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Background: Fatty acid ethyl esters (FAEEs) are nonoxi- dative ethanol ..... oleate, and ethyl linoleate; and ions 79, 91, and 117 for ethyl arachidonate, ethyl ...
Clinical Chemistry 48:1 77– 83 (2002)

Drug Monitoring and Toxicology

Liver and Adipose Tissue Fatty Acid Ethyl Esters Obtained at Autopsy Are Postmortem Markers for Premortem Ethanol Intake Majed A. Refaai, Phan N. Nguyen, Thora S. Steffensen, Richard J. Evans, Joanne E. Cluette-Brown, and Michael Laposata*

Background: Fatty acid ethyl esters (FAEEs) are nonoxidative ethanol metabolites that have been implicated as mediators of alcohol-induced organ damage. FAEEs are detectable in the blood after ethanol ingestion, and on that basis have been proposed as markers of ethanol intake. Because blood is not always available at autopsy, in this study we quantified FAEEs in human liver and adipose tissue as potential postmortem markers of premortem ethanol intake. Methods: Twenty-four sets of samples were collected at the Massachusetts State Medical Examiner’s Office, and 7 sets of samples were obtained from the Pathology Department of Massachusetts General Hospital. Samples of liver and adipose tissue were collected at autopsy, and FAEEs were isolated and quantified from these organs as mass per gram of wet weight. Postmortem analysis of blood involved assessment for ethanol and other drugs. Results: The study shows a substantial difference in FAEE concentrations in liver and adipose tissue of patients with detectable blood ethanol at the time of autopsy vs those with no detectable blood ethanol, who were either chronic alcoholics or social drinkers. In addition, a specific FAEE, ethyl arachidonate, was found at concentrations >200 pmol/g almost exclusively in the liver and adipose tissue of individuals with detectable blood ethanol at the time of death, providing an additional FAEE-related marker for prior ethanol intake. Conclusions: The mass of FAEEs in liver and adipose tissue and the presence of ethyl arachidonate can serve

as postmortem markers of premortem ethanol intake when no blood sample can be obtained. © 2002 American Association for Clinical Chemistry

The esterification products of ethanol and fatty acids are known as fatty acid ethyl esters (FAEEs) (1–3 ). The toxic effects of these products have been shown in several in vitro and in vivo studies (4 – 6 ). FAEEs have also been demonstrated to serve as long-term markers of ethanol intake: their presence in serum and plasma has been documented in clinical studies as long as 24 h after ethanol intake has been discontinued and at a time when ethanol is no longer detectable in the blood (7, 8 ). In some cases, there is a question of premortem ethanol intake when the individual is deceased. In this situation, evaluation of ethanol intake presents additional challenges. The peripheral blood may be coagulated by the time the autopsy is performed, making it necessary to attempt a blood collection from a large blood vessel or the heart. If the blood in either of these two locations is not coagulated and is used as a sample, an accurate interpretation of ethanol in this blood sample is often not possible. It is known that the generation of ethanol by bacteria postmortem can lead to distribution of ethanol into the blood of a deceased individual and produce an artifactual blood ethanol value. In these cases, urine and/or vitreous humor ethanol concentrations are often used as an additional assessment of premortem ethanol intake. If these samples are positive for ethanol, the conclusion is that the ethanol in the blood sample was indeed reflective of premortem ethanol intake. Given the complications of this testing and the uncertainties associated with the interpretation, it would be most useful if there were additional direct markers to indicate premortem ethanol intake in the tissues that are readily available at autopsy as opposed to blood, which may be coagulated. We have recently shown that rats receiving ethanol have significantly greater concentra-

Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114. *Address correspondence to this author at: Room 235, Gray Building, Massachusetts General Hospital, Boston, MA 02114. Fax 617-726-3256; e-mail [email protected] Received August 7, 2001; accepted October 17, 2001.

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tions of FAEEs in liver and adipose tissue than control animals who received no ethanol (9 ). In this study, we obtained liver and adipose samples from 31 patients who were deceased and autopsied at the Medical Examiner’s Office in Boston, MA or the Pathology Department of the Massachusetts General Hospital in Boston, MA. We studied a group of individuals who were acutely intoxicated at the time of death and may or may not have been chronic alcoholics, a group of known chronic alcoholics with a negative blood ethanol at the time of autopsy, and a group of social drinkers. Pieces of liver and adipose tissue were collected to determine FAEE concentration in these organs. The results showed a correlation between the blood ethanol concentration and total FAEEs in the liver and the adipose. Individuals with detectable blood ethanol at the time of autopsy were readily differentiated from the chronic alcoholics and the social drinkers by the total amount of FAEEs in the liver; and ethyl arachidonate (⬎200 pmol/g) in the liver and the adipose was also able to effectively identify those individuals with a detectable blood ethanol at the time of autopsy.

