Total Fatty Acid Ethyl Esters Research Articles

The Effect of Prolonged Storage Time on the Stability of Fatty Acid Ethyl Esters in Hair Samples.

The advantages of analysis of drugs in hair samples are recognized for the long window of detection, alongside easy sampling and long stability after sample collection. Alcohol markers, ethyl glucuronide (EtG) and total fatty acid ethyl esters (FAEEs) in hair, are widely used for monitoring alcohol consumption for clinical and forensic purposes. Although stability of drugs and EtG in hair samples is documented to a certain extent, stability of FAEEs in hair samples after collection has not been reported. This study covered hair samples that had been tested for FAEEs on the day of arrival at the laboratory and retested between 4 and 80months later. The statistical analysis of the data set reveals significant lower FAEEs levels including ethyl palmitate (EtPa) ester levels when samples were retested for the second time after 6days of storage under ideal conditions. Specifically, the results suggest that when measuring total FAEEs or solely EtPa in hair samples, the elapsed time between sample collection and analysis of the sample needs to be considered when interpreting the results. The recommendation is that whenever hair samples need to be tested for total FAEEs or EtPa, the analytical procedure needs to be performed within 1week after collection in order to obtain meaningful results. The study results substantiate the case for the use of hair samples solely for the analysis of EtG, in conjunction with other measurements such as full blood count, carbohydrate-deficient transferrin test, liver function test or phosphatidylethanol alongside clinical assessment for a more effective evaluation of alcohol consumption.

Protective Effect of Polyunsaturated Fatty Acids against Experimental Lung Injury Induced by Acute Ethanol Inhalation

Ethanol-induced diseases include oxidative mechanisms by which prolonged endoplasmic reticulum (ER) stress results in genesis and accumulation of cytotoxic total fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol). FAEEs participate in the pathogenesis of alcoholic lung disease. Polyunsaturated fatty acids (PUFA) offer a possible protective effect against damage induced by ethanol inhalation. The present study aimed to investigate the protective effect of flaxseed and fish oils administration against toxicity induced by ethanol inhalation. Forty healthy female albino rats were divided into four groups (control, ethanol, flaxseed and fish oils). Lung superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. Plasma advanced oxidation end product (AOPP) and phosphatidylinositol 3- kinase (PI3K) were determined. Erythrocyte membrane fatty acids were extracted and fractionated by HPLC. Ethanol inhalation results in significant increase in lung MDA, plasma AOPP and erythrocyte membrane arachidonic acid (AA), linolenic acid (LA), and oleic acid (OA) along with a significant decrease in erythrocyte membrane alpha-linolenic acid (ALA), lung SOD, and plasma PI3K while pretreatment with flaxseed and fish oils daily (1.2 ml/kg) significantly attenuated these parameters. Supplementation of marine PUFAs reduced the oxidative stress induced by ethanol inhalation in experimental animals.

Determination of fatty acid ethyl esters in dried blood spots by LC-MS/MS as markers for ethanol intake: application in a drinking study.

The forensic utility of fatty acid ethyl esters (FAEEs) in dried blood spots (DBS) as short-term confirmatory markers for ethanol intake was examined. An LC-MS/MS method for the determination of FAEEs in DBS was developed and validated to investigate FAEE formation and elimination in a drinking study, whereby eight subjects ingested 0.66-0.84 g/kg alcohol to reach blood alcohol concentrations (BAC) of 0.8 g/kg. Blood was taken every 1.5-2 h, BAC was determined, and dried blood spots were prepared, with 50 μL of blood, for the determination of FAEEs. Lower limits of quantitation (LLOQ) were between 15 and 37 ng/mL for the four major FAEEs. Validation data are presented in detail. In the drinking study, ethyl palmitate and ethyl oleate proved to be the two most suitable markers for FAEE determination. Maximum FAEE concentrations were reached in samples taken 2 or 4 h after the start of drinking. The following mean peak concentrations (c̅(max)) were reached: ethyl myristate 14 ± 4 ng/mL, ethyl palmitate 144 ± 35 ng/mL, ethyl oleate 125 ± 55 ng/mL, ethyl stearate 71 ± 21 ng/mL, total FAEEs 344 ± 91 ng/mL. Detectability of FAEEs was found to be on the same time scale as BAC. In liquid blood samples containing ethanol, FAEE concentrations increase post-sampling. This study shows that the use of DBS fixation prevents additional FAEE formation in blood samples containing ethanol. Positive FAEE results obtained by DBS analysis can be used as evidence for the presence of ethanol in the original blood sample.

