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Fatty Acid Ethyl Esters: Potentially Toxic Products of Myocardial Ethanol Metabolism
J Mol Cell Cardiol 30, 2487–2494 (1998) Article No. mc980812
Fatty Acid Ethyl Esters: Potentially Toxic Products of Myocardial Ethanol Metabolism Mary E. Beckemeier∗ and Puran S. Bora∗† ∗Veterans Affairs Medical Center, GRECC Service, St. Louis, Missouri, 63108 and †Department of Internal Medicine, Division of Cardiology, Saint Louis University Medical Center, St. Louis, Missouri, 63110, USA. (Received 7 May 1998, accepted in revised form 25 August 1998) M. E. B and P. S. B. Fatty Acid Ethyl Esters: Potentially Toxic Products of Myocardial Ethanol Metabolism. Journal of Molecular and Cellular Cardiology (1998) 30, 2487–2494. The chronic consumption of alcohol has proven detrimental to heart tissue and can lead to alcohol-induced heart muscle disease, a condition which may result in arrhythmias, cardiomegaly, and congestive heart failure. A search for the molecular mechanism underlying observed alcohol-induced end-organ damage, such as that seen in heart, has lead to the discovery of a nonoxidative pathway for the metabolism of alcohol in several human tissues including heart, brain, pancreas, and liver. It has been revealed that nonesterified fatty acids are esterified with ethanol to produce fatty acid ethyl esters (FAEE), neutral molecules which can accumulate in mitochondria and impair cell function. The observation that FAEEs are synthesized at high rates in the heart, and other organs that lack oxidative ethanol metabolism, provides a plausible link between the observed tissue damage, the ingestion of alcohol, and the subsequent development of alcohol-induced heart muscle disease. The synthesis of FAEEs are catalyzed by FAEE synthase enzyme, four of which have been characterized and purified to homogeneity from the human myocardium. Further analysis of these FAEE synthase enzymes opens up a new possibility to characterize and map a gene for alcohol-induced end-organ damage, such as that observed in heart and other organs. FAEEs have been found to be important metabolites of alcohol and are most commonly accumulated in those organs which are damaged by alcohol abuse, i.e. heart. It may now be important to establish a genetic link between alcohol abuse and alcohol-induced heart muscle disease in order to understand the mechanism of alcohol-induced 1998 Academic Press cardiomyopathy. K W: Metabolism; Alcohol; Mitochondria; Nonoxidative; Cardiomyopathy; Fatty Acids.
Introduction The chronic abuse of alcohol has been known to adversely affect the health of many individuals, causing life-threatening conditions in numerous organs including heart, brain, pancreas, and liver. With respect to the heart, alcohol abuse has proven to be a major cause of non-ischemic cardiomyopathy in Western society (Anderson and Waagstein, 1993; Roberts et al., 1987). These cases have come to be collectively known as alcoholinduced heart muscle disease, a condition in which
heart muscle is plagued with abnormal contractile function and energy metabolism that sometimes results in arrhythmias, cardiomegaly, and congestive heart failure (Asokan et al., 1972). On a cellular level, alcohol-induced myocardial damage includes loss and/or disruption of myocytes, myofibrils, muscle fibers, sarcolemma, sarcoplasmic reticulum, the Na+–K+–ATPase pump, and mitochondria (Alexander et al., 1967; Burch et al., 1971; Hibbs et al., 1965; Kupari et al., 1991; Noren et al., 1983; Urbano-Marquez et al., 1989; Vikhert et al., 1986). In terms of a cellular mechanism for the
Please address all correspondence to: Dr Puran S. Bora, Department of Medicine, Division of Cardiology, Saint Louis Medical Center, 3635 Vista at Grand, USA.