Materials and Methods This study was performed using samples collected from autopsied cases at the Massachusetts Medical Examiner’s Office and the Pathology Department in Massachusetts General Hospital. The study was approved by both the Massachusetts Medical Examiner Office Committee and the Massachusetts General Hospital Pathology Quality Assurance Committee. The postmortem interval between death and autopsy ranged from 5 to 29 h, with a mean of 16 h (Table 1). The FAEE analysis was performed without any knowledge of the clinical details. Liver and adipose tissue samples were collected at the time of autopsy, labeled by case number, and stored at 4 °C for up to 10 h. Immediately after arrival in the laboratory, samples were stored at ⫺80 °C until FAEE analysis was performed. Medical history, history of ethanol ingestion (obtained from the organ bank, treating physician, health insurance records, and/or relatives), and the blood ethanol concentration at autopsy were obtained in each case. Only cases with available blood ethanol concentrations were included in this study because the hypothesis was that FAEE concentrations in liver and adipose tissue would predict those with a positive blood ethanol. After the FAEE analysis was completed, cases were evaluated and classified into three groups. Individuals who had detectable blood ethanol at the time of autopsy, with or without a history of ethanol abuse, were grouped as “positive for ethanol”. This group most likely included chronic alcoholics and/or social drinkers who ingested an excessive amount of ethanol before death. Because the goal of the diagnostic test in question involves assessment of ethanol intake before death, the inclusion or exclusion of chronic alcoholics in this group is not a confounding variable. In this group, 10 of 15 cases were found to have organ damage from ethanol abuse (in 5 cases, ethanol

abuse was accompanied by abuse of other drugs), 3 cases were involved in automobile accidents, 1 case was associated with carbon monoxide poisoning, and 1 was an apparent sudden cardiac arrest. Individuals in the second group were chronic alcoholics with a negative blood ethanol at the time of death, with chronic alcoholism diagnosed according to the Diagnostic and Statistical Manual-IV (DSM-IV) (10 ). In this group, three of seven individuals showed organ damage from ethanol and drug abuse, three had cardiac and liver abnormalities, and one was cirrhotic. Some medical records did not contain sufficient information to make a definitive diagnosis of chronic alcoholism by the DSM-IV criteria alone. To limit the number of patients who were excluded because of an incomplete medical history, we developed non-DSM criteria to diagnose chronic alcoholism. A person received a diagnosis of chronic alcoholism if he or she met at least one of the following non-DSM criteria: (a) two or more hospital visits within a 2-year time period with a detectable blood ethanol concentration and abnormal liver function tests and/or pancreatic function tests; (b) admitted to being dependent on ethanol (a history of more than six drinks/ day for more than 2 years); (c) survived a blood ethanol concentration ⬎4000 mg/L; (d) had a hospital visit with a detectable blood ethanol concentration and signs of ethanol withdrawal or other clinical signs of prolonged ethanol intake, such as cirrhosis, esophageal varices, or pancreatitis; (e) admitted to being a member of Alcoholics Anonymous; or (f) had a documented medical history of at least one ethanol detoxification. The third group was a collection of social drinkers. A social drinker, as defined by the National Institute for Alcohol Abuse and Alcoholism, is an individual who consumes 14 drinks or fewer per week (11 ). In this group, five of nine cases had coronary artery disease as a cause of death. There was also one case of hepatitis, one with seizure disorder, one case with head trauma, and one individual with carbon monoxide and isopropanol toxicity.

faee extraction and quantification A portion of each tissue was harvested, weighed, and immediately placed on ice, then homogenized (1 g of tissue in 10 mL of buffer) in protease inhibitor buffer containing 10 mmol/L HEPES, 20 mg/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, and 0.1 g/L soybean trypsin inhibitor (pH 7.34) in a Fisher PowerGen 125 Homogenizer (Fisher Scientific) equipped with a 10 ⫻ 195 mm sawtooth generator. An internal standard of 500 pmol of ethyl heptadecanoate (Nu Chek Prep) was added to 1 mL of the homogenate along with 2 mL of cold acetone. The sample was then vortex-mixed for 1 min and centrifuged for 5 min at 170g at 4 °C, and the supernatant was transferred to a separate tube. Hexane (6 mL) was then added to each sample. The mixture was vortexmixed for 1 min and centrifuged at 170g for 5 min at 4 °C.