Dose-response and time-response analysis of total fatty acid ethyl esters in meconium as a biomarker of prenatal alcohol exposure

Little is known on how the dose and timing of exposure co-influence the cumulative concentration of fatty acid ethyl esters (FAEEs) in meconium. The objective of the study was to assess the cumulative concentration of FAEEs in meconium as a biomarker of light, moderate, or heavy prenatal alcohol exposure occurring at either first, second, or third trimesters of pregnancy. History of prenatal alcohol exposure was obtained in the 34th week of gestation from 294 pregnant women. Meconium was collected from their babies within the first 6 to 12 h after birth and examined for the presence of nine FAEEs. No significant differences were identified between the cumulative levels of FAEEs in the meconium from the babies born to abstainers and those born to mothers with history of light-to-moderate prenatal alcohol exposure during their pregnancy. Light-to-moderate prenatal alcohol exposure cannot be reliably predicted by the cumulative FAEE concentrations in meconium of exposed babies. A cumulative FAEE level of >10 nmol/g would be required to consider that prenatal alcohol exposure during the second to third trimesters occurred at risky levels in the absence of reliable maternal history of ethanol exposure.

Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3months. Blood ethanol concentration was 180mg% in ethanol fed mice, compared to <1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6μg/g in the lungs of ethanol-fed mice as compared to 1.5μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease.

Meconium Fatty Acid Ethyl Esters as Biomarkers of Late Gestational Ethanol Exposure and Indicator of Ethanol-Induced Multi-Organ Injury in Fetal Sheep

BackgroundMeconium fatty acid ethyl esters (FAEE) constitute a biomarker of heavy fetal ethanol exposure. Our objective was to measure meconium FAEE in fetal sheep following daily, relatively moderate-dose ethanol exposure in late gestation, and to evaluate their utility in identifying fetal organ-system injury.MethodsPregnant ewes received ethanol (0.75 g/kg; n = 14) or saline (n = 8) via 1-h IV infusion daily during the third trimester equivalent, while additional pregnant sheep served as untreated controls (n = 6). The daily ethanol regimen produced similar maximal maternal and fetal plasma ethanol concentrations of 0.11–0.12 g/dL. Ewes and fetuses were euthanized shortly before term, and meconium was collected and analyzed for FAEE (ethyl palmitate, stearate, linoleate, and oleate).ResultsMeconium total FAEE concentration was significantly higher in ethanol-exposed fetuses compared with controls, and a positive cut-off of 0.0285 nmol total FAEE/g meconium had 93.3% sensitivity and specificity for detecting fetal ethanol exposure. When the studied animals (ethanol-exposed and controls) were classified according to meconium FAEE concentration, FAEE-positive and FAEE-negative groups frequently differed with respect to previously examined pathological endpoints, including nephron endowment, lung collagen deposition, cardiomyocyte maturation, and tropoelastin gene expression in cerebral vessels. Furthermore, in all studied animals as a group (ethanol-exposed and controls combined), meconium FAEE concentration was correlated with many of these pathological endpoints in fetal organs.ConclusionsWe conclude that, in fetal sheep, meconium FAEE could serve as a biomarker of daily ethanol exposure in late gestation and could identify fetuses with subtle ethanol-induced toxic effects in various organs. This study illustrates the potential for using meconium FAEE to identify neonates at risk for dysfunction of major organs following in-utero ethanol exposure that does not result in overt physical signs of ethanol teratogenicity.

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.

Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol

This study investigated ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentration in meconium and in maternal and neonatal hair (HEtG and HFAEEs, respectively) as potential markers of intrauterine exposure to ethanol together with meconium fatty acid ethyl esters (FAEEs) in a cohort of 99 mother–infant dyads, 49 coming from the Arcispedale of Reggio Emilia (Italy) and 50 from the Hospital del Mar of Barcelona (Spain). FAEEs, EtG and EtS were measured in meconium samples using liquid chromatography–tandem mass spectrometry. A head space-solid phase microextraction-gas chromatography–mass spectrometry was used to test HEtG and HFAEEs in hair samples from mothers and their newborns. Eighty-two meconium samples (82.8%) tested positive for EtG, 19 (19.2%) for EtS while 22 (22.2%) showed FAEEs levels higher than 2 nmol/g, the cut-off used to differentiate daily maternal ethanol consumption during pregnancy from occasional or no use. Although EtG and EtS in meconium did not correlate with total FAEEs concentration, a good correlation between EtG, EtS and ethyl stearate was observed. Moreover, EtG correlated well with ethyl palmitoleate, while EtS with ethyl laurate, myristate and linolenate. Neither maternal nor neonatal hair appears as good predictors of gestational ethanol consumption and subsequent fetal exposure in these mother–infant dyads. In conclusion, these data show that meconium is so far the best matrix in evaluating intrauterine exposure to ethanol, with EtG and EtS being potentially good alternative biomarkers to FAEEs.

The Effect of Hair Pigment on the Incorporation of Fatty Acid Ethyl Esters (FAEE)

The objective of the current study was to determine whether FAEE incorporation is affected by hair pigmentation. Black hooded LE rats were injected intraperitoneally daily with ethanol. Prior to dosing, black and white patches of fur were shaved and analyzed for baseline levels of FAEE using an adapted extraction procedure and GCMS method. Once the shaved 'patches' had grown back they were re-sampled along with hair outside the 'patches', referred to as 'no patch' hair, and tested for post-treatment FAEE levels in the same manner. Blood was also sampled for pharmacokinetic analysis of ethanol. Total FAEE levels were significantly higher in post-treatment hair (black and white) compared to baseline (pre-treatment) levels. Total FAEE levels were also significantly higher in post-treatment 'patch' hair (black and white) compared to 'no patch' hair. No significant differences were found between post-treatment black and white hair. The FAEE profiles were similar between black and white hair, with FAEE levels being highest for ethyl myristate, followed by ethyl stearate, palmitate, and then oleate. FAEE incorporation into hair does not appear to be affected by hair pigment, which is in congruence with what is known about the chemistry of drug-melanin interactions. This is important in avoiding potential bias and discrimination in the interpretation of alcohol abuse based on hair color.

Alarming Prevalence of Fetal Alcohol Exposure in a Mediterranean City

The prevalence of gestational ethanol exposure and subsequent fetal exposure has been assessed in a cohort of mother-infant dyads in a Mediterranean city (Barcelona, Spain) by meconium analysis of fatty acid ethyl esters (FAEEs) after showing in this population a high prevalence of meconium opiates (8.7%), cocaine (4.4%), and cannabis (5.3%). Of the 353 meconium samples analyzed for FAEEs, 159 (45%) contained a total amount of seven FAEEs equal or above 2 nmol/g meconium, the cutoff internationally accepted to differentiate heavy maternal alcohol consumption during pregnancy from occasional use or no use at all. No parental sociodemographic differences or maternal features differentiated exposed from unexposed newborns. The prevalence of gestational consumption of ethanol was similar between women using and not using drugs of abuse during pregnancy (45.7% and 44.7% of samples with total FAEEs equal or higher than 2 nmol/g meconium, respectively). Meconium samples from newborns exposed in utero to ethanol, and positive for at least one illicit drug (cocaine, opiates, or cannabis), had total FAEEs and five of nine individual FAEEs statistically higher than the meconium samples that were negative for the most frequently used illicit drugs of abuse. Among the most prevalent FAEEs, oleic acid ethyl ester showed the best correlation to total FAEE concentration followed by palmitoleic acid ethyl ester . This study, which highlights a 45% ethanol consumption during pregnancy in a low socioeconomic status cohort, may serve as an eye opener for Europeans that gestational alcohol exposure is not endemic only in areas outside of Europe.

