Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.     

Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters

Myocardial homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of coenzyme A or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, has been purified 5400-fold to homogeneity [Mogelson, S., & Lange, L. G. (1984) Biochemistry (preceding paper in this issue)]. To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, we determined thermodynamic parameters relevant to the kinetics and equilibria of this reaction and specifically characterized (1) the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and (2) the physical properties of the product, ethyl oleate, in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (delta G) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, delta Senz - delta S uncat = 23.9 cal/(mol.deg). Rate constants in the presence and absence of enzyme at 37 degrees C were 6 X 10(-2) s-1 and 7.8 X 10(-11) M-1 s-1, respectively, indicating a catalytic power of at least 10(8)M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg.s) (37 degrees C). The equilibrium constant was calculated for the reaction oleate + ethanol in equilibrium ethyl oleate and was 0.095 M-1 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red blood cell fatty acid ethyl esters: a significant component of fatty acid ethyl esters in the blood

Although alcohol abuse is known to cause an array of ethanol-induced red blood cell (RBC) abnormalities, the underlying molecular mechanisms remain poorly understood. Fatty acid ethyl esters (FAEEs) are toxic, nonoxidative ethanol metabolites that have been found in blood, plasma, and tissues. Because FAEEs have been shown to be incorporated into phospholipid bilayers, we conducted a controlled ethanol intake study to test the hypothesis that FAEEs accumulate and persist within RBCs following ethanol ingestion. We demonstrated that RBC FAEEs account for approximately 5% to 20% of total whole-blood FAEEs, and that the fatty acid composition of FAEEs in RBCs and plasma are different and vary differently over time. These data indicate that a significant percentage of FAEEs in the blood is associated with RBCs and that the metabolism of RBC FAEEs and that of plasma FAEEs (bound to albumin or lipoproteins) are largely independent.     

Effects of acetyl-L-carnitine on the formation of fatty acid ethyl esters in brain and peripheral organs after short-term ethanol administration in rat

Increasing evidence suggests that Fatty acid ethyl esters (FAEE) play a central role in ethanol induced organ damage. In the current study we measured FAEE formation in rats after short-term oral administration of ethanol, in the presence and absence of pre-treatment with acetyl-L-carnitine. Ethanol treatment caused a significant increase in the levels of FAEE, particularly in the brain and heart, but also in the kidney and liver. Increases in FAEE were associated with a significant increase in FAEE synthase activity, GSH transferase activity, and lipid hydroperoxide levels. Pre-treatment with acetyl-L-carnitine resulted in a significant reduction of FAEE accumulation, decrease in FAEE synthase and GSH transferase activities, and lipid hydroperoxide levels. Administration of acetyl-L-carnitine greatly reduced the metabolic abnormalities due to non-oxidative ethanol metabolism, through an increment in lipid metabolism/turnover and by the modulation of the activities of enzymes associated with FAEE synthesis. These results suggest a potentially important pharmacological role for acetyl-L-carnitine in the prevention of alcohol-induced cellular damage.     

Concentration and fatty acid composition of cholesteryl esters of normal human brain

Abstract— —Cholesteryl esters were isolated from the cerebral cortex and white matter of human brains at different ages, and their concentration and composition determined. The esters were separated from other lipids by chromatography on silicic acid and finally purified by TLC. The fatty acids were converted to the methyl esters by alkaline trans-methylation and analysed by GLC. A TLC method was elaborated for quantitative determination of small amounts of cholesteryl esters in the presence of free cholesterol. The concentration of cholesteryl esters was only 0·1–0·2 per cent of the total cholesterol content of cerebral tissue in older children and adults. During early myelination the concentration was many times greater, especially in the white matter but it never exceeded 2 per cent of the total cholesterol in any subject. The major fatty acids of human brain cholesteryl esters were oleic, palmitic, palmitoleic and arachidonic acid. After completion of myelination, arachidonic acid constituted the major fatty acid. There were fairly small differences in the fatty acid pattern of the cholesteryl esters between grey and white matter, but the concentration of polyunsaturated fatty acids was larger in the grey matter. Cholesteryl esters appear to play an important role in the metabolism of the phosphoglyceride fatty acids in cerebral tissue.     