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Table 1. Descriptions of individuals in this study. Total FAEEs, pmol/g

Individuals with detectable blood ethanol at the time of autopsy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Chronic alcoholism (according to the medical record) 1 2 3 4 5 6 7 Social drinkers (according to the medical record) 1 2 3 4 5 6 7 8 9 a b

Age, years

Sex

Ethanol at the time of autopsy, mg/L

Liver

35 47 46 25 43 43 63 35 63 30 35 37 47 30 47

F M M M F F M M M M M M M M F

1820 1480 2220 240 1460 960 270 1670 2260 2320 1800 160 1400 1880 540

105 269 71 436 226 152 1235 102 916 40 219 14 521 100 062 101 588 568 723 193 511 69 125 322 447 183 298 185 168

15 878 NA 61 539 3611 51 430 5102 2723 16 844 44 310 75 699 9005 25 296 19 720 15 306 2987

12 10 10 ⬎20 15 9 ⬎11 6 8 7 10 15 9 6 8

45 42 46 35 46 47 44

M F M F M M M

0 0 0 0 0 0 0

853 871 5315 193 196 0 2226

17 287 271 869 569 787 2003 94 983

12 12 10 11 24 3 23

Cirrhosis ETOH and drug abuse/hospitalized Cardiomegaly and endocarditis Asthma, ETOH and drug abuse/hospitalized Cardiomegaly and hepatomegaly CAD and fatty liver ETOH and drug abuse, cirrhosis/hospitalized

42 63 49 46 23 42 41 40 40

F M M M M M M M M

0 0 0 0 0 0 0 0 0

988 0 306 1034 0 73 5485 402 3389

0 0 460 274 83 640 145 0 254

4 5 10 8 3 27 15 8 47

CAD, asthma, diabetes CAD CAD Hepatitis C Seizure disorder Cardiac arrest CO and isopropanol toxicity/depression CAD Blunt head trauma

Adipose PMI,a h

Relevant clinical and toxicology findings

Depression/ETOH abuse/suicide ETOH abuse/cirrhosis Heroin use/ETOH abuse Acute narcotic intoxicationb/ETOH abuse ETOH and cocaine abuse ETOH abuse and CAD Suicide/CO poisoning Opiate and ETOH intoxication Auto accident Auto accident ETOH and cocaine abuse Depression and heavy drinker Cardiac arrest Auto accident ETOH abuse

PMI, postmortem interval; ETOH, ethanol; CAD, coronary artery disease; CO, carbon monoxide; NA, not applicable. The toxicology findings for this individual were 6-monoacetylmorphine (13 ␮g/L), free morphine (295 ␮g/L), and ethanol (240 mg/L).

The hexane layer was transferred to a separate tube, and the aqueous phase was reextracted with an additional 2 mL of hexane. The wash was pooled with the original hexane layer, evaporated to dryness under nitrogen, and resuspended in 200 ␮L of hexane. A recent report showed improved recovery of FAEEs when smaller sample volumes were used for extraction (12 ). FAEEs were isolated from the lipid extract by solidphase extraction. Briefly, aminopropyl columns (BondElut LRC; Varian Diagnostics) were placed on a Vac-Elut vacuum apparatus (Varian Diagnostics) set at 10 kPa. The columns were preconditioned with 4 mL of hexane, followed by 4 mL of dichloromethane. The 200-␮L aliquot

of lipid extract was then applied, and FAEEs were eluted from the column with an additional 4 mL of hexane and 4 mL of dichloromethane. The eluate was next evaporated to a volume of 50 ␮L, and a 1-␮L aliquot was injected into a Hewlett-Packard 5971 mass spectrometer equipped with a Supelcowax 10 capillary column (Supelco, Inc.). The injector and detector were maintained at 260 and 280 °C, respectively. The oven program was initially maintained at 150 °C for 2 min, then ramped to 200 °C at 10 °C/min for 4 min, ramped again at 5 °C/min to 240 °C and held for 3 min, and finally ramped to 270 °C at 10 °C/min and held for 5 min. Carrier gas flow rate was maintained at a constant 0.75 mL/min throughout. Sin-

