Fatty acid ethyl esters in hair as markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers, and teetotalers.

BACKGROUND: Fatty acid ethyl esters (FAEEs) are products of nonoxidative ethanol metabolism. After incorporation in hair, they should be suitable long-term markers of alcohol abuse. METHODS: Hair samples from 19 alcoholics in a treatment program, 10 fatalities with verified excessive alcohol consumption, 13 moderate social drinkers who consumed up to 20 g ethanol/day, and 5 strict teetotalers were analyzed in 1-12 segments for four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) by external degreasing with n-heptane, extraction with a dimethyl sulfoxide-n-heptane mixture, headspace solid-phase microextraction of the extracts, and gas chromatography-mass spectrometry with deuterated internal standards. The n-heptane washings were analyzed in the same way for FAEEs from the hair surface. RESULTS: The sum of the four ester concentrations in hair calculated for the proximal 0-6 cm segment was 2.5-13.5 ng/mg (mean, 6.8 ng/mg) for the fatalities, 0.92-11.6 ng/mg (mean, 4.0 ng/mg) for 17 of the alcoholics in treatment, 0.20-0.85 ng/mg (mean, 0.41 ng/mg) for the moderate social drinkers, and 0.06-0.37 ng/mg (mean, 0.16 ng/mg) for the teetotalers. In almost all cases the segmental concentrations increased from proximal to distal. There was no agreement between the self-reported drinking histories of the participants and the FAEE concentrations along the hair length. Ethyl oleate was the dominant ester in all samples. CONCLUSIONS: FAEEs are deposited in hair mainly from sebum. Despite large individual differences, FAEE hair concentrations can be used as markers for excessive alcohol consumption with relatively high accuracy.     

Preanalytical variables affecting the quantification of fatty acid ethyl esters in plasma and serum samples

BACKGROUND: Fatty acid ethyl esters (FAEEs) are cytotoxic nonoxidative ethanol metabolites produced by esterification of fatty acids and ethanol. FAEEs are detectable in blood up to 24 h after ethanol consumption. The objective of this study was to assess the impact of gender, serum or plasma triglyceride concentration, time and temperature of specimen storage, type of alcoholic beverage ingested, and the rate of ethanol consumption on FAEE concentrations in plasma or serum. METHODS: For some studies, subject were recruited volunteers; in others, residual blood samples after ethanol quantification were used. FAEEs were isolated by solid-phase extraction and quantified by gas chromatography-mass spectrometry. RESULTS: For weight-adjusted amounts of ethanol intake, FAEE concentrations were twofold greater for men than women (P /=24 h. The type of alcoholic beverage and rate of consumption did not affect FAEE concentrations. CONCLUSION: These studies advance plasma and serum FAEE measurements closer to implementation as a clinical test for ethanol intake.     

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

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.     

Prevalence of fatty acid ethyl esters in meconium specimens

BACKGROUND: Fetal alcohol syndrome (FAS), alcohol-related birth defects (ARBDs), and alcohol-related neurodevelopment disorders (ARNDs) in neonates are often the result of maternal alcohol consumption during pregnancy. Facial characteristics are associated with FAS, but ARBDs and ARNDs are more difficult to diagnose. Fetal exposure to alcohol can cause central nervous system dysfunction, pre- and postnatal growth problems, cardiac defects in neonates, and attention deficit disorders and mental retardation in older children. To date, diagnosis of fetal alcohol effect has depended largely on maternal interview, although clinical tests are becoming more widely used. Fatty acid ethyl esters (FAEEs) are formed in the body by esterification of ethanol with free fatty acids and trans-esterification of glycerides and have been detected in the meconium of newborns. This report estimates the prevalence of fetal alcohol exposure in two populations by detecting FAEEs in meconium. METHODS: We analyzed the prevalence of FAEEs in the meconium of two separate groups of neonates by use of solid-phase extraction and analysis by gas chromatography-mass spectrometry in the chemical ionization mode. In the first study, meconium samples were taken anonymously from babies born in a large, regional perinatal center in Hawaii. In the second study, specimens were obtained from infants admitted to six different newborn intensive care units within the state of Utah. RESULTS: In the first study, 73 of 436 (16.7%) meconium specimens tested were considered positive for FAEEs. When broken down into quartiles, the mean total FAEEs measured were 1,059, 3,133, 6,628, and 62,115 ng/g. In the second study, 35 of 289 (12.1%) specimens were considered positive. When broken into quartiles, the mean total FAEEs were 1,139, 3,067, 7,674, and 50,143 ng/g. The overall FAEE profiles of the two study sets were remarkably similar. CONCLUSION: In an adequate meconium specimen, a total FAEE concentration >10,000 ng/g may indicate that the newborn has been exposed to significant amounts of alcohol during pregnancy.     

