Alcohol and Alcoholism Vol. 35, No. 3, pp. 283-285, 2000
© 2000 Medical Council on Alcoholism
ETHYL GLUCURONIDE IN HUMAN HAIR
Institute of Legal Medicine and Traffic Medicine, Voßstr. 2, 69115 Heidelberg and
1 Institute of Legal Medicine, Am Pulverturm 3, 55131 Mainz, Germany
Received 8 September 1999; first review notified 4 November 1999; accepted 1 December 1999
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethyl glucuronide (EtG) is considered to be a promising candidate marker of alcohol consumption, but exhibits a short window of detection in blood or urine. Keratinized tissues are known to retain foreign substances and to provide a greater retrospective window of detection than body fluids. Therefore, post-mortem hair, skin swabs, and stratum corneum samples were collected from four subjects with a reported history of alcohol misuse and from seven subjects with a report of regular, socially accepted drinking behaviour, and were investigated for EtG. Additionally, certain specimens were collected from three children, who had not yet consumed any alcoholic beverages. EtG was detectable in most of the hair and stratum corneum samples as well as in perspiration stains from alcohol-consuming subjects. The results indicated that EtG might be formed locally in very small and highly variable amounts. The most important finding was that EtG cannot be expected to be generally detectable in keratinized tissues or perspiration stains from alcohol-drinking subjects, whereas a positive result is always associated with recent alcohol consumption.
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethyl glucuronide (EtG) is a phase II metabolite of ethanol and is considered to be a promising candidate marker of alcohol consumption (Besserer and Schmidt, 1983
; Aderjan et al., 1994; Wurst et al., 1995; Schmitt et al., 1997; Seidl et al., 1998). About 0.5–1.6% of the total elimination pathway of ethanol is attributed to conjugation of ethanol with activated glucuronic acid (Kamil et al., 1952). The formation of EtG has been shown to depend on the serum ethanol concentration. EtG peaks 2 to 3.5 14;h later in blood than ethanol, and subsequently its concentration decreases exponentially. The half-life of EtG ranges from 2 to 3 14;h (Schmitt et al., 1997). EtG was reported to be detectable in urine up to 80 14;h after heavy alcohol consumption (Schmitt et al., 1997; Wurst et al., 1999). Therefore, EtG can be regarded as a diagnostic tool for recent alcohol use. However, EtG is of limited value for abstinence control due to its rapid elimination and its narrow time frame for detection, compared with other potential markers of alcohol misuse, such as carbohydrate-deficient transferrin, the mean corpuscular volume of erythrocytes, or liver enzyme activities.
Keratinized tissues, such as hair fibres, are known to retain foreign substances and to provide a greater retrospective window of detection than body fluids. The purpose of the present study was to supplement preliminary data on positive EtG findings in hair (Skopp et al., 1995; Sachs, 1997).
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MATERIALS AND METHODS |
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Study design
The study was limited to those cases (n = 11) in which morphological investigations of the hair samples did not indicate cosmetic treatments, such as colouring or bleaching. Hair samples were obtained from all subjects, whereas skin swabs from the chest (skin area of 25 14;cm2, Salivette, Sarstedt, Nümbrecht, Germany) could only be taken from seven subjects who were fully dressed and thus for whom alteration prior to sampling could be excluded. Sufficient amounts of stratum corneum (sole) could be obtained from six subjects. All subjects except subject 1 were influenced by alcohol at the time of death (blood-ethanol concentration: 110–335 14;mg/dl, femoral blood).
Skin swabs and horny layer were collected prior to post-mortem examination. Detailed medical reports on heavy drinking behaviour were available in subjects 1–4; this could be corroborated by pathomorphological findings, such as fatty degeneration or cirrhosis of the liver as well as diseases of the pancreas and varicosis of the oesophagus. Medical data were not available for subjects 5–11. In these cases, a history of socially accepted, regular alcohol consumption was reported by eyewitnesses. Post-mortem findings did not reveal morphological correlates of harmful alcohol use. As a control group for ethanol abstinence, hair fibres, skin swabs and horny layer preparations were collected from children (n 14 ;= 14;3), who had never consumed alcoholic beverages.