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gle-ion monitoring was performed to quantify appropriate base ions for individual FAEEs: ions 67, 88, and 101 for ethyl palmitate, ethyl heptadecanoate, ethyl stearate, ethyl oleate, and ethyl linoleate; and ions 79, 91, and 117 for ethyl arachidonate, ethyl eicosapentaenoate, and ethyl docosahexaenoate. FAEEs were quantified by interpolation of the slope of the calibration curve, generated by plotting FAEE/ethyl heptadecanoate peak-height ratios to concentration ratios. Total FAEE mass was determined by totaling the masses of the individual FAEEs listed above. All manipulations were performed under nitrogen to make any losses of FAEEs with polyunsaturated fatty acids negligible.

quantification of blood ethanol from autopsy specimens Ethanol was assayed in a gas chromatograph with a flame ionization detector and an electronic integrator. The serum was diluted with internal standard (an aqueous solution of 1-propanol). The mixture was injected directly into the gas chromatograph (13 ).

Results Descriptions of the patients in the three groups are given in Table 1. The individuals with detectable blood ethanol at the time of autopsy had extremely high FAEE concentrations in the liver and the adipose tissue. The concentrations in the liver nearly always exceeded the concentrations in the adipose tissue in this group. The one patient whose adipose concentration exceeded that of the liver had a cause of death that was labeled as “narcotic intoxication”. This patient was an outlier according to both markers, which correctly identified those individuals with positive blood ethanol at the time of autopsy. It is possible that this outlier may actually represent an individual whose blood ethanol was not reflective of premortem ethanol intake or that one of the illicit substances that the patient had taken somehow gave a false-positive blood ethanol test. Five of seven individuals with chronic alcoholism and negative blood ethanol at the time of autopsy showed higher FAEE concentrations in adipose tissue than in liver. This is not unexpected for chronic alcoholics because adipose tissue is a storage organ for FAEEs (14 ). The documentation of chronic alcoholism was established by fixed criteria in a review of the medical records. The social drinkers had the lowest FAEE concentrations in both liver and adipose tissue. Correlations of blood ethanol concentration to FAEE mass in the liver and in the adipose tissue were significant. The correlation coefficient (r) was 0.751 in liver and 0.721 in adipose tissue, showing the metabolic connection between blood ethanol and its nonoxidative ethanol derivatives, FAEEs, in the liver and adipose. The total-FAEE distribution of the individuals in each of the groups is shown in Fig. 1, which also shows that, with the exception of the outlier, there was a complete separation in total FAEEs between the individuals with

Fig. 1. Comparison of total-FAEE concentrations in liver samples from individuals with detectable blood ethanol at the time of autopsy (n ⫽ 15), chronic alcoholics (n ⫽ 7), and social drinkers (n ⫽ 9). Error bars, ⫾ SE.

detectable blood ethanol at the time of autopsy and the chronic alcoholics and social drinkers. This indicates that total FAEEs in the liver are extremely effective markers in a postmortem setting for premortem ethanol intake. It should be noted that the y axis in Fig. 1 is on a logarithmic scale. The highest value for total FAEEs in liver for chronic alcoholics and for social drinkers was 5485 pmol/g, and the lowest value for the individuals with detectable blood ethanol at the time of autopsy, other than the one outlier, was 14 521 pmol/g. This is approximately a threefold difference between the highest concentration in the chronic alcoholics and social drinkers vs the lowest concentration in the individuals with detectable blood ethanol at the time of death. The sensitivity for this test to identify individuals with detectable blood ethanol at the time of autopsy vs chronic alcoholics and social drinkers with negative blood ethanol at the time of autopsy was 93%, with a specificity of 100%. The total-FAEE concentrations in adipose tissue for the individuals in each group are shown in Fig. 2. There was some overlap between the chronic alcoholics and the individuals with detectable blood ethanol at the time of autopsy. This is not surprising because FAEEs have been

Fig. 2. Comparison of total-FAEE concentrations in adipose tissue from individuals with detectable blood ethanol at the time of autopsy (n ⫽ 14), chronic alcoholics (n ⫽ 7), and social drinkers (n ⫽ 9). Error bars, ⫾ SE.