Fatty acid ethyl esters in liver and adipose tissues as postmortem markers for ethanol intake

BACKGROUND: Fatty acid ethyl esters (FAEEs) are nonoxidative metabolites of ethanol. FAEEs are found in liver, pancreas, and adipose tissues up to 24 h after consumption of ethanol, and on that basis, they are potentially useful markers for ethanol intake. In this study with rats, we investigated the efficacy of using FAEEs in liver and in adipose tissue as postmortem markers for premortem ethanol ingestion. METHODS: An animal study was conducted in which test rats received injections of ethanol and control rats received injections of normal saline. The rats were killed 2 h after the injections. The bodies of the animals were stored at 4 degrees C up to 12 h, and samples of liver and adipose tissues were collected at different time intervals and processed for FAEE quantification. In another set of experiments, the rats received injections and were killed as described above, but bodies of animals from both groups were stored at 4, 25, or 37 degrees C for up to 72 h, and liver samples were collected and processed for FAEE quantification. RESULTS: FAEEs were detected up to 12 h after death in liver and adipose tissue samples from the bodies of ethanol-treated animals stored at 4 degrees C; negligible amounts were detected in the bodies of animals that received normal saline. Adipose tissues contained higher amounts of FAEEs than liver, as well as more species: eight FAEE species in adipose tissue and five in liver tissue. Higher concentrations of FAEEs were detected in livers of treated animals stored at 25 degrees C for up to 48 h than in livers of controls stored under the same conditions. CONCLUSIONS: For at least 12 h after death, FAEEs in liver and adipose tissues are useful postmortem markers of premortem ethanol ingestion.     

Citalopram decreases desirability, liking, and consumption of alcohol in alcohol-dependent drinkers.

In previous studies serotonin uptake inhibitors such as citalopram decreased alcohol consumption in alcoholics. The mechanism of the effect is not fully understood. This study tested the hypothesis that it is mediated by changes in desire to drink and alcohol effects. After a 1-week baseline period, subjects (13 men and three women; aged 26 to 69 years; healthy, nondepressed, alcohol-dependent drinkers [mean, 6.6 drinks per day]) were randomized in a double-blind fashion to receive 40 mg/day citalopram and placebo for 1 week each, separated by a 1-week washout period. Daily standard alcoholic drinks (13.6 gm ethanol), nonalcoholic drinks, and tobacco use were recorded; evening urine samples were taken; and interest, desire, craving, and liking for alcohol were rated. Medical status, depression, and anxiety were assessed weekly, but no other treatment or advice was given. Daily alcoholic drinks significantly decreased during citalopram treatment (mean +/- SEM = 4.6 +/- 0.6) compared with placebo (5.7 +/- 0.8; p = 0.01), and the average decrease was 17.5%. Percentage of days abstinent increased during citalopram administration (27.7% +/- 5.7%) compared with placebo (15.5% +/- 3.7%; p less than 0.01). Citalopram decreased interest, desire, craving, and liking for alcohol (all p less than 0.05). There was clear internal validation of these measures in that variations in each correlated with alcohol consumption (all r greater than 0.5, p less than 0.05). Nonalcoholic drinks, self-reports of cigarettes smoked (daily smokers), and body weight did not change significantly. In experimental bar sessions, after the citalopram and placebo periods, subjects were required to consume as many of 18 minidrinks as possible (equivalent to six standard drinks) at 5-minute intervals. Subjects rated their desire for alcohol, intoxication, and mood. Citalopram had no significant effects on the desirability of alcohol or subjective feelings of intoxication. The findings indicate that serotonin uptake inhibitors may act by decreasing the urge to drink and the reinforcing effects of alcohol. Also, a naturalistic outpatient trial is a sensitive, simple, and economic procedure for detecting these drug effects.     

Beta-hexosaminidase isoenzyme profiles in serum, plasma, platelets and mononuclear, polymorphonuclear and unfractionated total leukocytes