Chemicals and reagents
EtG was prepared from d-glucurono-3,6-lactone as previously described (Schmitt et al., 1995). Methyl glucuronide was chosen as the internal standard (Sigma, Deisenhofen, Germany) and ethanol standard solutions were from Medichem (Stuttgart, Germany). N-Methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA, from Sigma, Deisenhofen, Germany) was used for derivatization. All chemicals (chloroform, methanol, n-hexane, from Merck, Darmstadt, Germany) were of analytical grade.
Preparation of hair samples for analysis
The hair samples were washed twice (5 14;ml ether, 5 14;ml acetone, 10 14;min) and the proximal 3-cm segments were pulverized in a ball mill (Retsch, Haan, Germany). To 50 mg of the powder, 50 ng methyl glucuronide, 0.25 ml distilled water, and 1.0 ml methanol were added. After incubation at ambient temperature (5 h) and ultrasonication (3 h, 30°C) the mix-ture was centrifuged (14 14;000 g, 10 min) and 0.8 ml of the supernatant was filtrated (membrane filtration device, Spartan 13/30, Schleicher & Schuell, Dassel, Germany) and evaporated to dryness under nitrogen. The residue was derivatized (30 µl MSTFA, 60 min, 70°C), evaporated to dryness and redissolved in 70 µl of n-hexane. One microlitre was injected into a GC/MS gas chromatography/mass spectrometry system. A calibration curve of 2, 5, 10, 15, 20, 30, and 40 ng/mg hair was established by adding a methanolic solution of EtG to powdered hair collected from infants. Powdered hair samples were also investigated without adding methyl glucuronide prior to sample processing.
Preparation of skin swabs for analysis
The absorption material of the Salivette (500 mg) was incubated with 500 ng methyl glucuronide as the internal standard and 2.0 ml methanol for 2 h at ambient temperature. After centrifugation (4300 g, 10 min), 1.60 ml of the supernatant was processed as described above. A calibration curve of 10, 50, 100, 200, 300, 400, and 600 ng/Salivette was established by adding a methanolic solution of EtG onto the cotton roll of unused sampling devices. The recovery of the analytes from the adsorbing material of the saliva collection device was also determined (n = 5, 300 ng EtG/Salivette). Additionally, skin swabs were investigated for the absence of methyl glucuronide.
Preparation of stratum corneum specimens for analysis
Stratum corneum samples were washed with acetone (5 ml, 10 min), air dried and chopped with a scalpel. To 50 mg of the sample, 500 ng methyl glucuronide, 0.4 ml distilled water, and 3.0 ml methanol were added. After incubation at ambient temperature (24 h) and ultrasonication (3 h, 30°C), the mixture was centrifuged (14 000 g, 10 min), 1.0 ml of the supernatant was filtrated (Spartan 13/30, membrane filtration device, Schleicher & Schuell, Dassel, Germany), drawn through a solid phase extraction column (C8, Bond Elut, Varian, Harbor City, CA, USA) and processed as described above. A calibration curve of 5, 10, 20, and 30 ng EtG/mg horny layer was established by adding a methanolic solution of EtG to chopped stratum corneum from teetotallers. In addition, samples were investigated without adding methyl glucuronide as the internal standard.
Determination of EtG
Analysis was performed using a HP 5890 series II gas chromatograph equipped with a HP 5972 mass selective detector and a HP 5693 autoinjector (Hewlett Packard, Waldbronn, Germany). Samples were eluted from a capillary column (CP-Sil 5, 12 m x 0.25 mm i.d., 0.4 µm film thickness, Chrompack, Middelburg, The Netherlands), which was temperature programmed from 60°C (1 min hold) to 320°C (1 min hold) at 20°C/min. The detector was operated in the selected ion monitoring mode. The ions monitored were: m/z 261, 292, 375, 405 (target) for EtG, and m/z 391 (target) for methyl glucuronide.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methyl glucuronide was chosen as the internal standard for the following reasons. This compound could not be detected in any of the samples investigated, and deuterium-labelled EtG revealed the same diagnostic fragmentation pattern as the undeuterated compound. The retention time was 9.14 min for methyl glucuronide and 9.33 min for EtG. The validation data of the analytical method and the concentration ranges of the calibration curves are summarized in Table 1
. The linear correlation coefficients were >0.995 for the five- and seven-point standard curves of spiked horny layer, cotton rolls, and hair powder. The limit of detection (LOD) and of quantitation (LOQ) was calculated according to DIN 32645 (1994), using software written in house (B.E.N.). Interfering peaks from blank horny layer and hair specimens from teetotallers or from cotton rolls were not observed.
