FATTY ACID ETHYL ESTERS IN SCALP, PUBIC, AXILLARY, BEARD AND BODY HAIR AS MARKERS FOR ALCOHOL MISUSE

Sven Hartwig, Volker Auwärter and Fritz Pragst*

1 Institute of Legal Medicine, Humboldt-University, Hannoversche Straße 6, 10115 Berlin, Germany

Received 5 August 2002; in revised form 11 October 2002; accepted 28 October 2002


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Aims: This study examined the possibility of using hair samples other than scalp hair for analysis of fatty acid ethyl esters (FAEE) as markers for alcohol misuse. Methods: Samples of scalp and pubic hair and, if available, axillary, beard and body hair of one teetotaller, five moderate social drinkers and 22 fatalities were analysed for FAEE by head-space solid-phase microextraction and gas chromatography–mass spectrometry. The sum of the concentrations of ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate (CFAEE) in the hair samples was compared with information about the drinking behaviour of the individuals. Results: Although there were large differences in the analyte concentrations in hair from different sites in the same individual, cases of chronic excessive alcohol consumption were characterized by CFAEE > 1.0 ng/mg in almost all samples. Conclusion: FAEE concentrations in hair other than scalp hair can be used as markers of chronic alcohol misuse.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Hair fatty acid ethyl ester (FAEE) concentrations can be used as markers of chronic excessive alcohol ingestion (Auwärter et al., 2001Go; Pragst et al., 2001Go). These products of non-oxidative alcohol metabolism are formed in blood and almost all tissues after consumption of alcoholic beverages (Laposata, 1998Go, 1999Go). In blood they can be detected only up to 24 h after the end of drinking (Doyle et al., 1996Go; Laposata, 1997Go). It is assumed that they are incorporated in hair mainly from sebum (Auwärter et al., 2001Go). From the analysis of scalp hair from teetotallers, social drinkers, patients undergoing treatment for alcohol withdrawal, and fatalities who were known to drink excessively, it has been established that a total ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate concentration (CFAEE) above 1 ng/mg is a strong indicator of chronic excessive alcohol consumption. In teetotallers, CFAEE was <=0.4 ng/mg, and for moderate social drinkers (<=20 g ethanol/day) CFAEE was generally <0.8 ng/mg. CFAEE was little affected by usual hair care/hair cosmetics, but regular treatment of the scalp with hair lotions containing high concentrations of ethanol leads to higher results [1.5–6.2 ng/mg after 3 months daily treatment with a hair lotion containing 62.5% (v/v) ethanol; Hartwig et al., 2003Go].

The analysis of human hair for illicit and therapeutic drugs is now a routine procedure in many laboratories. Comprehensive reviews have been published about the anatomical, physiological and biochemical basis of hair analysis as well as the analytical methods used and the application of this methodology (Kintz, 1996Go; Pragst et al., 1998Go; Sachs and Kintz, 1998Go). Usually scalp hair is investigated. The use of hair from additional or alternative sites may be helpful if: (1) the head has been shaved (no scalp hair available); (2) the scalp hair is too short or it has been treated frequently by invasive cosmetics (dyeing, bleaching, permanent wave); (3) an additional sample is needed to confirm the scalp hair results; or (4) there is a suspicion of external contamination with the substances to be analysed (Mangin, 1996Go). In most such cases, pubic hair was analysed, but beard, axillary or body hair has been used only on occasions. However, the analyte concentrations in these alternative samples may be different from those in scalp hair, since there are substantial inter-site differences in hair growth rate and growth cycle, in the nature and activity of sweat and sebum glands, and in hair thickness (Ebling, 1987Go). Furthermore, there are differences with respect to atmospheric exposure and other external influences, as well as to hair care/use of cosmetics.

Drug concentrations in scalp, pubic and axillary hair have been compared by several authors (Mangin and Kintz, 1993Go; Offidani et al., 1993Go; Kintz and Mangin, 1994Go; Mangin, 1996Go; Kosuge et al., 1998Go). As a rule, the concentrations of opiates and cocaine decreased in the order pubic > scalp > axillary hair. Although beard hair was analysed several times for drugs in the context of the time of appearance of the analyte after drug intake (e.g. Cone, 1990Go), no comparison of analyte concentrations in this site with hair from other sites has been described.

In order to investigate the extent to which hair samples from sites other than scalp hair could be analysed in cases of suspected alcohol misuse, FAEE concentrations in scalp and pubic hair from 22 deaths, of which 18 had an alcohol anamnesis, and of one teetotaller and five moderate social drinkers were measured. Axillary hair, beard hair and different body hair was also analysed in some cases.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Hair samples
Hair samples from different sites were collected during post-mortem examinations at the Institute of Legal Medicine, Humboldt-University. In most cases the deceased had a history of heavy alcohol consumption, as estimated from police reports. The cadavers were not putrefied and were autopsied up to 5 days after death. Furthermore, hair samples of one teetotaller and five moderate social drinkers were analysed. The post-mortem samples were first washed with deionized water, in order to remove water-soluble impurities, and then dried. Subsequently, they were washed twice with heptane in order to remove any external lipids. The samples from the volunteers were taken after shampooing and were washed with heptane. CFAEE is not markedly decreased by washing with water or by shampooing (Hartwig et al., 2003Go). The analysis was carried out within 1 week after sampling. It was established with storage experiments that CFAEE did not change markedly during this period.