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Fig. 5. Ethyl arachidonate concentrations in liver samples from individuals with detectable blood ethanol at the time of autopsy (n ⫽ 15), chronic alcoholics (n ⫽ 7), and social drinkers (n ⫽ 9). Fig. 3. Mean concentrations of individual FAEE species (by percentage) in liver samples from individuals with detectable blood ethanol at the time of autopsy (n ⫽ 15), chronic alcoholics (n ⫽ 7), and social drinkers (n ⫽ 9). Error bars, ⫾ SE. 16:0, ethyl palmitate; 18:0, ethyl stearate; 18:1, ethyl oleate; 18:2, ethyl linoleate; 20:4, ethyl arachidonate.

shown to be stored in adipose tissue in chronic alcoholics (15 ). If these individuals had ingested alcohol within days before their deaths, FAEEs would likely have still been present in the adipose tissue. In contrast, total FAEEs in the adipose tissue of social drinkers were well below the concentrations found in individuals with detectable blood ethanol at time of autopsy. The sensitivity of the test to detect ethanol ingestion before death by the concentration of total FAEEs in adipose tissue was 100%, with a specificity of 88%. The distribution of the individual fatty acids in the FAEEs in the liver are shown in Fig. 3, and the distribution of the fatty acids in the FAEEs in adipose tissue is shown in Fig. 4. There are several findings of note. Ethyl arachidonate in both liver and adipose tissue was markedly higher in individuals with detectable blood ethanol at the time of autopsy than in chronic alcoholics and social

Fig. 4. Mean concentrations of individual FAEE species (by percentage) in adipose tissue from individuals with detectable blood ethanol at the time of autopsy (n ⫽ 14), chronic alcoholics (n ⫽ 7), and social drinkers (n ⫽ 9). Error bars, ⫾ SE. 16:0, ethyl palmitate; 18:0, ethyl stearate; 18:1, ethyl oleate; 18:2, ethyl linoleate; 20:4, ethyl arachidonate.

drinkers. The data in Figs. 5 and 6 show the ethyl arachidonate concentrations in individual cases. The presence of ethyl arachidonate at a concentration ⬎200 pmol/g in either liver and adipose tissue also differentiated individuals with detectable blood ethanol at the time of autopsy.

Discussion The results of this study indicate that the total FAEEs in liver and adipose tissue and the presence of ethyl arachidonate in both the liver and adipose are useful autopsy markers of premortem ethanol intake. Because measuring the ethanol concentration in the blood, fluids, and/or organs is not always possible, many biologic markers other than ethanol itself serve as either short- or long-term markers for ethanol intake. Some of these markers that have been evaluated in different studies for ethanol ingestion include FAEEs (6 ), carbohydratedeficient transferrin (16 ), acetaldehyde (17 ), phosphatidylethanol (18 ), ethylglucuronide (19 ), and 5-hydroxytryptophol (20 ). FAEEs have also been found in the hair of alcoholics, in the extractable hair lipids originating mainly from sebum but also from structural lipids of cell membranes (21 ). Ethyl palmitate, ethyl stearate, and ethyl oleate were unambiguously identified in hair samples from three alcoholics (22 ). For FAEE analysis, the liver was selected over the

Fig. 6. Ethyl arachidonate concentrations in adipose tissue from individuals with detectable blood ethanol at the time of autopsy (n ⫽ 14), chronic alcoholics (n ⫽ 7), and social drinkers (n ⫽ 9).

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pancreas because the pancreas degrades rapidly after death and the liver tends to remain intact for a longer period. Adipose tissue was selected because it was expected that there would be a longer half-life of the FAEEs

in the adipose and that there would be multiple sites for sampling adipose tissue to allow determination of FAEEs. An algorithm indicating how FAEE concentrations in the liver and adipose might be used diagnostically is

Fig. 7. Algorithm indicating how FAEE concentrations in liver and adipose tissue can be used diagnostically. ETOH, ethanol.

Clinical Chemistry 48, No. 1, 2002

shown in Fig. 7. This study adds an important diagnostic test for tissue concentrations of FAEEs to the already published assays for FAEEs in the blood because blood is not always available for analysis. The algorithm begins with a test for blood ethanol. If the blood ethanol is ⬎100 mg/L in an autopsy from which a sample for blood ethanol is available, the question of whether the detected ethanol was generated by bacteria can be further verified with urine and/or vitreous ethanol concentrations or liver and adipose FAEE concentrations. A value ⬎10 000 pmol/g for liver FAEEs provides evidence of premortem ethanol intake. Because ethyl arachidonate was found only in individuals with detectable blood ethanol at the time of autopsy, a measured value ⬎ 200 pmol/g in liver and/or adipose tissue also indicates premortem ethanol intake (9 ). The histologic changes in liver and pancreas should assist in differentiating chronic alcohol abusers from social drinkers. In conclusion, we describe two new indices that can be used as postmortem markers for premortem ethanol intake, either in cases in which there is no blood available to determine the ethanol concentration or when there is a desire to confirm the blood ethanol concentration.

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