Fatty acid ethyl esters, nonoxidative ethanol metabolites, synthesis, uptake, and hydrolysis by human platelets

The consumption of alcohol is known to have both positive and negative effects on the functioning of the cardiovascular system in general, and on platelet function in particular. Fatty acid ethyl esters (FAEEs) are non-oxidative metabolite of ethanol that may mediate the ethanol effect on platelet function leading to either bleeding or clotting. The aim of the current study was to investigate the synthesis, uptake, and hydrolysis of FAEEs by human platelets. Isolated platelets were incubated with ethanol for various times, and FAEE synthesis were measured by gas chromatography mass-spectrometry (GC-MS). In addition, platelets were incubated with (14)C-ethyl oleate, and FAEE uptake and hydrolysis were measured. There was significant synthesis of FAEEs by human platelets within 30 min of exposure to ethanol. The major FAEE species formed by human platelets exposed to ethanol were ethyl palmitate and ethyl stearate. FAEE uptake by human platelets showed maximum uptake by 60 s. The majority of FAEEs (50-80%) incorporated into platelets remained intact for up to 10 min. FAEE hydrolysis led to an increase in free fatty acids, with minimal subsequent esterification of the free fatty acids into phospholipids, triglycerides, and cholesterol esters. These studies show that FAEEs, non-oxidative metabolite of ethanol, can be incorporated into, synthesized, and hydrolyzed by human platelets.     

Identification and quantitation of fatty acid ethyl esters in biological specimens

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.     

Quantitation of fatty acid ethyl esters in human meconium by an improved liquid chromatography/tandem mass spectrometry

This paper reports the development and validation of an improved assay for quantitation of fatty acid ethyl esters (FAEEs) in human meconium using liquid chromatography/tandem mass spectrometry (LC–MS/MS). FAAEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) and the internal standard (I.S.), ethyl heptadecanoate, were separated by reverse phase HPLC and quantified by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ionization mode. The absolute recovery of FAEEs varied from 55 ± 10% for 0.33 nmol/g (100 ng/g) of ethyl linoleate up to 86 ± 8% for 1.55 nmol/g (500 ng/g) of ethyl miristate. The LODs and LOQs varied from 0.01 to 0.08 nmol/g and from 0.02 to 0.27 nmol/g, respectively. The assay has been successfully applied to examine the FAEE levels in 81 meconium samples from babies born to mothers reporting alcohol consumption, to varying degrees, during pregnancy.     

Thermotropic behavior of fatty acid ethyl esters in phospholipid liposomes.

The thermotropic behavior of a series of synthetic fatty acyl ethylesters (FAEE) in multilamellar liposomes has been studied by differential scanning calorimetry and monitoring the changes in polarization emitted by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Their thermotropic behaviour has been compared to that of the homologous fatty acids (FA) from which they are synthesized in vivo in the presence of ethanol. Compared to the correspondent FA, saturated FAEE show, depending on the chain length, a minor rigidifying effect or even a slight fluidizing effect on phospholipid bilayers. Unsaturated FAEE show, compared to the homologous FA, slightly greater fluidizing properties. The difference between FA and FAEE is more evident in single component phospholipid liposomes in the gel phase, and in mixed liposomes of two lipids at temperatures at which microdomains of gel and liquid zones coexist. The calorimetric data suggest that FAEE are completely miscible with phospholipids both in the gel and liquid phases; they appear to destabilize the bilayer wherein the ethoxy head group interferes with the intrinsic phospholipid interactions.     