0022–2828/98/112487+08 $30.00/0
1998 Academic Press
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observed ethanol-induced tissue daage, past attempts have been hampered due to a lack of evidence of a metabolic, or biochemical, alcohol metabolizing pathway in the heart. Ethanol has been shown to be metabolized oxidatively creating a toxic product known as acetaldehyde, yet it was revealed that only the liver substantially metabolizes alcohol in this manner (Goldstein, 1983). However, a nonoxidative pathway for ethanol metabolism has been discovered in heart, brain, pancreas, and several tissues including blood (Bora et al., 1989a, b, c, 1991, 1992a, 1996a; Bora and Lange, 1990, 1991a, b; Lange et al., 1981; Gorski et al., 1996). This metabolic ethanol pathway has a potentially toxic product known as fatty acid ethyl ester (FAEE), a neutral family of molecules that are thought to accumulate in the mitochondria of the cell and facilitate the uncoupling of oxidative phosphorylation. The uncoupling of the energy transduction process of the mitochondria can result in an inefficiency of energy production as well as cell damage, providing a link between ethanol ingestion and the subsequent development of alcohol-induced heart muscle disease as well as other end-organ damage (Lange and Sobel, 1983a; Bora et al., 1996a). The synthesis of FAEE in the body is catalyzed by an enzyme known as FAEE synthase. In the human heart four FAEE synthase enzymes have been characterized and purified to homogeneity. These myocardial synthase enzymes are known as synthase-I, synthase-II, synthase-III, and synthase/carboxylesterase (Bora et al., 1989a, b, c, 1991, 1992a, 1996b; Bora and Lange, 1990, 1991a, b). Activity of the FAEE synthase enzymes is highest in heart, brain, and pancreas, the extrahepatic organs most commonly damaged by alcohol abuse (Laposata and Lange, 1986; Laposata et al., 1987). Because these organs lack or have minimal oxidative ethanol metabolism and because a genetic component to selectivity of alcohol-induced end-organ damage exists for some of these syndromes, gene products that modulate ethanol-associated tissue injury must reside within these organs. Thus, FAEE synthase may be at least one candidate for the gene product producing alcoholinduced heart muscle disease. A better understanding of the FAEE synthase enzymes could provide for regulation of the toxic effects of FAEE as well as insight into a possible genetic predisposition for alcohol-induced heart muscle disease.
Fatty Acid Ethyl Esters Alcohol is easily absorbed and distributed into tissues high in water content and blood flow, i.e. heart (Goldstein, 1983). The acute and chronic
administration of alcohol in several human and animal models has demonstrated depressed cardiac function (Ettinger et al., 1976; Gimeno et al., 1962; Hastillo et al., 1980; Regan et al., 1969; Pachinger et al., 1973; Sarma et al., 1976). Furthermore, electron-microscopic and histochemical analysis have revealed shrunken, disorganized myocytes, loss of myofibrils, a dilated and disordered sarcoplasmic reticulum, the excessive accumulation of lipids, and enlarged mitochondira with chaotic cristae (Alexander et al., 1967; Burch and De Pasquale, 1971; Hibbs et al., 1965; Kupari et al., 1991; Urbano-Marquez et al., 1989; Vikhert et al., 1986). It has been found that chronic alcohol administration decreases activity of the Na+–K+–ATPase pump which functions in providing the necessary ionic concentrations for membrane potentials needed for cardiac muscle contraction (Noren et al., 1983). Also, several weeks of chronic alcohol consumption by rats demonstrated in the myocytes a leaky sarcolemma and increases in cardiac intracellular calcium (Noren et al., 1983). The observed compromising of the heart cell membrane can have deleterious effects on heart muscle contraction. Studies have also found disturbances in sarcoplasmic reticulum, responsible for Ca2+ release and uptake during contraction and relaxation (Bing et al., 1974; Williams et al., 1975; Sarma et al., 1976; Segal et al., 1975; 1981). Most notable for the purposes of this review is the observation of decreased myocardial mitochondrial function which includes, as Piano and Dorie (Piano and Dorie, 1994) have pointed out in their 1994 review of alcoholic heart disease, a drop in mitochondrial oxidative enzyme activity and a drop in levels of ATP and oxygen consumption (Pachinger et al., 1973; Sarma et al., 1976; Williams et al., 1975). The mitochondria manufactures most of the cell’s energy currency, ATP, via the tricarboxylic acid cycle (TCA cycle), b-oxidation of fatty acids, and oxidative phosphorylation. The very high energy requirements of the heart muscle are mostly met by b-oxidation of fatty acids (Lopaschuk et al., 1994). Fatty acids bound to albumin, or from chylomicrons and very-low-density lipoproteins, enter the myocyte where they are converted, after a series of steps, to acetyl CoA through b-oxidation in the mitochondrial matrix. Acetyl CoA then enters the TCA cycle, the products of which will enter the electron transport chain of the inner mitochondrial membrane where most of the cell’s ATP is produced (Lopaschuk et al., 1994). It has been established, through the work of Lange and Sobel (Lange and Sobel, 1983a, b), that esters formed in the heart after alcohol consumption
Fatty Acid Ethyl Esters
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Figure 1 Nonesterified fatty acids (FAs) bound in the cell cytosol react with ethanol (EtOH) to form FAEEs by FAEE synthase. The neutral FAEEs accumulate and bind to the inner mitochondrial membrane where they cause damage by uncoupling oxidative phosphorylation. Additionally, some of the FAEEs are hydrolyzed to FAs. Since FAs are a known uncoupler of oxidative phosphorylation, the FAs generated after FAEE hydrolysis can also cause cell damage (Lange and Sobel, 1983a).
