1 Psychiatric University Hospital and 2 Department Central Laboratory, University of Basel, Switzerland, 3 Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Switzerland, 4 Department of Medical Neurochemistry, Lund, Sweden, 5 State Mental Hospital Bayreuth, 6 Department of Legal Medicine, University of Magdeburg, Magdeburg and 7 Institute of Legal Medicine, Humboldt University, Berlin, Germany and 8 Pacific Institute for Research and Evaluation, Calverton, MD, USA
* Author to whom correspondence should be addressed at: Psychiatric University Hospital, University of Basel, Wilhelm Klein Strasse 27, CH-4025 Basel, Switzerland. Tel.: +41 61 325 5512; Fax: +41 61 325 5583; E-mail: friedrich.wurst{at}pukbasel.ch
(Received 17 February 2003; first review notified 11 April 2003; in revised form 23 September 2003; accepted 30 September 2003)
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ABSTRACT |
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INTRODUCTION |
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Especially during the last decade, three non-oxidative direct ethanol metabolites have attracted attention. Promising markers of this type include fatty acid ethyl esters (FAEE) (Wada et al., 1971; Doyle et al., 1994
, 1996
; Dan and Laposata, 1997
; Diczfalusy et al., 1999
, 2001
), ethyl glucuronide (EtG) (Schmitt et al., 1995
; Alt et al., 2000
; Seidl et al., 2001
; Wurst et al. 1999a
,b
, 2000
, 2002
, 2003
), and phosphatidyl ethanol (PEth) (Alling et al., 1983
, 1984
; Hansson et al., 1997
; Gunnarsson et al., 1998
; Varga et al., 1998
, 2000
). Each of these remains positive in serum and urine for a characteristic time spectrum after cessation of alcohol intake: FAEE in serum up to 24 h, EtG in urine up to 5 days, PEth in whole blood more than 2 weeks.
Recent studies have supported the use of phosphatidyl ethanol (PEth) in blood as a marker of alcohol misuse. Chronic alcohol-dependent patients admitted for detoxification had mean PEth levels of 13.2 µmol/l on the first day, and levels remained detectable up to 14 days after admission (Hansson et al., 1997). Using liquid chromatography electrospray mass spectrometric detection (HPLCELSD), PEth has been detected in extracts of blood from alcoholics (Gunnarsson et al., 1998
). These patients had PEth levels of 513 µmol/l, which remained detectable up to 3 weeks after the beginning of an alcohol-free period. A third study on chronic alcoholics showed mean PEth levels of 2.5 and 5.1 µmol/l in two different groups respectively (Varga et al., 2000
). A study on healthy volunteers revealed that a single dose of ethanol (3247 g) does not produce measurable amounts of PEth (Varga et al., 1998
). However, out of twelve volunteers who consumed between 624 and 2134 g of ethanol during a 3 week period, eight persons produced levels of PEth of 1.02.1 µmol/l. A threshold of total ethanol intake yielding detectable PEth seems to be around 1000 g, with a mean daily intake of about 50 g. Thus far, analysis of PEth has been performed using whole blood, however, a recent study on blood from chronic alcoholics showed that almost all PEth was found in the erythrocyte fraction (Varga et al., 2000
).
Fatty acid esters of ethanol (FAEE) have been implicated as possible mediators for at least some of the toxic effects associated with alcohol consumption, as short term markers of ethanol intake in serum and as long term markers in hair. Direct toxic effects of FAEE have not been demonstrated (Laposata and Lange, 1986; Doyle et al., 1996
; Dan and Laposata, 1997
; Soderberg et al., 1999
), although these esters have been shown to uncouple mitochondrial oxidative phosphorylation in rabbit heart (Lange and Sobel, 1983
), inhibit protein synthesis and cell proliferation in human hepatoblastoma cells (Szczypiorkowski et al., 1995
) and render pancreatic lysosomes more fragile (Haber et al., 1993
).
Two enzymatic activities catalyse the formation of FAEE: acyl-coenzyme A:ethanol O-acyltransferase (AEAT) and fatty acid ethyl ester (FAEE) synthases. FAEE synthases have been purified from several sources, including rabbit myocardium, human brain and rat adipose tissue, and two of these FAEE synthases were found to be identical to rat liver carboxylesterase (Tsujita and Okuda, 1992; Bora et al., 1996
). Furthermore, a variety of other enzymes (e.g. pancreatic lipase, lipoprotein lipase and gluthathione transferases) reveal FAEE synthase activity (Tsujita and Okuda, 1992
; Bora et al., 1989
; Riley et al., 1990
; Tsujita and Okuda, 1994
; Chang et al., 1997
). At present there is no evidence for the existence of a specific FAEE synthase and the formation of FAEE from free fatty acids seems to result from the interplay of a host of enzymes and other functions. The enzyme responsible for most of the AEAT activity in rat liver is localized at the lumenal side of the endoplasmic reticulum (Polokoff and Bell, 1978
), but has not yet been identified or purified. Characterization of AEAT and FAEE synthase activities using isolated rat liver microsomes and human tissue homogenates suggests that AEAT is quantitatively the more important of these two activities (Diczfalusy et al., 1999
, 2001
).
FAEE have also been suggested as markers of ethanol intake. In serum and erythrocytes, FAEE can be detected up to 24 h after drinking. They are not stable in blood samples due to continued enzyme activity, but are incorporated into hair and can be analysed in this medium even after several months (Pragst et al., 2001). In these investigations, the sum of the concentrations (CFAEE) of the four esters: ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate, was used as an alcohol intake marker. The incorporation was found to occur mainly from sebum into the completed hair, which results in an increase of CFAEE by accumulation from proximal to distal hair sections (Auwärter et al., 2001
). Beside the effects of inter-individual differences in the sebum production, variables such as the kind and frequency of hair care and hair cosmetics can influence the efficiency of deposition (Hartwig et al., 2003a
). The differences in CFAEE between hair samples from different sites of the same individual can be explained in the same way (Hartwig et al., 2003b
).