Enzymatic synthesis of fatty acid ethyl esters by utilizing camellia oil soapstocks and diethyl carbonate

This study was reported on a novel process for fatty acid ethyl esters preparation by transesterification and esterification from renewable low-cost feedstock camellia oil soapstocks and friendly acyl acceptor diethyl carbonate. The main components of product were 83.9% ethyl oleate, 8.9% ethyl palmitate, 4.7% ethyl linoleate and 2.1% ethyl stearate, which could be used as eco-friendly renewable resources or additives of industrial solvent and fossil fuel. The effects of molar ratio of diethyl carbonate to soapstocks oil, lipases, organic solvent, reaction temperature and time were investigated, and process conditions were optimized. The yield was up to 98.4% in solvent-free system with molar ratio of diethyl carbonate to soapstocks oil 3:1 and 5% Novozym 435 (based on the weight of soapstocks oil) at 50 °C and 180 rpm for 24 h. Moreover, there was no obvious loss in the yield after lipases were reused for 10 batches without treatment under optimized conditions.     

Steroidal fatty acid esters

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3β-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3β-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estrodiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 and C-17β. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.     

Continuous lipase-catalyzed production of fatty acid ethyl esters from soybean oil in compressed fluids

This work investigates the continuous production of fatty acid ethyl esters from soybean oil in compressed fluids, namely carbon dioxide, propane and n-butane, using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of temperature in the range of 30–70 °C, from 50 to 150 bar, oil to ethanol molar ratio of 1:6–1:18 and solvent to substrates mass ratio of 4:1–10:1. In contrast to the use of carbon dioxide and n-butane, results showed that lipase-catalyzed alcoholysis in a continuous tubular reactor in compressed propane might be a potential route to biodiesel production as high reaction conversions were achieved at mild temperature (70 °C) and pressure (60 bar) conditions in short reaction times.     

Modulation of lipid peroxidation and antioxidant enzymes in murine salivary gland by dietary fatty acid ethyl esters

The present study was undertaken to investigate the effect of n-9, n-6, and n-3 dietary fatty acid ethyl esters on basal (uninduced) and Fe2+/ascorbate (induced) lipid peroxidation (LPO) in salivary gland (SG) of mice. Feeding n-3 ethyl ester polyunsaturated fatty acids (PUFA) increased the uninduced and induced LPO in SG homogenates. In contrast, feeding olive oil ethyl esters (n-9) significantly lowered the induced and uninduced LPO in SG tissue. Salivary gland susceptibility to LPO increased in the order of: olive oil < corn oil < safflower oil < n-3 ethyl esters. Olive oil esters in the diet increased primarily the 18:1 levels in SG tissue. Whereas feeding n-3 PUFA notably increased the superoxide dismutase (SOD) and catalase activities in SG homogenates, no significant changes were seen between n-9 and n-6 PUFA-fed mice. Lower levels of Vitamin E (Vit E) in the tissues of n-3 PUFA-fed mice indicate that the higher the dietary lipid unsaturation, the higher the requirement for Vit E in the diet. Our results indicate that, similar to other organs, salivary gland susceptibility to uninduced or induced oxidation depends on the source of dietary PUFA. In conclusion, feeding olive oil increases the resistance of SGs to induced and uninduced LPO.     

Docosahexaenoic acid ethyl esters from Isochrysis galbana

In order to produce docosahexaenoic acid (DHA), a culture of the microalgal strain Isochrysis galbana was implemented. In Erlenmeyer flasks, a natural seawater medium, the Provasoli 1/3 medium, was compared to the classical Jones medium for DHA production. The Provasoli 1/3 medium stimulated growth (0.44 d(-1)), but influenced DHA accumulation negatively (0.240 pg cell(-1)). However, DHA production per liter of culture medium were of the same order of magnitude with both media (0.961 mg l(-1)). In a 2-l bioreactor, DHA production per liter of culture medium did not increase significantly between 4 and 8 days of culture. With a view to optimize DHA productivity, cells should be harvested at the end of exponential phase i.e. after 4 days of culture. Two strategies were then attempted to produce DHA ethyl esters. First, lipids from I. galbana were submitted to lipase-catalyzed transesterification with ethanol. Secondly, fatty acids from I. galbana were submitted to lipase catalyzed esterification with ethanol. In both cases, lipase from Candida antarctica was shown to be the best candidate, among the five tested, with conversion yields of 20 and 60% after 24 h of transesterification and esterification respectively.     