The protocol was approved by the ethics commission of the University Hospital Charité of Humboldt University.

Sample preparation and measurement of FAEE by gas chromatography–mass spectrometry (GC–MS) and head-space solid-phase microextraction (HS-SPME)
The analytical procedure and the mass spectra of the four FAEE (the myristate, palmitate, oleate and stearate ethyl esters) and of the corresponding deuterated internal standards (d5-FAEE) have been described in detail (Pragst et al., 2001Go). Briefly, 20–50 mg of the washed hair sample were cut to pieces of about 1 mm length and weighed. Then, 0.5 ml of dimethylsulphoxide, 2 ml of heptane and 40 ng of each of the four d5-FAEE were added in 20 µl of chloroform. The mixture was shaken (15 h, 25°C), centrifuged, the heptane phase separated, the solvent evaporated to dryness in a nitrogen stream, and the residue analysed by HS-SPME and GC-MS-SIM (selected ion monitoring). The limits of detection and limits of quantification were: ethyl myristate, 0.015 and 0.05 ng/mg; ethyl palmitate, 0.02 and 0.07 ng/mg; ethyl oleate, 0.04 and 0.12 ng/mg; and ethyl stearate, 0.01 and 0.04 ng/mg, respectively. Assay reproducibility was assessed by replicate analysis (n = 10) of the one hair sample. The standard deviations were: ethyl myristate (0.067 ng/mg), 15.7%; ethyl palmitate (0.796 ng/mg), 6.6%; ethyl oleate (0.387 ng/mg), 5.9%; and ethyl stearate (0.133 ng/mg), 3.5%.


    RESULTS AND DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The four esters were detected in all hair samples. The results (CFAEE together with the analysed hair lengths) are given in Table 1Go. With the exception of some axillary samples, the ratio of the concentrations of the four esters was similar to that described previously, with higher concentrations of ethyl oleate and ethyl palmitate, and clearly smaller concentrations of ethyl myristate and ethyl stearate (Auwärter et al., 2001Go). Three examples are shown in Fig. 1Go.


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Table 1. Concentrations of fatty acid ethyl esters (FAEE) in scalp, pubic, axillary, beard and body hair
 


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Fig. 1. Concentrations of fatty acid ethyl esters in hair from different sites of the moderate social drinker and two alcoholic patients. The moderate social drinker was VA03 (male aged 30 years, drinking ~6 g ethanol/day) and the two alcoholic fatalities were S317/01 (male aged 31 years, alcoholic in withdrawal treatment, death from subdural haematoma after accident in withdrawal delirium) and S330/02 (male aged 49 years, alcoholic, found dead in his apartment, liver cirrhosis, no other cause of death found at post-mortem).

 
As expected, the FAEE concentrations in the scalp hair of the teetotaller and the social drinkers were below 0.8 ng/mg. In 18 of the 22 scalp hair samples from the cadavers, CFAEE was clearly above 1.0 ng/mg (maximum 16.3 ng/mg). In these latter cases, information about excessive alcohol consumption was found in police reports (Table 1Go). Moreover, in most of these cases, typical pathological signs of chronic alcohol misuse were found at autopsy (e.g. fatty degeneration of the liver, liver cirrhosis, ascites, degeneration of pancreas, gastritis and gastric haemorrhage, atrophy of testes). Six cases had a post-mortem blood alcohol concentration above 1.0 mg/ml. There was no indication for heavy alcohol misuse in the last months before death in the police reports in the four cases with low FAEE concentrations [S083/01 (drug fatality), S271/01 (septic infection), S220/01 (pulmonary embolism) and S484/01 (previous alcoholic, paralysed after a stroke, living in a nursing home for 3 years before death, respectively)]. With the exception of the teetotaller and of post-mortem case S235/01, all subjects were male. In previous work, no major differences were observed between samples from male and female subjects (Auwärter et al., 2001Go).

From the study data in Table 1Go, it is clear that, in most cases, the FAEE concentrations in scalp hair differed considerably from the concentrations in hair from other sites in the same subject, but, nevertheless, excessive alcohol consumption was clearly indicated—this is shown for a moderate social drinker and two alcoholics in Fig. 1Go. Some features of hair growth and hair properties, which could contribute to the explanation of these differences in FAEE concentrations between samples from different sites, are given in Table 2Go. Human hair follicles and their associated sebaceous and apocrine glands are generally regulated by androgens and do not behave identically, but vary greatly between body site (Takayasu et al., 1980Go; Ebling, 1986Go). Different growth rate and growth cycle, as well as different size and activity of the glands, could explain the differences in FAEE concentrations observed.


