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; in revised form 4 November 1999; accepted 1 December 1999
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ABSTRACT |
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INTRODUCTION |
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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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Skin swabs and horny layer were collected prior to post-mortem examination. Detailed medical reports on heavy drinking behaviour were available in subjects 14; 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 511. 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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DISCUSSION |
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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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REFERENCES |
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