OBJECTIVES: The relative proportion in percentage of the isoenzyme A of beta-hexosaminidase (Hex) is the single discriminatory function most frequently used for the biochemical screening of heterozygote Tay-Sachs disease carriers. It has been suggested that the assay of the Hex isoenzymes in homogeneous cell preparations is preferable to that in mixed total leukocytes which present greater interindividual variation. The major aim of our study was the evaluation of this hypothesis. DESIGN AND METHODS: Total Hex and its Hex A and Hex B isoenzymes were determined in different samples of serum and plasma (n = 81) as well as in lysates of platelets (n = 75), and mononuclear (n = 81), polymorphonuclear (n = 81) and mixed total leukocytes (n = 33). RESULTS: The interindividual variations found for % Hex A in the different biological samples were: plasma (CV = 23.4%), platelets (CV = 10.2%), mononuclear (CV = 5.7%), polymorphonuclear (CV = 5.3%) and total leukocytes (CV = 7.1%). Although the relative proportion of Hex A was significantly greater in polymorphonuclear than in mononuclear leukocytes (P < 0.001), a statistical significance was not attained for the correlation between the relative proportions of blood polymorphonuclear cells and Hex A in mixed total leukocytes (r = 0.220). CONCLUSIONS: The use of total leukocyte lysates does not appear to introduce a significant increase for the interindividual variation of the Hex A isoenzyme relative proportion in relation to the use of homogeneous cell preparations.     

Simultaneous gas-chromatographic measurement of rhamnose, lactulose and sucrose and their application in the testing gastrointestinal permeability

BACKGROUND: As it is important to test gastric and intestinal permeability simultaneously in gastrointestinal disorders such as Celiac disease, we developed a gas-chromatographic (GC) method to estimate rhamnose (L-rh), lactulose (Lacl) and sucrose (Suc) in urine. METHODS: The method is based on the use of alditol acetate derivatives giving a lower number of GC peaks than reducing sugars do. Acetate derivatives are more stable and less expensive than GC silylates and keep the flame-detector cleaner. We checked the chemical stability of alditol acetates by verifying the reproducibility of the standard curve of a sugar derivative sample which had been stored for 2 months at -20 degrees C. RESULTS: The calibration proved linear over the range 0.1-1 microg of sugar injected. Analytical sugar recovery was 88%+/-19.4% (mean+/-S.D.) for rhamnose, 105%+/-7.4% for sucrose and 102%+/-2.4% for lactulose. Mean within-day precision (CV) was 7.7% for rhamnose, 5.7% for sucrose and 1.9% for lactulose, and between-day (CV) was 6.7% for rhamnose, 3.9% for sucrose and 1.6% for lactulose. The rhamnose, lactulose and sucrose as the lactulose/rhamnose ratio clearly differentiated 25 healthy controls from 36 patients with active gluten-sensitive enteropathy. CONCLUSIONS: A fast, reliable and cheap gas-chromatographic method is presented here to evaluate gastric and intestinal permeability.     

Identification of a satellite fatty acid ethyl ester synthase from human myocardium as a glutathione S-transferase.

Nonoxidative alcohol metabolism catalyzed by fatty acid ethyl ester (FAEE) synthases may contribute to extrahepatic injury resulting from alcohol abuse. Unlike rabbit myocardial FAEE synthase, that from human heart has a satellite minor synthase (I) eluting from DEAE cellulose at a conductivity of 5 mS. Synthase I was purified 1,118-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and Superose-12 fast-protein liquid chromatographies. SDS-PAGE showed a single polypeptide with a molecular mass of 26 kD and gel permeation chromatography indicated a molecular mass of 52 kD for the active enzyme. Homogeneous synthase I catalyzed ethyl ester synthesis at highest rates with unsaturated octadecanoic fatty acid substrates. The amino acid composition of synthase I was highly homologous to that of human myocardial major synthase, recently identified as an acidic glutathione (GSH) S-transferase. Antibody raised against homogeneous human heart major synthase cross-reacted with the 26-kD synthase I. FAEE synthase co-chromatographed with GSH S-transferase on DEAE cellulose, Sephadex G-100 and S-hexylglutathione agarose, and also displayed GSH S-transferase activity in catalyzing the conjugation of GSH with nitrobenzene-containing carcinogens. Thus, human myocardium contains a satellite peak of FAEE synthase activity and it is a neutral GSH S-transferase.     

Arteriovenous differences of blood alcohol concentrations after celiac plexus block.

After a celiac plexus block with ethyl alcohol, patients sometimes complain of symptoms of alcohol intoxication. We studied the consecutive changes of arterial and venous blood alcohol concentrations in 11 patients and investigated whether an arteriovenous difference exists. We performed a celiac plexus block with 10 ml absolute ethyl alcohol. The sampling sites were radial artery and internal jugular vein. Blood samples were collected at 0, 5, 10, 15, 30, 60, 120, 240 and 480 minutes after the block. The maximum level was reached 15 minutes after injection in both arterial and venous blood, 29.9 +/- 19.4 and 27.7 +/- 21.8 mg/dl (means +/- SD), respectively. Arteriovenous differences were observed 5 and 10 minutes after ethyl alcohol injection (p less than 0.01). There was a significant negative correlation between the ratio of arteriovenous differences to venous sampling and the time elapsed after the block (r = 0.41, p less than 0.01).