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Table 2. Data about growth rate, growth cycle and possible exposure of human hair from different sitesa
 
Pubic hair
The FAEE concentrations in pubic hair as a function of the concentrations in scalp hair are shown in Fig. 2Go. The pubic hair/scalp hair ratio varied from 0.13 (case S063/01) to 3.3 (VA08), with a mean of 0.94. There was not a good correlation between FAEE concentrations (R2 = 0.519), but using a cut-off value of CFAEE = 1.0 ng/mg would only in two cases (S072/01 and S177/01, strong alcohol misuse known in both cases) have led to a false negative interpretation. Because of the physiological differences and other variables listed in Table 2Go , precise agreement or proportionality between concentrations in the two samples cannot be expected. Very large differences could be explained by the different time period in which the hair under investigation has grown. Thus, in case VA08, the social drinker ceased drinking several months before sampling. He shaved his head 1 month before sampling. Therefore, the scalp hair sample represents only the abstinence of the last 1 or 2 months, whereas the 5 cm long pubic hair sample, which was left in its natural state and had a portion of more than 50% telogen hair, originated during a 1 year period of higher alcohol consumption. On the other hand, the density and size of sebaceous glands in the scalp region is much higher than at other body sites (Ebling, 1986Go). This could explain the lower FAEE concentrations in pubic hair in the majority of the cases.



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Fig. 2. Relationship between the fatty acid ethyl ester (FAEE) concentrations of scalp hair and pubic hair of 28 subjects. Circles, teetotaller (TT), social drinkers (SD) and fatalities with no evidence of excessive alcohol consumption (NE); diamonds, fatalities with known excessive alcohol consumption (AL). The suggested cut-off of CFAEE = 1.0 ng/mg is indicated by the dotted lines. CFAEE in ng/mg (hair length in cm)

 
Axillary hair
With the exception of the volunteer SH02 (daily use of a deodorant) the concentrations in the 10 axillary hair samples were lower than in corresponding scalp hair samples in all cases (mean axillary hair/scalp hair ratio, 0.62). Furthermore, in some of the cases, a lower proportion of ethyl oleate was present, as compared with the saturated esters (Fig. 1Go, cases S317/01 and VA03). A peculiarity of the axillary region is the presence of apocrine and apoeccrine sweat glands with a special lipid metabolism (Barth et al., 1989Go; Sato et al., 1989Go; Porter, 2001Go). Furthermore, deodorants which may contain ethanol are frequently applied at these sites and this may lead to false positive results by topical formation of FAEE (Hartwig et al., 2003Go). Nevertheless, apart from case S093/01, excessive alcohol consumption was recognized from this material using the CFAEE cut-off of 1.0 ng/mg.

Beard hair
All 16 beard hair samples were collected from the chin. Of these, seven showed lower FAEE concentrations (beard/scalp FAEE concentration ratio, 0.45–0.89) and nine higher FAEE concentrations (beard/scalp, 1.2–5.7), compared with scalp hair. With alcoholics particularly (generally much higher FAEE concentrations in beard hair, e.g. S247/01), it is possible that the beard is frequently wetted by alcoholic beverages from which ethanol could be absorbed topically and transformed into FAEE in the sebum glands. A similar phenomenon may occur after use of hair lotions containing high ethanol concentrations (Hartwig et al., 2003Go). Assuming again a cut-off of 1.0 ng/mg, the interpretation of the beard hair concentrations with respect to excessive alcohol consumption leads to the same 11 positive results as with scalp hair.

Body hair
In eight cases, hair samples from the chest, arms or legs, and in one case also from the eyebrow, were analysed. Of all kinds of human hair, body hair varies most in growing conditions, length and diameter (Ebling, 1990Go). The FAEE concentrations indicated excessive alcohol consumption in four cases, in the same way as the samples from the other sites by CFAEE > 1.0 ng/mg. However, the number of samples was too small for firm conclusions to be drawn.

In conclusion, FAEE contain the ethyl group of ethanol and, therefore, are direct markers of ethanol exposure. They are deposited in all kinds of human hair after alcohol consumption. Chronic excessive alcohol consumption can be detected reliably by the presence of high FAEE concentrations in scalp, pubic, axillary, beard as well as body hair, although the actual values obtained vary markedly between different sites in the same subject. The analysis of one or more hair samples from different body sites serves to confirm scalp hair results and helps to avoid errors in interpretation caused by the use of hair cosmetics. We believe that the results of this preliminary study warrant further research on larger samples of both alcoholic and control subjects.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The authors thank the Deutsche Forschungsgemeinschaft (DFG) for the generous support of these investigations.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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