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Liver and adipose tissue fatty acid ethyl esters obtained at autopsy are postmortem markers for premortem ethanol intake.

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 concentrations 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 FREE 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 FREE 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 [degrees]C for up to 10 h. hninediately after arrival in the laboratory, samples were stored at -80 [degrees]C until FREE 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 FREE 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-W) (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 x 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 1708 at 4 [degrees]C, and the supernatant was transferred to a separate tube. Hexane (6 mL) was then added to each sample. The mixture was vortex-mixed for 1 min and centrifuged at 1708 for 5 min at 4 [degrees]C. 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 [micro]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-[micro]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 elutee was next evaporated to a volume of 50 [micro]L, and a 1-[micro]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 [degrees]C, respectively. The oven program was initially maintained at 150 [degrees]C for 2 min, then ramped to 200 [degrees]C at 10 [degrees]C/min for 4 min, ramped again at 5 [degrees]C/min to 240 [degrees]C and held for 3 min, and finally ramped to 270 [degrees]C at 10 [degrees]C/min and held for 5 min. Carrier gas flow rate was maintained at a constant 0.75 mL/min throughout. Single-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 FREE/ethyl heptadecanoate peak-height ratios to concentration ratios. Total FREE 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 FREE 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[degrees]. 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 FREE 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 FREE concentrations in both liver and adipose tissue.

Correlations of blood ethanol concentration to FREE 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-FREE 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 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%.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The total-FREE 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 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%.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

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 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.

[FIGURE 5 OMITTED]

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), carbohydrate-deficient 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).

[FIGURE 6 OMITTED]

For FREE analysis, the liver was selected over the 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.

[FIGURE 7 OMITTED]

An algorithm indicating how FREE concentrations in the liver and adipose might be used diagnostically is 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 FREE 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.

Received August 7, 2001; accepted October 17, 2001.

References

(1.) Newsome WH, Rattray JBM. The enzymatic esterification of ethanol with fatty acids. Can J Biochem 1965;43:1223-33.

(2.) Goodman DS, Deykin D. Fatty acid ethyl ester formation during ethanol metabolism in vivo. Proc Soc Exp Biol Med 1963;113:65-70.

(3.) Lange LG, Bergmann SR, Sobel RE. Identification of fatty acid ethyl esters as products of myocardial ethanol metabolism. J Biol Chem 1981; 256:12968-73.

(4.) Laposata M, Szczepiorkowski ZM, Cluette-Brown JE. Fatty acid ethyl esters: non-oxidative metabolites of ethanol. Prostaglandins Leukotrienes Essent Fatty Acids 1995;52:87-91.

(5.) Laposata EA, Lange LG. Presence of nonoxidative ethanol metabolism in human organs commonly damaged by ethanol abuse. Science 1986;231:497-9.

(6.) Laposata M. Fatty acid ethyl esters: ethanol metabolites which mediate ethanol induced organ damage and serve as a marker of ethanol intake. Prog Lipid Res 1998;37:307-16.

(7.) Doyle KM, Bird DA, AI-Salihi S, Hallaq Y, Cluette-Brown JE, Goss KA, Laposata M. Fatty acid ethyl esters are present in human serum after ethanol ingestion. J Lipid Res 1994;35:428-37.

(8.) Doyle KM, Cluette-Brown JE, Dube DM, Brenhardt TG, Morse CR, Laposata M. Fatty acid ethyl esters in the blood as markers for ethanol intake. JAMA 1996;276:1152-6.

(9.) Salem, R0, Refaai M, Cluette-Brown JE, Laposata M. Fatty acid ethyl esters in liver and adipose tissues as postmortem markers for ethanol intake. Clin Chem 2001;47:722-5.

(10.) American Psychiatric Association. Substance-related disorders. In: Diagnostic and statistical manual of mental disorders-IV, 4th ed. Washington: American Psychiatric Association, 1994:175-273.

(11.) Meister KA, Whelan EM, Kava R. The health effects of moderate alcohol intake in humans: an epidemic review. Crit Rev Clin Lab Sci 2000;37:261-96.

(12.) Zybko WC, Cluette-Brown JE, Laposata M. Improved sensitivity and reduced sample size in serum fatty acid ethyl ester analysis. Clin Chem 2001;47:1120-1.

(13.) Porter WH. Clinical toxicology. In: Burtis CA, Ashwood ER, eds. Tietz textbook of clinical chemistry, 3rd ed. Philadelphia: WB Saunders, 1999:906-81.

(14.) Laposata EA, Scherrer DE, Lange LG. Fatty acid ethyl esters in adipose tissues as a laboratory marker for alcohol related death. Arch Pathol Lab Med 1989;113:762-6.

(15.) DePergola G, Kjellstrom C, Holm C, Conradi N, Pettersson P, Bjorntorp P. The metabolism of ethyl esters of fatty acids in adipose tissue of rats chronically exposed to ethanol. Alcohol Clin Exp Res 1991;15:184-9.

(16.) Bean P. Carbohydrate-deficient transferring: what have we learned in the last decade? Am Clin Lab 2001;20:8-10.

