Presence of nonoxidative ethanol metabolism in human organs commonly damaged by ethanol abuse.

Acetaldehyde, the end product of oxidative ethanol metabolism, contributes to alcohol-induced disease in the liver, but cannot account for damage in organs such as the pancreas, heart, or brain, where oxidative metabolism is minimal or absent; nor can it account for the varied patterns of organ damage found in chronic alcoholics. Thus other biochemical mediators may be important in the pathogenesis of alcohol-induced organ damage. Many human organs were found to metabolize ethanol through a recently described nonoxidative pathway to form fatty acid ethyl esters. Organs lacking oxidative alcohol metabolism yet frequently damaged by ethanol abuse have high fatty acid ethyl ester synthetic activities and show substantial transient accumulations of fatty acid ethyl esters. Thus nonoxidative ethanol metabolism in addition to the oxidative pathway may be important in the pathophysiology of ethanol-induced disease in humans.

Mitochondrial dysfunction induced by fatty acid ethyl esters, myocardial metabolites of ethanol.

Mechanisms responsible for alcohol-induced heart muscle disease have been difficult to elucidate partly because of previously obscure, demonstrable cardiac metabolism of ethanol. Recently, fatty acid ethyl esters were identified in our laboratory and found to be myocardial metabolites of ethanol. In the present study, they have been shown to induce mitochondrial dysfunction. Incubation of isolated myocardial mitochondria with fatty acid ethyl esters led to a concentration-dependent reduction of the respiratory control ratio index of coupling of oxidative phosphorylation and decrement of maximal rate of oxygen consumption. Furthermore, fatty acid ethyl esters were demonstrated to bind to mitochondria in vitro, and, importantly, 72% of intracellularly synthesized ethyl esters were found to bind to mitochondria isolated from intact tissue incubated with ethanol. Protein binding of fatty acid ethyl esters was markedly less than that of fatty acids. Because uncoupling of mitochondrial oxidative phosphorylation correlated with the cleavage of fatty acid ethyl ester shown to be initially bound to mitochondria, with resultant generation of fatty acid, a potent uncoupler, in a locus in or near the mitochondrial membrane, fatty acid ethyl esters may contribute to a potentially toxic shuttle for fatty acid with transport from physiological intracellular binding sites to the mitochondrial membrane; direct effects of fatty acid ethyl esters may also be deleterious. Operation of this shuttle as a result of ethanol ingestion and subsequent accumulation of fatty acid ethyl esters may account for the impaired mitochondrial function and inefficient energy production associated with toxic effects of ethanol on the heart.

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.

Oxygen at physiological concentrations. A potential, paradoxical mediator of reperfusion injury to mitochondria induced by phosphate.

Cellular injury induced by reperfusion after myocardial ischemia is manifested by striking mitochondrial damage as well as other hallmarks such as contraction band necrosis. Calcium has been implicated as a mediator of irreversible cellular injury in several systems. To identify other potential mediators of the mitochondrial injury associated with reperfusion, interactions between inorganic phosphate, oxygen, and mitochondria harvested from rabbit hearts were evaluated in vitro. Mitochondria exhibited rapid inactivation of oxidative phosphorylation after preincubation at 25 degrees C when phosphate and oxygen were present. Inactivation was partially but not completely precluded by EDTA, EGTA, magnesium, diltiazem, or ruthenium red, results in concert with findings of others suggesting involvement of a deleterious influx of calcium into mitochondria; exogenous calcium enhanced inactivation. However, the present data indicate that inactivation is prevented by incubation of mitochondria in the absence of oxygen, and demonstrate for the first time that injury elicited by phosphate is dependent on oxygen at physiological concentrations either because calcium and/or phosphate influx is linked to aerobic metabolism or because oxygen exerts deleterious effects on mitochondria, which may render them particularly susceptible to calcium influx. Since intracellular inorganic phosphate concentration increases markedly with ischemia, reperfusion with oxygenated medium may paradoxically augment mitochondrial injury in this setting. Thus, in the presence of increased intracellular concentrations of calcium and phosphate induced by ischemia, subsequent reestablishment of physiological levels of intracellular oxygen tension may promote mitochondrial damage, which is known to increase with reperfusion.