Based on earlier investigation of hair samples from fatalities with known excessive alcohol consumption, patients in withdrawal treatment, social drinkers and teetotallers, a cut-off value for CFAEE of 1.0 ng/mg has been suggested to establish heavy alcohol use (Auwärter et al., 2001; Hartwig et al., 2003b
).
Our aim in this project is to further evaluate FAEE in hair as an indicator of chronic intake of high amounts of ethanol and to consider the effect of using a lower cut-off to identify heavy drinking/alcohol dependence by comparing FAEE in hair with self reports, PEth in whole blood and traditional markers (GGT, MCV, CDT).
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SUBJECTS AND METHODS |
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Data on alcohol use were obtained by using the timeline follow back procedure. The alcohol intake of the last month was reported to be representative for the last half year. Blood samples were taken at day 1 and hair samples at day 7 of hospitalization.
For comparison, previously published data from 10 social drinkers and 10 teetotallers (Pragst et al., 2001) were employed. The comparison sample included seven male and 13 female subjects with a mean age of 32.9 (SD 17.9) years. The self reported ethanol consumption in grams per day was 52 (mean, SD12.3, mean 60) for the social drinkers and zero for the teetotallers. For the teetotallers, CFAEE was 0.0 mean (SD 0.0, median 0.0) and for the social drinkers 0.21 mean (SD 0.1, mean 0.7) [ng/mg]. In the Kruskal Wallis test the three groups (teetotallers, social drinkers, alcoholics) were significantly different (P < 0.0001).
The study was reviewed by the Bavarian Chamber of Physicians Ethical Committee and all patients gave written informed consent.
Methods
FAEE in hair
The analytical determination of the four FAEE was performed by external decontamination of the hair with n-heptane, liquid extraction with a dimethylsulphoxide/ n-heptane mixture, separation and evaporation of the n-heptane layer, solid phase micro-extraction of the residue and gas chromatography-mass spectrometry using deuterated standards of all four esters. The experimental details, limits of detection, assay reproducibility and standard deviations have been described elsewhere (Pragst et al., 2001; Hartwig et al., 2003b
).
Phosphatidyl ethanol in whole blood
PEth was measured in heparinized whole blood as described elsewhere by high pressure liquid chromatography (HPLC) combined with an evaporative light-scattering detector (ELSD) method (Varga et al., 2000).
%CDT in serum
CDT estimation was performed on duplicate serum samples using the %CDT turbidimetric immunoassay kit (Bio-Rad Laboratories, Philadelphia, PA, USA) according to the manufacturer's instructions. This method is based on micro anion-exchange chromatography followed by turbidimetric measurement. Isoelectric focusing to exclude rare genetic D-variants was undertaken as previously described followed by semi-quantitative evaluation by means of a scanner (Bean and James, 1994; Kuchheuser et al., 1995
).
Cut-off values The cut-off of 1.0 ng/mg for CFAEE was compared with 0.4 ng/mg, as suggested by data from several studies and from ROC curve analysis. In addition, the conventional cut-off (0.4 ng/mg) for ethyl palmitate, which usually accounts for about 40% of CFAEE, was compared with a cut-off of 0.16 ng/mg.
Statistical analysis
For statistical analysis (descriptive statistics, Spearman rank correlation, Kruskal Wallis test) SPSS 11 was used (SPSS Inc., Chicago, IL).
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RESULTS |
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Correlations were not significant between CFAEE and total grams of ethanol consumed last month, blood-alcohol concentration at admission to hospital, hair length, CDT, MCV, GGT, age, number of previous detoxifications, number of previous admissions to hospital, age of first hospitalization, age of onset of disorder, years of duration of disorder and total amount of clomethiazole required for the treatment of withdrawal symptoms.
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DISCUSSION |
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ROC curve analysis indicated a sensitivity for CFAEE of 100% and a specificity of 90% at a cut-off of 0.29 and sensitivity of 94.4% and a specificity of 90%, respectively, for a cut-off of 0.46 ng/mg in distinguishing teetotallers/social drinkers and heavy drinkers/alcohol dependency (AUC = 0.983). In contrast, only 55.6% of alcohol-dependent patients with previous chronic ethanol intake were identified with the cut-off of 1.0 ng/mg.
As in previous investigations, concentrations of FAEE were also found in hair of teetotallers (Auwärter et al., 2001), but CFAEE was never above 0.4 ng/mg. The origin of FAEE in these cases is not yet clear. Generally social drinkers have CFAEE <0.4 ng/mg, but there were also some cases of social drinkers with CFAAE up to 0.8 ng/mg. However, the possibility can not be dismissed that some of those individuals, although currently being non-dependent social drinkers, drank at levels comparable to those of alcohol-dependent patients. Furthermore, it is seen from the cases described in this study, as well as from the patients on withdrawal treatment described previously (Auwärter et al., 2001
), that a cut-off of 1.0 ng/mg leads to excessive numbers of negative results. In contrast to the post-mortem cases with clearly higher CFAEE, the patients investigated here during inpatient detoxification washed their hair regularly, thus decreasing FAEE incorporation from sebum.
Interesting as well and meriting further research attention is the near-comparable performance of ethyl palmitate in isolation to CFAEE in this study. Nevertheless, CFAEE is the more usual criterion for ethanol consumption and additionally, possible external contamination can more likely be excluded by the ratio of the four esters.
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