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.     

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.     

Detection and quantification of fatty acid ethyl esters in meconium by headspace-solid-phase microextraction and gas chromatography–mass spectrometry

Meconium fatty acid ethyl esters (FAEEs) are currently used as biomarkers to detect heavy prenatal alcohol exposure. We introduce a novel technique to quantify FAEEs in meconium using headspace-solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS). This method improves on previous approaches by decreasing sample preparation time, eliminating the need for organic solvents, and reducing the required sample size. Using 50 mg of meconium, the detection limits of FAEEs ranged from 0.05 to 0.16 nmol/g and had good reproducibility making it ideal for routine analysis of clinical samples.     

Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol.     

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.     

Occurrence of Fatty Acid Short‐Chain‐Alkyl Esters in Fruits of Celastraceae Plants

Small amounts of a mixture of fatty acid short‐chain‐alkyl esters (FASCAEs) were obtained from the fruits of twelve plant species of Celastraceae family, and in five of them the FASCAEs were present not only in the arils but also in the seeds. These mixtures contained 32 individual FASCAE species, which formed four separate fractions, viz. FA methyl, ethyl, isopropyl, and butyl esters (FAMEs, FAEEs, FAIPEs, and FABEs, resp.). The FASCAE acyl components included the residues of 16 individual C₁₄–C₂₄ saturated, mono‐, di‐, and trienoic FAs. Linoleic, oleic, and palmitic acids, and, in some cases, also α‐linolenic acid predominated in FAMEs and FAEEs, while myristic acid was predominant in FAIPEs. It can be suggested that, in the fruit arils of some plant species, FAMEs and FAEEs were formed at the expense of a same FA pool characteristic of a given species and were strongly different from FAIPEs and FABEs esters regarding the mechanism of their biosynthesis. However, as a whole, the qualitative and quantitative composition of various FASCAE fractions, as well as their FA composition, varied considerably depending on various factors. Therefore, separate FASCAE fractions seem to be synthesized from different FA pools other than those used for triacylglycerol formation.     

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.     

Placental Fatty Acid Ethyl Esters Are Elevated with Maternal Alcohol Use in Pregnancies Complicated by Prematurity

The accumulation of fatty acid ethyl esters (FAEEs) in meconium of term newborns has been described as one potential biomarker of maternal alcohol use during pregnancy. FAEEs accumulate in multiple alcohol-exposed fetal tissues and in the placenta. Limited research has focused on the identification of the premature newborn exposed to alcohol in utero. We hypothesized that maternal alcohol use occurs in a significant proportion of premature deliveries and that this exposure can be detected as elevated placental FAEEs. The goals of this study were to 1) determine the prevalence of maternal alcohol use in the premature newborn and 2) investigate whether placental FAEEs could identify those newborns with fetal alcohol exposure. This prospective observational study evaluated 80 placentas from 80 women after premature delivery. Subjects were interviewed for alcohol intake and placental FAEEs were quantified via GC/MS. Receiver Operator Characteristic (ROC) Curves were generated to evaluate the ability of placental FAEEs to predict maternal drinking during pregnancy. Adjusted ROC curves were generated to adjust for gestational age, maternal smoking, and illicit drug use. 30% of the subjects admitted to drinking alcohol during pregnancy and approximately 14% answered questions indicative of problem drinking (designated AUDIT+). The specific FAEEs ethyl stearate and linoleate, as well as combinations of oleate + linoleate + linolenate (OLL) and of OLL + stearate, were significantly (p<0.05) elevated in placentas from AUDIT+ pregnancies. Adjusted ROC Curves generated areas under the curve ranging from 88–93% with negative predictive values of 97% for AUDIT+ pregnancies. We conclude that nearly one third of premature pregnancies were alcohol-exposed, and that elevated placental FAEEs hold great promise to accurately determine maternal alcohol use, particularly heavy use, in pregnancies complicated by premature delivery.     

Occurrence of fatty acid lower-alkyl esters in euonymus fruits

Small amounts of a mixture of fatty acid lower-alkyl esters (FALAEs) were obtained from chloroform extracts of the fruit arils and seeds of four euonymus species (Euonymus sp., Celastraceae). The FALAEs were the products of biosyntheses in the cell rather than experimental artifacts. By using GC-MS, this mixture was shown to contain a total of 19 individual FALAE species comprising four separate fractions, viz. methyl (FAMEs), ethyl (FAEEs), n-propyl, and n-butyl FA esters. Fruit FALAEs included mainly FAEEs and, to a lesser extent, FAMEs, while the n-propyl and n-butyl FA esters, which occurred less frequently, were found here for the first time as the plant products. The FALAE acid components included C₁₄-C₁₈ saturated, mono-, di-, and trienoic FAs with the predominance of ubiquitous linoleic, oleic, palmitic, and, in some cases, also α-linolenic acid. The indices of qualitative and quantitative composition of separate FALAE fractions varied considerably depending on the plant species, fruit part (aril or seed), and the extent of fruit maturity. It can be supposed that, in some euonymus species, FAMEs and FAEEs are formed at the expense of the same FA pool characteristic for a given species. As a whole, euonymus FALAEs and triacylglycerols seem to be synthesized from different FA pools. Discussed is the physiological significance of FALAE biosynthesis in plant metabolism, possible pathways of this biosynthesis, as well as the perspectives of further investigations of FALAEs of plant origin.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

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.     

Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with <Emphasis Type="Italic">OPI1</Emphasis> deletion

During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.     

Fatty Acid Ethyl Esters Induce Intestinal Epithelial Barrier Dysfunction via a Reactive Oxygen Species-Dependent Mechanism in a Three-Dimensional Cell Culture Model

Background & Aims

Evidence is accumulating that ethanol and its oxidative metabolite, acetaldehyde, can disrupt intestinal epithelial integrity, an important factor contributing to ethanol-induced liver injury. However, ethanol can also be metabolized non-oxidatively generating phosphatidylethanol and fatty acid ethyl esters (FAEEs). This study aims to investigate the effects of FAEEs on barrier function, and to explore the role of oxidative stress as possible mechanism.

Methods

Epithelial permeability was assessed by paracellular flux of fluorescein isothiocyanate-conjugated dextran using live cell imaging. Cell integrity was evaluated by lactate dehydrogenase release. Localization and protein levels of ZO-1 and occludin were analyzed by immunofluorescence and cell-based ELISA, respectively. Intracellular oxidative stress and cellular ATP levels were measured by dichlorofluorescein and luciferase driven bioluminescence, respectively.

Results

In vitro, ethyl oleate and ethyl palmitate dose dependently increased permeability associated with disruption and decreased ZO-1 and occludin protein levels, respectively, and increased intracellular oxidative stress without compromising cell viability. These effects could partially be attenuated by pretreatment with the antioxidant, resveratrol, pointing to the role of oxidative stress in the FAEEs-induced intestinal barrier dysfunction.

Conclusions

These findings show that FAEEs can induce intestinal barrier dysfunction by disrupting the tight junctions, most likely via reactive oxygen species-dependent mechanism.     

Simplifying Hansen Solubility Parameters for Complex Edible Fats and Oils

Hansen solubility parameters (HSPs), often used to predict the miscibility between two compounds, are an alternative tool in evaluating the ability of the solvent to interact via dispersion, dipole-dipole, and hydrogen bonding interactions. The aim of this paper is to find a simple way to calculate HSPs for complex mixtures of triglycerides (TAGs). HSPs were calculated using two approaches: the first assumes that the contributions to the dispersion, dipole-dipole, and hydrogen bonding interactions may be subdivided into larger functional moieties (i.e., fatty acids and fatty acid methyl esters) that are additive, while the second approach assumes that vegetable oils are comprised of mixtures of simple TAGs in the same mass fractions as the fatty acids. The HSPs obtained using the two approaches are compared to reference values determined using the “Hansen Solubility Parameters in Practice” software (HSPiP) considering the complex TAG profile for each vegetable oil.HSPs for vegetable oils, obtained with the HSPiP software, did not correspond well to the HSPs obtained from the group contribution approach, when using fatty acids, fatty acids + glycerol or fatty acid methyl esters. In contrast, the HSPs calculated for vegetable oils, assuming that all TAGs are simple and in the same mass fractions as the fatty acids, provide similar values to the HSPs obtained from the HSPiP software. Therefore, it is possible to calculate the HSPs for complex oils by simply knowing the fatty acid composition. Knowledge of HSPs may be used to rationalize the ability of certain low molecular weight molecules to develop organogels in vegetable oils as well as the crystallization of triglycerides.     

Engineering Escherichia coli for biodiesel production utilizing a bacterial fatty acid methyltransferase

The production of low-cost biofuels in engineered microorganisms is of great interest due to the continual increase in the world's energy demands. Biodiesel is a renewable fuel that can potentially be produced in microbes cost-effectively. Fatty acid methyl esters (FAMEs) are a common component of biodiesel and can be synthesized from either triacylglycerol or free fatty acids (FFAs). Here we report the identification of a novel bacterial fatty acid methyltransferase (FAMT) that catalyzes the formation of FAMEs and 3-hydroxyl fatty acid methyl esters (3-OH-FAMEs) from the respective free acids and S-adenosylmethionine (AdoMet). FAMT exhibits a higher specificity toward 3-hydroxy free fatty acids (3-OH-FFAs) than FFAs, synthesizing 3-hydroxy fatty acid methyl esters (3-OH-FAMEs) in vivo. We have also identified bacterial members of the fatty acyl-acyl carrier protein (ACP) thioesterase (FAT) enzyme family with distinct acyl chain specificities. These bacterial FATs exhibit increased specificity toward 3-hydroxyacyl-ACP, generating 3-OH-FFAs, which can subsequently be utilized by FAMTs to produce 3-OH-FAMEs. PhaG (3-hydroxyacyl ACP:coenzyme A [CoA] transacylase) constitutes an alternative route to 3-OH-FFA synthesis; the coexpression of PhaG with FAMT led to the highest level of accumulation of 3-OH-FAMEs and FAMEs. The availability of AdoMet, the second substrate for FAMT, is an important factor regulating the amount of methyl esters produced by bacterial cells. Our results indicate that the deletion of the global methionine regulator metJ and the overexpression of methionine adenosyltransferase result in increased methyl ester synthesis.     