Metabolism of very low density lipoproteins after cessation of cholesterol feeding in rabbits. A factor potentially contributing to the slow regression of atheromatous plaques.

Aortic atheromatous plaques regress slowly in cholesterol-fed rabbits that have been returned to normal laboratory diet. To delineate metabolic factors potentially responsible for persistence of atherosclerosis under these conditions, the physical, chemical, and metabolic characteristics were determined for lipoproteins of d less than 1.006 g/ml; such lipoproteins are thought to be the major determinant of progression of atherosclerotic lesions in cholesterol-fed rabbits. At the time of return to a normal laboratory diet regimen after 3 mo of feeding with cholesterol-enriched laboratory diet, plasma cholesterol concentrations were 2,275 +/- 252 mg/dl, mostly attributable to cholesteryl ester-rich very low density lipoproteins (VLDL). On the hypercholesterolemic diet, fractional catabolic rates of plasma clearance of 125I-labeled VLDL were reduced (0.011 +/- 0.002 vs. 0.151 +/- 0.015 h-1), but the total VLDL catabolic rate was increased considerably (17.1 +/- 2.2 vs. less than 1.2 +/- 0.4 mg of protein/kg X d), because of the expansion of the endogenous pool of cholesteryl ester-rich VLDL. The total catabolic rate of VLDL was maintained above estimated control values (5.8 +/- 0.7 mg protein/kg X d) even 10 wk after return of the rabbits to a normal chow regimen, an effect attributable to continued high rates of cholesteryl ester-rich VLDL synthesis in liver. Accumulation of cholesteryl ester-rich VLDL into aortic tissue persisted at a high rate. Thus the persistence of aortic atheromatous lesions after cessation of cholesterol feeding was attributable in part to continued high rates of hepatic production of cholesteryl ester-rich VLDL and its persistent delivery into the aortic wall.

Effects of acetyl glyceryl ether of phosphorylcholine (platelet activating factor) on ventricular preload, afterload, and contractility in dogs.

Acetyl glyceryl ether of phosphorylcholine (AGEPC), platelet activating factor, is a potent hypotensive agent that may mediate changes in blood pressure during anaphylaxis and may be involved in blood pressure variations of renal origin. This study was designed to characterize the hemodynamic mechanisms responsible for hypotension induced by this recently identified phospholipid. Intravenous administration of AGEPC to anesthetized open-chest dogs (n = 5) produced hemodynamic alterations which, for the purpose of analysis, were divided into three phases based on changes in the mean systemic blood pressure. During phase I (5-30 s) mean systemic blood pressure decreased to levels 5 to 10% below baseline values in association with a rise in cardiac output and a decrease in systemic vascular resistance. Phase II (30-90 s) consisted of a substantial reduction in systemic blood pressure to its nadir, 50% of baseline values, together with a decrease of similar magnitude in cardiac output and a rise in systemic vascular resistance. Phase III (90 s-60 min) exhibited a gradual recovery of mean systemic blood pressure toward normal with a several-fold rise in systemic vascular resistance and a continued low cardiac output. On the right side of the circulation, the predominant effect of AGEPC was a marked transient increase in pulmonary artery pressure in phase I, associated with an elevation of pulmonary resistance during phase II. Diethylcarbamazine blocked virtually all of these hemodynamic changes induced by AGEPC; FPL 55712 substantially blocked the rise in systemic vascular resistance in phase III. These results suggest that leukotrienes may mediate at least some of the hemodynamic effects induced by AGEPC, but further studies will be required when more specific leukotriene blocking agents become available. As assessed during phase III with the end-systolic pressure-dimension relation, myocardial performance itself was diminished. The occurrence of an AGEPC-induced negative inotropic effect was further confirmed in isolated Krebs-perfused guinea pig hearts and isolated blood-perfused rabbit hearts. The results indicate that the mechanism of AGEPC-induced hypotension is complex, affecting both vascular tone and the inotropic state of the myocardium.