can attach to the mitochondria and disrupt the energy process. This family of molecules, created by the nonoxidative metabolism of ethanol, are known as fatty acid ethyl esters (FAEEs) and have been found in those tissues damaged by ethanol abuse (Laposata and Lange, 1986). These potentially dangerous molecules are synthesized at high rates in myocardium, as well as in brain, pancreas, and blood, as a response to the ingestion of alcohol (Bora et al., 1989a, b, c, 1991, 1992a, 1996; Bora and Lange, 1990, 1991a, b; Lange et al., 1981; Gorski et al., 1996). In terms of molecular mechanism for FAEEs, it has been shown that fatty acids bound in the cytosol of the myocyte react with ethanol to form neutral FAEE molecules which bind to and accumulate in the mitochondrial inner membrane (Fig. 1) (Lange et al., 1981; Lange and Sobel, 1983b). The mitochondria then hydrolyze some of the FAEEs, by way of carboxylesterase enzyme (Bora et al., 1996b), to fatty acids (Lange et al., 1981; Lange and Sobel, 1983a; Bora et al., 1996b). Free fatty acids are a known uncoupler of oxidative phosphorylation in concentrations as low as 5 l (Lange and Sobel, 1983a; Borst et al., 1962; Jezek and Freisleben, 1994). Hence, the free fatty acids, as well as the esters themselves, cause uncoupling of oxidative phosphorylation, damaging the ATP synthesis activity of the mitochondria, providing for cellular dysfunction and possible cell death (Lange and Sobel, 1983a). Thus, a mechanism for extrahepatic damage due to alcohol abuse has been provided via the nonoxidative metabolism of ethanol and its products, FAEEs. The biological half-life of FAEE spans from 20–24 h, with FAEEs accumulating in the tissues of those individuals who chronically abuse alcohol
(Lange and Sobel, 1983a). This exposes the heart and other tissues to the potentially toxic effects of FAEEs for long periods of time, increasing the risk for possible end-organ damage (Bora et al., 1996a; Lange and Sobel, 1983a). The normal concentration of FAEEs in the heart muscle is less than 0.001 lmol/l, yet in human hearts obtained at autopsy of persons who were either acutely intoxicated or where chronic ethanol abusers concentrations reached as high as 115 lmol/l (Lange and Sobel, 1983a, b). Moreover, to illustrate the damage caused by FAEEs, rat pancreatic lysosomes incubated with the esters became unstable and fragile (Haber et al., 1993) and the incubation of mouse brain synaptosomes with FAEEs caused a disordering of the membrane lipid bilayer (Hungund et al., 1988). Additionally, our laboratory has shown that by injecting small amounts of FAEE into rat myocardium the histopathological changes, noted through light microscopy, were significant and dose dependent (Bora et al., 1996a). The damage was evident in the myocardial tissue, four and 30 days after injection of the FAEE, as swollen and deformed cells (Table 1). The results coincide with a study done by Szczepiorkowski et al., 1995, in which intact HepG2 liver cells exposed to FAEE showed marked histological differences when compared to a control. However, more studies are needed to determine the damage caused by FAEEs, including the development of transgenic and knockout animal models. We have also shown that FAEEs have binding capabilities to the mitochondria (Bora et al., 1996a). Our results demonstrate that the binding of ethyl oleate to the mitochondria increases linearly with time, providing the evidence that FAEEs can bind to
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Table 1 Myocardial cell damage analysis by a computer software program with digital microscopic planimetry Days
Injection amount (lmol)
Damage area (lm2 )
30 30 30 50 50 50
270±2.1 932±0.9 3692±0.4 418±1.9 1298±1.5 5263±0.8
Table 2 GST Activity of synthase I, II, III and synthase/ carboxylesterase enzymes#
Enzyme 2 4 30 2 4 30
Values are mean±...