(17.) Hernandez-Munoz R, Ma XL, Baraona E, Lieber CS. Method of acetaldehyde measurement with minimal artifactual formation in red blood cells and plasma of actively drinking subjects with alcoholism. J Lab Clin Med 1992;120:35-41.

(18.) Hansson P, Caron M, Johnson G, Gustavsson L, Alling C. Blood phosphatidylethanol as a marker of alcohol abuse: levels in alcoholic males during withdrawal. Alcohol Clin Exp Treat 1997; 21:108-10.

(19.) Wurst FM, Kempter C, Metzger J, Seidl S, Alt A. Ethyl glucuronide: a marker of recent alcohol consumption with clinical and forensic implications. Alcohol 2000;20:111-6.

(20.) Helander A, Beck 0, Jones AW. Laboratory testing for recent alcohol consumption: comparison of ethanol, methanol, and 5-hydroxtyptophol. Clin Chem 1996;42:618-24.

(21.) Robbins CR. Chemical and physical behavior of human hair. New York: Springer Verlag, 1994:71,79-84.

(22.) Pargst F, Spiegel K, Sporkert F, Bohnenkamp M. Are there possibilities for the detection of chronically increased alcohol consumption by hair analysis? Forensic Sci Int 2000;107:201-23.

MAJED A. REFAAI, PHAN N. NGUYEN, THORA S. STEFFENSEN, RICHARD J. EVANS, JOANNE E. CLUETTE-BROWN, and MICHAEL LAPOSATA *

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 mlaposata@partners.org.
Table 1. Descriptions of individuals in this study.

 Ethanol
 at the
 time of Total FAEEs, pmol/g
 Age, autopsy,
 years Sex mg/L Liver Adipose

Individuals
with detectable
blood ethanol
at the time of
autopsy

1 35 F 1820 105 269 15 878
2 47 M 1480 71 436 NA
3 46 M 2220 226 152 61 539
4 25 M 240 1235 3611
5 43 F 1460 102 916 51 430
6 43 F 960 40 219 5102
7 63 M 270 14 521 2723
8 35 M 1670 100 062 16 844
9 63 M 2260 101 588 44 310
10 30 M 2320 568 723 75 699
11 35 M 1800 193 511 9005
12 37 M 160 69 125 25 296
13 47 M 1400 322 447 19 720
14 30 M 1880 183 298 15 306
15 47 F 540 185 168 2987

Chronic
alcoholism
(according to
the medical
record)

1 45 M 0 853 17 287
2 42 F 0 871 271
3 46 M 0 5315 869
4 35 F 0 193 569
5 46 M 0 196 787
6 47 M 0 0 2003
7 44 M 0 2226 94 983

Social drinkers
(according to
the medical
record)

1 42 F 0 988 0
2 63 M 0 0 0
3 49 M 0 306 460
4 46 M 0 1034 274
5 23 M 0 0 83
6 42 M 0 73 640
7 41 M 0 5485 145
8 40 M 0 402 0
9 40 M 0 3389 254

 PMI,
 (a) h Relevant clinical and toxicology findings

Individuals
with detectable
blood ethanol
at the time of
autopsy

1 12 Depression/ETOH abuse/suicide
2 10 ETOH abuse/cirrhosis
3 10 Heroin use/ETOH abuse
4 >20 Acute narcotic intoxication2/ETOH abuse
5 15 ETOH and cocaine abuse
6 9 ETOH abuse and CAD
7 >11 Suicide/CO poisoning
8 6 Opiate and ETOH intoxication
9 8 Auto accident
10 7 Auto accident
11 10 ETOH and cocaine abuse
12 15 Depression and heavy drinker
13 9 Cardiac arrest
14 6 Auto accident
15 8 ETOH abuse

Chronic
alcoholism
(according to
the medical
record)

1 12 Cirrhosis
2 12 ETOH and drug abuse/hospitalized
3 10 Cardiomegaly and endocarditis
4 11 Asthma, ETOH and drug abuse/hospitalized
5 24 Cardiomegaly and hepatomegaly
6 3 CAD and fatty liver
7 23 ETOH and drug abuse, cirrhosis/
 hospitalized

Social drinkers
(according to
the medical
record)

1 4 CAD, asthma, diabetes
2 5 CAD
3 10 CAD
4 8 Hepatitis C
5 3 Seizure disorder
6 27 Cardiac arrest
7 15 CO and isopropanol toxicity/depression
8 8 CAD
9 47 Blunt head trauma

(a) PMI, postmortem interval; ETOH, ethanol; CAD, coronary artery
disease; CO, carbon monoxide; NA, not applicable.

(2) The toxicology findings for this individual were
6-monoacetylmorphine (13 [micro]g/L), free morphine
(295 [micro]g/L), and ethanol (240 mg/L).
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Title Annotation:Drug Monitoring and Toxicology
Author:Refaai, Majed A.; Nguyen, Phan N.; Steffensen, Thora S.; Evans, Richard J.; Cluette-Brown, Joanne E.
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Jan 1, 2002
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