Partial purification and product characterization of fatty acid ethyl ester synthases in rabbit myocardium.

Homogenates of rabbit ventricular myocardium synthesize fatty acid ethyl esters using as substrates nonesterified fatty acid and ethanol in the absence of coenzyme A and ATP. This catalytic activity resides in two soluble cytosolic enzymes accounting for 19 and 81% of total fatty acid ethyl ester synthetic capability. These enzymes have been separated and partially purified by anion exchange chromatography. Gas chromatographic/mass spectrometric analyses of the catalytic products formed by these enzymes from nonesterified fatty acid and ethanol confirm their identity as ethyl esters of fatty acids. Kinetic studies indicate apparent Km values for ethanol of 0.65 M and 0.75 M for the minor and major activities, respectively. These data confirm the presence of a myocardial pathway for nonoxidative ethanol metabolism and for a metabolism of fatty acids independent of coenzyme A.

Protein-stimulated exchange of phosphatidylcholine between intact erythrocytes and various membrane systems.

Phosphatidylcholine specific exchange protein from beef liver was found to catalyze the exchange of phosphatidylcholine between intact rat and human erythrocytes and various artificial membranes. Both multilamellar liposomes and single bilayer vesicles prepared from egg lechithin, cholesterol and phosphatidic acid (46:50:4, mol/mol) appeared to be effective phospholipid donor systems. Some merits and disadvantages of the various donor systems are discussed.

Immune cytokines and cardiac disease.

In conditions such as idiopathic dilated congestive cardiomyopathy associated with lymphocytic myocarditis and cardiac allograft rejection, the immune system can reversibly impair cardiac function. Cytokines interleukin-1 and tumor necrosis factor disrupt ß-adrenergic signal transduction and agonist stimulation of contractility. Identification of this reversible effect potentially offers a novel pathophysiologic mechanism for producing cardiac injury.

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).

Relationship of human pancreatic cholesterol esterase gene structure with lipid phenotypes.

Pancreatic cholesterol esterase is one of the enzymes that plays a pivotal role in cholesterol absorption. Differences in the genotype of this enzyme could affect the susceptibility of individuals to dyslipidemia and/or cardiovascular disease. We undertook this study to investigate if any correlation exists between restriction fragment length polymorphism in the human pancreatic cholesterol esterase gene and serum lipid levels. DNA from 96 healthy adults was restricted with Stu I, Southern blotted, and probed with cDNA of human pancreatic cholesterol esterase. Results revealed six distinct patterns which were classified as A, B, C, D, E, and F which had a population frequency of 1%, 34.5%, 49%, 12.5%, 1% and 2% respectively. Correlation of the distribution of lipid and lipoprotein levels by pattern and sex revealed a significant interaction between pattern type and HDL (p=0.03) in the most common group (group C) for males. Male patients of pattern C tended to have a lower LDL cholesterol than non-pattern C males (p=0.07); in addition, 80% of all males in the study population with LDL cholesterol under 100 mg/dl were found in pattern C. Thus, the most common Stu I RFLP genotype is associated with a favorable lipid phenotype. This report shows an association between the human pancreatic cholesterol esterase genotype and serum lipid levels. Further analysis of a larger study group with Stu I and alternative polymorphic restriction enzymes is warranted, to confirm this biologically plausible result.

Fatty acid ethyl esters in adipose tissue. A laboratory marker for alcohol-related death.

Fatty acid ethyl esters, a family of ethanol metabolites, are formed by esterification of ethanol with fatty acids and have been detected in human organs commonly damaged by ethanol abuse. Because alcohol-related deaths may occur up to six times as often as reported on death certificates, we undertook quantitation of these potentially longer-lived alcohol metabolites in postmortem human adipose tissue to assess their usefulness as a measure of recent ethanol exposure. After isolation and identification using sequential thin-layer and gas chromatography, fatty acid ethyl esters were present in adipose tissue of chronic alcoholics (mean +/- SEM equals 300 +/- 46 nmol/g), even though blood ethanol concentration at the time of death was undetectable. Unintoxicated nonalcoholic subjects who had no history of alcohol abuse had concentrations seven times lower (mean +/- SEM equals 43 +/- 13 nmol/g). In vitro studies demonstrate that fatty acid ethyl esters are synthesized by human adipose tissue in proportion to the ethanol concentration present and their half-life in adipose tissue of laboratory animals is 16 +/- 1.6 hours, ie, fourfold greater than that of alcohol. These results indicate that fatty acid ethyl esters are long-lived ethanol metabolites whose persistence and accumulation in adipose tissue may allow an accurate diagnosis of significant alcohol consumption even when ethanol has been completely eliminated from the body.

