended period of time after the complete elimination of the parent compound from the body: up to 18 h in blood and up to 80 h in urine. EtG can accumulate in hair and its first identification was reported by Sachs [2] in 1993. Due to its high volatility, ethanol itself cannot be used as an analyte, because it does not incorporate into the hair shaft. Detection of EtG in hair is considered to be associated with alcohol consumption, whereas a negative result does not unambiguously exclude the alcohol abuse [3]. Investigations on fatty acid ethyl esters (FAEE) has been proposed by Pragst and colleagues [4] to monitor alcohol consumption. FAEE are a group of more than 20 substances and are formed in presence of ethanol and free fatty acids, triglycerides, lipoproteins or phospholipids by FAEE synthases as well as by nonspecific enzymes such as carboxylesterase, lipoprotein lipase, carboxylester lipase or cholesterol esterase found in the liver but also in hair roots. FAEE determination is of interest as they appear
* Corresponding author. Tel.: +33 388266431; fax: +33 388272403. E-mail addresses: [email protected], [email protected] (P. Kintz). http://dx.doi.org/10.1016/j.forsciint.2014.03.012 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
responsive to alcohol-induced organ damage. Four FAEE (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) are suitable markers for the detection of heavy alcohol consumption and show differential concentrations in hair of children, adult teetotalers and social drinkers in comparison with FAEE concentrations found in hair of alcoholics. Hydrophilic EtG is mainly formed in the liver cells and incorporated into the hair matrix by sweat. In contrast, lipophilic FAEE are built by esterification in blood and almost all other tissues. They are incorporated into the hair matrix by sebum [5]. Comparison with traditional indirect alcohol markers in blood such as CDT or gGT showed for FAEE and for EtG advantages in sensitivity and in specificity [6–8]. The Society of Hair Testing [9] provides guidelines for hair testing for chronic excessive alcohol consumption. The cut-off for EtG in hair to strongly suggest chronic excessive alcohol consumption is proposed at 30 pg/mg scalp hair measured in the 0–3 up to 0–6 cm proximal segment. The cut-off for the sum of the four FAEE esters in hair to strongly suggest chronic excessive alcohol consumption is proposed at 0.5 ng/mg scalp hair measured in the 0–3 cm proximal segment. If the proximal 0–6 cm segment is used the proposed cut-off is 1.0 ng/mg scalp hair. In both cases, if samples less than 3 cm are used the results should be interpreted with caution. It has been proposed to use, for mutual confirmation, both EtG and FAEE analyses at the same time [10–12]. Unfortunately, only 69–75% of the results (n = 1657) were in agreement in the first study [10]. There were more positive results (18.5%) with FAEE than with EtG (positive FAEE and negative EtG), probably due to
P. Kintz, D. Nicholson / Forensic Science International 243 (2014) 44–46
hair cosmetic treatments, since hair spray increases the portion of positive FAEE results, and bleaching and dying increase the portion of negative EtG results, mostly in women. The possibility of having positive EtG results associated to negative FAEE results, representing 10% of the cases, was not documented. However, given the legal consequences of a positive result, it was suggested by some teams not to anymore perform hair analyses for alcohol markers [13]. According to the SoHT on point 18 of its consensus [9], it is not advisable to use the results of hair testing for alcohol markers in isolation. The present paper deals with the interpretation of 97 cases where EtG and FAEE were simultaneously measured in human hair and the discrepancies that were observed. 2. Materials and methods Between January 2012 and August 2013, we received 97 files with a view to assessing if there is any evidence of historical alcohol abuse by the donor of the sample. The items were received sealed and the chain of custody was intact. The samples were logged onto the system and processed at the laboratory. At the request of the solicitor, the hair was examined, with the aid of scientific support staff for EtG and FAEE over a 0–3 or 0–6 month period. The hair length was always the same for both tests. All analyses were achieved under ISO17025 accreditation, using previously published methods. EtG was tested by liquid chromatography–tandem mass spectrometry after solid-phase extraction and separation on a hydrophilic interaction liquid chromatography column [14]. For the determination of FAEE a procedure based on headspace solid-phase micro extraction followed by gas chromatography–mass spectrometry was applied [15]. All the hair specimens met the SoHT criteria in terms of anatomical origin (head hair) and length of the submitted segment (3 or 6 cm). Self-reported alcohol consumption was not known. For some subjects, the current medical treatment was available, but this was not considered in the discussion, given the chemical profile of the involved pharmaceuticals. The use of cosmetics and hair sprays or lotions was sometimes indicated but cannot be considered as a general rule. 3. Results and discussion It is published in the literature [5,10–12] that FAEE and EtG are complementary to each other also with respect to their different sensitivity to hair cosmetics. False positive FAEE results due to ethanol containing sprays or lotions would not be ‘‘confirmed’’ because the same specimen would have a negative EtG result, and false negative results caused by bleaching or dyeing will be compensated by a positive FAEE finding. From the data pool of individuals analyzed over the 20 month period for alcohol markers in hair, only those in accordance with the SoHT consensus were included in this evaluation. In all the 97 cases, EtG and FAEE were measured at the same time. In Table 1, the percentages of the four possible EtG-FAEE result combinations are shown. The results were in agreement in 68% of the cases (positive EtG and positive FAEE, negative EtG and negative FAEE). This is close to the 69–75% agreement published by Suesse et al. [10]. There were 27.8% of cases where the results for EtG were negative while the results for FAEE were positive, when applying the SoHT cut-offs. This is presented Table 2. The influence of the Table 1 Distribution of the results after combined testing of EtG and FAEE. Pos EtG/pos FAEE
Neg EtG/neg FAEE
Pos EtG/neg FAEE
Neg EtG/pos FAEE
22.7%
45.4%
4.1%
27.8%
45
Table 2 Individual results in case of neg EtG and pos FAEE. Case
Gender
Age
Length of segment
EtG (pg/mg)
FAEE (ng/mg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
F Fa F F F Fb Mb F M M Fb M F Fa Ma M Fb M Fb F M Ma F Fb M F Fb
44 34 24 25 20 23 25 36 34 36 43 36 20 18 25 50 34 36 20 27 38 47 35 41 40 20 34
6 cm 3 cm 3 cm 6 cm 3 cm 3 cm 3 cm 3 cm 3 cm 3 cm 6 cm 3 cm 3 cm 3 cm 3 cm 3 cm 6 cm 3 cm 3 cm 3 cm 3 cm 3 cm 6 cm 6 cm 3 cm 6 cm 3 cm