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Six hair samples obtained from alcohol-drinking subjects were found to be positive for EtG whereas in five hair samples EtG could not be detected (Table 2
). Positive results were mostly close to the limit of detection. Hair specimens obtained from children who had never consumed alcohol were negative (Table 2). The results could not be correlated with morphological parameters of the hair strands. EtG was present in skin swabs from five subjects, whereas it could not be detected in two subjects (Table 2). Stratum corneum samples were positive for EtG in three of six subjects (Table 2). Materials obtained from the alcohol-abstaining group were all negative.
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The most important finding of the present investigation was the observation that EtG cannot be expected to be generally detectable in hair samples obtained from people who drink alcohol. The results obtained from skin swabs and the horny layer of the skin indicate that detection of EtG might fail even in people who are actually under the influence of alcohol.
Though the results were established from post-mortem material, it seemed appropriate to draw conclusions on EtG disposition in hair fibres and the stratum corneum. EtG is a non-volatile solid and concentration changes of a drug substance in keratinized tissues have not been observed to change within a few days. The present findings relativized results formerly established by analyses on a very few samples using external standardization (Skopp et al., 1995). It was found that a negative result for EtG in hair may not indicate that the person abstained from alcoholic beverages. However, positive results were always associated with alcohol consumption.
The lack of correlation between the detection of EtG in hair and drinking behaviour suggests that EtG might be formed locally. The hair follicle is a moderately metabolically active tissue and glucuronosyl transferase activities have been observed in the outer root sheet (Pham et al., 1990). In addition, the other known ethanol-metabolizing enzymes were detected in the hair root cells (Goedde et al., 1980). As a result, ethanol would be expected to be preferentially oxidized.
The low concentrations of EtG in the hair shaft are in accordance with the phenomenon that basic substances are found to be preferentially incorporated into hair, compared to neutral or acidic molecules. Keratinized hair is known to be acidic in nature with an isoelectric point close to 6 (Robbins, 1988), which implies a poor binding of EtG to the hair fibre and facilitates its removal by routine washing and hair care procedures. Previous experiments have shown that hair fibres exposed to an aqueous solution of EtG did not readily accumulate the substance, which is in contrast to opiates (Skopp et al., 1995).
Although the source of EtG in hair fibres remains uncertain, it can be concluded from the present results that keratinized tissues cannot serve as specimens providing long-term information on alcohol consumption. The detection of EtG in hair samples may, however, help to identify some subjects who have consumed alcohol.
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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* Author to whom correspondence should be addressed at: Institut für Rechtsmedizin und Verkehrsmedizin im Klinikum der Universität Heidelberg, Voßstr. 2, 69115 Heidelberg, Germany.
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REFERENCES |
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Aderjan, R., Besserer, H., Sachs, H., Schmitt, G. and Skopp, G. (1994) Ethyl glucuronide — a non volatile metabolite in human hair. Proceedings of the TIAFT-SOFT Joint Meeting, Abstract 12. Tampa, FL.
Besserer, K. and Schmidt, V. (1983) Ein Beitrag zur renalen Ausscheidung von Äthylglucuronid nach oraler Alkoholaufnahme. Zentralblatt Rechtsmedizin 25, 369.
DIN 32645 (1994) Nachweis-, Erfassungs- und Bestimmungsgrenze. Beuth Verlag, Berlin.
Goedde, H. W., Agarwal, D. P. and Harada, S. (1980) Genetic studies on alcohol metabolizing enzymes: detection of isoenzymes in human hair roots. Enzyme 25, 281–286.[Web of Science][Medline]
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