Nonoxidative ethanol metabolism: formation of fatty acid ethyl esters by cholesterol esterase.

The recent identification of myocardial metabolites of ethanol--fatty acid ethyl esters--suggests that some of the pathophysiological derangements associated with alcohol-induced heart muscle disease may be a consequence of products of myocardial ethanol metabolism. The donor of the fatty acid moiety in the formation of fatty acid ethyl esters has been identified as nonesterified fatty acid. Fatty acid esterification with ethanol is shown to be mediated by cholesterol esterase (sterol-ester acylhydrolase, EC 3.1.1.13), a finding that identifies a singular nonoxidative ethanol metabolism by an enzyme. A potential basis for the protective effect of ethanol ingestion on atherogenesis is also suggested because fatty acid ethyl esters inhibit cholesterol esterification catalyzed by pancreatic cholesterol esterase and hepatic and aortic microsomal fatty acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26).

Immune mechanisms of cardiac disease.

It is evident that cellular infiltration can affect cardiac structure and function in a variety of disease states. Myocardial contractility can be impaired by cell-mediated injury or local release of cytokines. The study of immune cardiac disease has entered a period of rapid expansion that should be characterized by delineation of the mechanisms by which immune cells and factors localize in the myocardium, modulate myocyte function, and remodel myocardial architecture (Fig. 2). This new knowledge should result in the ability to target specifically both the pathways by which cardiac contractility is impaired by chronic inflammation and the sustained immune reactivity to cardiac antigens that underlies chronic myocardial inflammation. Nonspecific therapeutic interventions directed at congestive heart failure, currently the only acceptable approach to the treatment of immune myocarditis, should then serve a more ancillary function in the context of the use of rationally designed drugs. Such drugs could, for example, be specifically targeted to inhibiting the trafficking of leukocytes into the heart or the effects of their subsequent activation within the myocardium.

Molecular mechanism of ethanol metabolism by human brain to fatty acid ethyl esters.

Ethanol metabolism in the human brain has been documented to occur with the formation of fatty acid ethyl esters. These neutral lipids can disorder membranes and interrupt mitochondrial function. Their formation is under the control of three synthases, localized to grey matter and purified to homogeneity. cDNA cloning demonstrates two of these enzymes to be GSH S-transferases and has enabled initiation of genetic studies of alcohol-induced CNS injury.

Myocardial metabolites of ethanol.

Because of the importance of alcohol-induced heart muscle disease and the obscurity of its pathogenesis, this study was undertaken to determine whether fatty acid ethyl esters, myocardial metabolites of ethanol recently described in our laboratory to be synthesized in cell-free extracts of rabbit myocardium, accumulate in hearts of human subjects exposed to ethanol in vivo. Lipid extracts were prepared from left ventricular samples obtained at necropsy from six subjects who had been exposed to ethanol acutely or chronically. Fatty acid ethyl esters were present in each extract in concentrations ranging from 9 to 115 microM. In contrast, they were consistently absent from analogous samples obtained from hearts of abstainers (n = 5). In parallel studies in experimental animals, we found that fatty acid ethyl esters are formed not only in the heart but also in the pancreas and liver--targets of injury associated with chronic alcohol abuse. These results demonstrate the presence in human myocardium of a novel metabolite of ethanol that potentially may serve as a marker for exposure to alcohol and that could be relevant to the pathophysiology of excessive alcohol consumption leading to cardiac abnormalities.

Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases.

A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.