Controlling for Serum Albumin Level Improves the Correlation Between Serum Fatty Acid Ethyl Esters and Blood Ethanol Level

Fatty acid ethyl esters (FAEEs) are generated by the nonoxidative metabolism of alcohol and correlate positively with blood alcohol levels (BAL). As FAEEs are produced predominantly in the liver and bind to albumin in plasma, blood FAEE concentrations may be affected by serum albumin levels. The aim of this exploratory study was to define the relationship of FAEE levels with BAL after adjustment for serum albumin concentration. Fatty acid ethyl ester, BAL, and albumin concentrations were measured from de-identified, ethanol-containing serum samples (N=18). The assay focused on 3 FAEE species ethyl palmitate, ethyl stearate, and ethyl oleate. The relationships of individual and total FAEE concentrations with BAL using albumin as a covariate were analyzed. Values for Pearson's r between the BAL and the natural log-transformed FAEE (ln FAEE) levels were calculated, and then compared with partial correlation coefficients when controlling for albumin. The impact of serum albumin levels on the relationship between ln FAEE and BAL was evaluated by simple linear regression analysis. Concentrations of total FAEE ranged from 632 to 20,166 nM, and BAL ranged from 9 to 375 mg/dL. In the 18 samples, the Pearson correlation coefficient between BAL and total FAEE levels was 0.868, and increased to 0.909 when controlling for albumin (p<0.0001). Similar statistically significant increases in the partial correlation coefficient occurred for each of the individual species of FAEE when controlling for albumin. In simple linear regression, albumin significantly reduced the variability in the model correlating BAL and FAEE for each of the individual species, as well as for total FAEE. The association between FAEE levels and BAL is enhanced by consideration of serum albumin concentrations. Our findings suggest that serum albumin should be included in any model attempting to define the relationship between BAL and FAEE.

FATTY ACID ETHYL ESTERS (FAEE); COMPARATIVE ACCUMULATION IN HUMAN AND GUINEA PIG HAIR AS A BIOMARKER FOR PRENATAL ALCOHOL EXPOSURE

To compare the incorporation rate (ICR) of fatty acid ethyl esters (FAEE) in hair between guinea pigs and humans, and to assess the relationship between ethanol exposure and FAEE concentrations in hair. Published data from pregnant guinea pigs, including maximum blood ethanol concentration (BEC), dosage regimen, and total hair FAEE concentration, were compared with published data from alcoholic patients, where dose of ethanol consumed and total hair FAEE concentration were reported. Mean values of ethanol Vmax for pregnant guinea pigs and humans were obtained from published data (26.2 and 24 mg/dl/h, respectively). Total and individual FAEE ICRs, defined as the ratio of hair FAEE to the area under the BEC-time curve (total systemic ethanol exposure), were found to be on average an order of magnitude lower in the guinea pig than in the human. The profiles of ester incorporation also differed slightly between species, with ethyl stearate being highly incorporated in guinea pig hair and less so in human hair. The results may reflect in the human greater FAEE production, greater FAEE deposition in hair, slower FAEE catabolism, differential sebum production and composition, or a combination thereof. Also, ethyl oleate was found to correlate with total systemic ethanol exposure for both guinea pigs and humans, correlation coefficients equalling 0.67 (P < 0.05) and 0.49 (P < 0.05), respectively. No other ethyl esters, nor total FAEE, were found to correlate with systemic ethanol exposure. When extrapolating FAEE concentrations in hair from guinea pigs to humans, an order of magnitude difference should be considered, with humans incorporating more FAEE per unit of ethanol exposure. Also, the results suggest caution should be taken when interpreting values of single esters because of their differential incorporation among species. Lastly, our findings suggest ethyl oleate may be of keen interest in FAEE hair analysis, particularly across species.

Chronic Prenatal Ethanol Exposure and Increased Concentration of Fatty Acid Ethyl Esters in Meconium of Term Fetal Guinea Pig

In humans, the occurrence of prenatal exposure to ethanol is difficult to validate objectively. Increased concentration of fatty acid ethyl esters (FAEE) in the meconium of the newborn may be a biomarker of prenatal ethanol exposure. The validity of this proposed biomarker was tested in pregnant guinea pigs that received chronic oral administration of 4 g ethanol/kg maternal body weight/day (n=8), isocaloric-sucrose/pair-feeding (n=8) or water (n=2) throughout gestation. At gestational day 65 (term, gestational day 66 to 69), each dam and her offspring were euthanized, and meconium was collected from the term fetal large intestine. Eight individual FAEE (lauric, myristic, palmitic, palmitoleic, stearic, oleic, linolenic and arachidonic AEE) were measured by gas chromatography--flame ionization detection and confirmed by gas chromatography--mass spectrometry. The chronic maternal ethanol regimen decreased fetal body weight and brain weight. There was virtually no measurable FAEE in the meconium for the water group (n=3 fetuses). For meconium of the ethanol offspring (n=25 fetuses) compared with the sucrose offspring (n=23 fetuses), the total FAEE concentration was 8-fold higher; and lauric, palmitic, stearic and oleic AEE concentrations were at least 5-fold higher for the ethanol group. The data indicate that fetal meconium FAEE constitute a biomarker of prenatal ethanol exposure for a maternal ethanol regimen that restricts fetal development, with an inverse relationship between meconium total FAEE concentration and both body weight and brain weight.