Divergent effects of eicosapentaenoic and docosahexaenoic acid ethyl esters, and fish oil on hepatic fatty acid oxidation in the rat

The physiological activity of fish oil, and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affecting hepatic fatty acid oxidation was compared in rats. Five groups of rats were fed various experimental diets for 15 days. A group fed a diet containing 9.4% palm oil almost devoid of n-3 fatty acids served as a control. The test diets contained 4% n-3 fatty acids mainly as EPA and DHA in the form of triacylglycerol (9.4% fish oil) or ethyl esters (diets containing 4% EPA ethyl ester, 4% DHA ethyl ester, and 1% EPA plus 3% DHA ethyl esters). The lipid content of diets containing EPA and DHA ethyl esters was adjusted to 9.4% by adding palm oil. The fish oil diet and ethyl ester diets, compared to the control diet containing 9.4% palm oil, increased activity and mRNA levels of hepatic mitochondrial and peroxisomal fatty acid oxidation enzymes, though not 3-hydroxyacyl-CoA dehydrogenase activity. The extent of the increase was, however, much greater with the fish oil than with EPA and DHA ethyl esters. EPA and DHA ethyl esters, compared to the control diet, increased 3-hydroxyacyl-CoA dehydrogenase activity, but fish oil strongly reduced it. It is apparent that EPA and DHA in the form of ethyl esters cannot mimic the physiological activity of fish oil at least in affecting hepatic fatty acid oxidation in rat.     

Enrichment of pinolenic acid from pine nut oil via lipase-catalyzed ethanolysis with an immobilized Candida antarctica lipase

Abstract

Pinolenic acid (PLA) enrichment as an ethyl ester from pine nut oil was successfully accomplished in a batch reactor by lipase-catalyzed ethanolysis using Novozym 435 lipase from Candida antarctica as a biocatalyst. PLA is predominantly an sn-3 substituent of the pine nut oil triacylglycerol (TAG), where it accounts for about 39 mol% of the fatty acids esterified at that position. In the presence of ethanol, Novozym 435 exhibited sn-3 regiospecificity with respect to the TAG of pine nut oil. The effect of the molar ratio of reactants on PLA enrichment by ethanolysis was investigated. The molar ratios of pine nut oil to ethanol were varied from 1:20 to 1:100. A fatty acid ethyl ester (FAEE) fraction with higher PLA content was obtained in the early stage of the reaction, although the yield of PLA was small. However, the PLA content of the FAEEs decreased with increasing reaction time, while the yield of PLA increased. The molar ratio of pine nut oil to ethanol that produced the optimum content and yield of PLA in FAEEs was 1:80.     

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.     

Fatty acyl esters potentiate fatty acid induced activation of protein kinase C

Purified rat pancreas protein kinase C (PKC) is activated by unsaturated free fatty acids (oleic and arachidonic). The ethyl esters of these fatty acids are ineffective as enzyme activators. However, when the ethyl esters are added in combination with a free fatty acid, there is significant enhancement of enzyme activation. Nearly optimal PKC activation was obtained when non-activating ethyl oleate or ethyl arachidonate was added to sub-optimally activating concentrations of oleic or arachidonic acids. In addition to the ethyl esters, 1-monooleylglycerol also had a potentiating effect on PKC activation by oleic acid. However, the degree of activation observed in the presence of a free fatty acid and an acyl ester of the fatty acid quantitatively never surpassed that produced by sn-1,2-dioleylglycerol. Our findings indicate that significant PKC activation can be achieved by presenting the enzyme with an environment which we believe approximates the structural characteristics of the endogenous activator, sn-1,2-diacylglycerol.     

Biomass to fuels via microbial transformations

The conversion of biomass to fuels is advancing on two fronts: first, to enhance the usability of biomass and second, to generate the best fuel molecule(s). Lignin generally presents a barrier to biomass utilization; this problem may be circumvented by the genetic alteration of lignin-producing plants. Plant cellulosic material will need to be utilized more efficiently, requiring a greater knowledge of the multiprotein complex, the cellulosome. Many biofuel types are being considered: alcohols, esters, ethers, and hydrocarbons. Intrinsic alcohol toxicity to cells may impose limitations on ethanol and n-butanol production. Biodiesel, or fatty acid esters, can now be synthesized via recombinant Escherichia coli and may offer an improvement over alcohol fermentations. Biopetroleum (hydrocarbons) is being considered as an alternative biofuel.