Fatty acid ethyl esters: recent observations

Fatty acid ethyl esters (FAEE), esterification products of fatty acids and ethanol, have been shown to be mediators of ethanol-induced cell injury and their presence in the blood and tissues is a marker of ethanol intake. Recently, it has been shown that FAEE are produced within seconds of infusion of ethanol into the heart, when using a protocol similar to that used for myocardial ablation. This raises the possibility that the mechanism for the death of myocytes in cardiac ablation involves the generation of toxic FAEE. It has also been recently demonstrated that chronic alcoholics have a high concentration of a specific FAEE species--ethyl oleate. The use of the serum ethyl oleate concentration may be helpful in differentiating binge drinkers from chronic alcoholics.     

Lipoproteins: carriers of dehydroepiandrosterone fatty acid esters in human serum

The association between lipoproteins, cholesterol and cholesteryl esters is very well known to facilitate both the transport in plasma and the entry of these non-polar compounds into the cellular compartment. However, recent observations suggest that in addition to cholesterol, lipoproteins contain several other steroids in their lipoidal metabolite forms which may be transported in the very low, low and high density lipoproteins in human serum. Using the important androgen and oestrogen precursor, dehydroepiandrosterone (DHEA), the biosynthetic formation of lipoidal DHEA was demonstrated in human serum. Serum was also fractionated into its lipoprotein components during the course of its incubation with tritiated DHEA. A progressive movement of the label from the fraction containing the conventional steroid binding-proteins to the lipoproteins was observed with the fraction containing the low density lipoproteins demonstrating the greatest incorporation of the label. This displacement occurred simultaneous to an extensive esterification of the labelled DHEA in serum. After 6 h of incubation, approx. 90% of the radioactivity in all the lipoprotein fractions was in the lipoidal form. Very little labelled lipoidal DHEA was associated with the serum protein fraction throughout the duration of incubation. These data suggest that lipoproteins act as the carriers of lipoidal DHEA following its formation from the non-conjugate parent steroid in serum.     

Cholesterol esters in developing rat brain: concentration and fatty acid composition

Abstract— The contents and the fatty acid composition of cholesterol esters were analysed in developing rat brain. The total content did not exceed 20 μg/brain throughout development. Elimination of serum by adequate perfusion was essential for accurate results. Two separate events appeared to affect the levels of cholesterol esters in developing rat brain, one probably reflecting general developmental changes and the other apparently related to myelination. On either a unit weight or a whole brain basis, the curves appeared to be a superimposition of the two events. There was an underlying developmental change, which was characterized on a unit weight basis by the highest level of cholesterol esters immediately after birth and a steady decline to the adult level by 30 days of age or which on the basis of whole brain was characterized by a steady increase throughout the development. A period of transient increase was superimposed on this underlying developmental change between the ages of 7 and 27 days and corresponded to the period of active myelination. The major fatty acids of rat brain cholesterol esters were palmitic, palmitoleic, oleic and arachidonic acids. Palmitic and palmitoleic acids decreased in proportion while oleic acid increased, as the animal matured. The fatty acid composition of serum cholesterol esters was distinctly different from that of brain cholesterol esters; those from serum contained much higher proportions of linoleic and arachidonic acids and much less palmitoleic and oleic acids.     

Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium

Non-oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5, 7 and 11 mS, respectively. The major synthase was purified 8900-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-human albumin affinity-chromatographies with an overall yield of 25%. SDS-PAGE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates (Vmax = 100 and 65 nmol/mg/h for oleate and linoleate, respectively). Km values for oleate, linoleate, arachidonate, palmitate and stearate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised or two identical, or nearly identical, subunits (Mr = 26000).