Fatty Acid Ethyl Esters in Human Mononuclear Cells: Production by Endogenous Synthesis Greatly Exceeds the Uptake of Preformed Ethyl Esters

Fatty acid ethyl esters (FAEE) are nonoxidative metabolites of ethanol. They are esterification products of ethanol and fatty acids. Fatty acid ethyl esters have been implicated as important mediators of ethanol-induced cytotoxicity, organ damage, and disease. In addition, they serve as specific and sensitive biomarkers for ethanol intake. Following ethanol consumption, FAEE are found in circulating blood bound to albumin or/and lipoproteins. Using a mononuclear fraction of white blood cells (WBC) exposed to ethanol, we investigated FAEE synthesis. We then determined the amount of uptake of preformed FAEE presented to the cells and compared the amounts of FAEE within the cells that were derived from endogenous synthesis with the amount derived from uptake of exposure FAEE. We also measured the persistence of FAEE within these cells and assessed the fate of the FAEE-associated fatty acid upon FAEE hydrolysis. A mononuclear fraction of human WBC was incubated with 25, 50, or 100 mM ethanol for 0.08 to 120 minutes, and FAEE synthesis was measured by gas chromatography/mass spectrometry. In other experiments, mononuclear cells were incubated with 25, 50, and/or 100 microM [3H]ethyl oleate, a representative FAEE species, for 0.08-120 minutes, and FAEE uptake and hydrolysis were measured. The total FAEE formed by treating the cells with 25 mM ethanol, which represents a physiologic dose achievable with excess alcohol intake, greatly exceeded the FAEE within cells derived from uptake of 100 microM ethyl oleate, which represents a supraphysiologic dose. There was hydrolysis of FAEE by human mononuclear cells, with free fatty acids as major metabolites of FAEE hydrolysis. Unlike any other cell type or homogenate studied, the only ethyl ester formed by human mononuclear cells exposed to ethanol was ethyl oleate. There is significant synthesis of FAEE by human mononuclear cells within seconds of exposure to physiologic doses of ethanol. The amount of FAEE in these cells derived from endogenous synthesis greatly exceeds the amount acquired by exogenous uptake.

Rapid, accurate, and sensitive fatty acid ethyl ester determination by gas chromatography-mass spectrometry

Fatty acid ethyl esters (FAEEs) are useful markers of ongoing alcohol use and may be associated with alcohol-induced damage to the liver and pancreas. In this article, we describe a novel method for rapid determination of the three major FAEEs found in human plasma. Internal standard, ethyl heptadecanoate, was added to plasma samples, and FAEEs were isolated by acetone precipitation, hexane lipid extraction, and amino-propyl silica solid phase extraction. FAEEs were quantitated by gas chromatography-mass spectrometry (GC-MS) using a nonpolar dimethylpolysiloxane column. The accuracy, precision, specificity, and sensitivity of the assay were defined from plasma samples from recently drinking and abstinent persons, with and without the addition of FAEEs. Individual FAEE peaks demonstrated excellent resolution. Instrument time was reduced by more than 60%. The lower limit of detection was 5 to 10 nM, and the lower limit of quantitation for each FAEE was 60 nM (for 22 samples with known concentration 60 nM, x +/-SD: 61 +/- 5.7, 57 +/- 5.7, and 57 +/- 5.9 nM, for ethyl palmitate, ethyl oleate, and ethyl stearate, respectively). Instrument precision (coefficient of variance, CV) for these three FAEEs was 0.3%, 0.4%, and 0.7%, respectively. Intra-assay precision (CV) for total FAEEs was less than 7%. FAEEs were absent in 49 samples from abstinent persons. FAEEs were detected in all 76 samples with associated positive blood alcohol levels. Our method of FAEE analysis is rapid and potentially useful in research and clinical studies. FAEE determination using this method is precise, accurate, sensitive, and specific and deserves broader application.

New Methods for Neonatal Drug Screening

After completing this article, readers should be able to: 1. Describe perinatal and neonatal complications of maternal use of cocaine, alcohol, opiates, and barbiturates in pregnancy. 2. List established cutoffs of conventional immunoassays of urine for common substances of abuse. 3. List the advantages of using meconium testing for substances of abuse. 4. Describe the role of fatty acid ethyl esters in detecting prenatal alcohol exposure. Maternal substance use during pregnancy can have direct consequences on both the developing fetus and the mother and is a significant public health concern. The prevalence of gestational use of drugs of abuse varies, depending on the study population and method of detection, but it generally ranges from 1% to 15%. (1) One of the largest epidemiologic studies conducted in the United States documented that although most pregnant women decrease the use of substances as the pregnancy progresses, a significant proportion of them remain regular users, even in the third trimester (eg, ethanol [13%], cannabis [9.3%], cocaine [10%], and heroin [1%]). (2) Various perinatal and neonatal complications can result from prenatal exposure to common drugs of abuse. (3) The use of cocaine during pregnancy is associated with increased risks of preterm labor, intrauterine growth retardation, and neurobehavioral abnormalities. Heavy alcohol use during pregnancy leads to fetal alcohol spectrum disorder, which is characterized by an array of congenital malformations, central nervous system abnormalities, and neurodevelopmental deficits. Neonates exposed to opiates, alcohol, and barbiturates in utero are at risk of developing neonatal abstinence syndrome, which is characterized by irritability, gastrointestinal dysfunction, respiratory distress, and other nonspecific symptoms. (1) Data in the literature conflict regarding adverse events in the immediate postnatal period that can be attributed to prenatal exposure to other illicit drugs. Thus, early identification of neonates in whom substance exposure is suspected is important for appropriate treatment intervention and …

Ethyl arachidonate is the predominant fatty acid ethyl ester in the brains of alcohol-intoxicated subjects at autopsy.

The role of fatty acid ethyl esters (FAEE), the nonoxidative ethanol metabolites, as mediators of alcohol-induced organ damage is increasingly being recognized. FAEE are detectable in the blood and in liver and adipose tissue after ethanol ingestion, and on that basis, FAEE can be used as markers of ethanol intake. In this study, 10 samples of human brain were collected at autopsy at the Massachusetts Medical Examiner's Office and analyzed for FAEE. FAEE were isolated and quantified as mass per gram of wet weight. The blood ethanol level was also obtained in each case along with the other drugs detected in routine postmortem toxicology screening tests. Ethyl arachidonate was the predominant FAEE species in the brain, representing up to 77.4% of total FAEE in the brain. The percent age of ethyl arachidonate of the total FAEE in the brain was significantly higher than what has been found in all other organs and tissues previously analyzed. Linoleate, the precursor of arachidonate, was a poor substrate for FAEE synthesis, as the percentage of ethyl linoleate of the total FAEE content was extremely low. Thus, this reflects preferred incorporation of arachidonate into newly synthesized FAEE in the brain. Since arachidonate is derived from linoleate, which is depleted in FAEE while arachidonate is enriched, the synthesis of FAEE may be linked to the desaturation and elongation of linoleate to arachidonate.

Prevalence of fatty acid ethyl esters in meconium specimens.

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. 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. 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. 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.

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.

Production of fatty acid ethyl esters by Pseudomonas fragi under conditions of gas stripping

Pseudomonas fragi biomass was produced on whey and whey permeate with and without supplementation with various concentrations of butyric or valeric acid. The biomass produced in whey supplemented with 0·1% butyric acid possessed the highest ethylating activity of valeric acid in the presence of ethanol. The performance of the ethylating activity of P. fragi biomass was studied in aqueous medium under conditions of in-situ product removal using gas stripping. From 1000 ppm valeric acid and 2000 ppm ethanol, resting cells of P. fragi produced more than 800 ppm total fatty acid ethyl esters of which valeric acid ethyl ester accounted for more than 90%. The use of a gas stripping technique resulted in a 3.6-fold and 4.4-fold increase in the formation of total fatty acid and valeric acid ethyl esters, respectively, as compared to bioconversions conducted under conditions of product accumulation.