the mitochondria (Bora et al., 1996a). Furthermore, we have shown that of the total ethyl oleate injected into the myocardium, 8 lmol oleate and 1 lmol ethyl oleate were bound to the mitochondria. The 8 lmol labeled oleate bound to the mitochondria suggests that the labeled ethyl loeate was broken down in the mitochondria to form fatty acid. This data is corroborated with a liver study in which 90% of the FAEE taken in by intact HepG2 cells was hydrolyzed to fatty acids (Szczepiorkowski et al., 1995). Taken together, this data provides evidence that the cell, possibly the mitochondria, breaks down FAEEs to fatty acids, a known uncoupler of oxidative phosphorylation (Lange and Sobel, 1983a; Borst et al., 1962; Jezek and Freisleben, 1994). Investigators have found FAEEs in human blood serum after the ingestion of ethanol (Doyle et al., 1994). Furthermore, FAEEs have been shown to be delivered to the tissues by way of low density lipoproteins (LDLs) in the bloodstream, affecting such organs as the liver (Szczepiorkowski et al., 1995). This study has revealed that LDLs are passing their FAEE component into human hepatoblastoma (Hep G2) cells and causing a marked decrease in protein synthesis and cell formation in the liver. The assertion that most of the extracted FAEE was hydrolyzed by the liver cells to ethanol and fatty acid has left undetermined whether the liver damage was directly caused by the FAEE, or by the suspected released fatty acid component. Nevertheless, this data supports FAEE as perhaps a new culprit in alcohol-mediated liver damage and provides general evidence of a role for FAEE in endorgan damage.
The Role of Myocardial Fatty Acid Ethyl Ester Synthase Enzymes The formation of FAEEs in the body is catalyzed by an enzyme known as FAEE synthase (Lange et al.,
Synthase I Synthase III Synthase/ Carboxylesterase Synthase II
FAEE synthase activity (nmol/mg/h)
∗GST Activity
200 100 370
225 315 0.03
140
0.02
(nmol/mg/h)
∗ GST activity was assayed according to Bora et al., 1989a. # Bora et al., 1996b J Mol Cell Cardiol 28: 2027–2032.
1982; Mogelson and Lange, 1984; Mogelson et al., 1984). FAEE synthase has been found in the liver and pancreas of rats (Hamamoto et al., 1991), as well as in the brain of mice (Hungund et al., 1998), and has been purified and characterized from rabbit myocardium (Mogelson and Lange, 1984; Mogelson et al., 1984). Additionally, four FAEE synthase enzymes have been purified and characterized from human myocardium (Bora et al., 1989a; b, c, 1990, 1991, 1992a, 1996b; Bora and Lange, 1989a, b). As mentioned earlier, these myocardial FAEE synthase enzymes are known as synthase-I, synthase-II, synthase-III and synthase/carboxylesterase. Synthase-I and synthase-III are dimers with a subunit molecular mass of 26 kDa, both possessing glutathione S-transferase (GST) enzyme activity (Bora et al., 1989a, b, c) (Table 2). The GSTs are a family of enzymes involved in detoxification and drug resistance (Mannervik et al., 1985). SynthaseIII, whose main function is FAEE synthesis, exhibits 98% homology to an isoenzyme of GST known as GSTP1, whose main function is detoxification (Smith et al., 1995). Sequence analysis revealed a six amino acid difference between the genes. This small differential region most likely confers the functional variations between synthase-III and GSTP1 (Bora et al., 1991). Yet, even with this small differential region in mind, evidence of homology and GST enzyme activity suggests that synthaseIII and synthase-I are likely members of the GST enzyme family. An amino acid residue in synthase-III (purified in human myocardium), histidine 72, has shown to be important for GST but not synthase activities (Bora et al., 1992b). This data offers significant information in designing selective inhibitors of FAEE synthase-III. Additionally, the gene structure for synthase-III has been isolated, determined, and localized to human chromosome 11q13 (Bora et al., 1997). The complete nucleotide sequence of synthase-III has also been determined. The promoter
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Fatty Acid Ethyl Esters
Table 3 Allelic frequencies of ATAAA repeat region of FAEE synthase-III gene in different groups∗
Type
Alleles size (bp)
Frequency healthy control
Frequency heart patient
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11
409 410 415 418 420 425 427 431 433 435 437
0.110 0.309 0.410 0.710 0.818 0.050 0.135 0.115 0.095 0.080 0.075
0.095 0.805 0.790 0.115 0.250 0.325 0.110 0.107 0.090 0.085 0.065
∗ Unpublished data (Bora et al., 1998).
Figure 2 PCR products of fatty acid ethyl ester synthaseIII gene. The figure shows the number of alleles present in four different representative cardiovascular patients (unpublished data, Bora et al., 1998).
region has several transcription regulatory sequences including a TATA box and two SP1 recognition sequences. It has been also been shown that the repeat region of the synthase-III gene is polymorphic when PCR was performed using the DNA from unrelated individuals (unpublished data by Bora et al.). Figure 2 shows the electrophoretic patterns of the PCR products and the 11 alleles observed in the unrelated individuals. Table 3 shows the allele sizes and frequencies. The allelic frequencies from 100 unrelated healthy controls (no history of drinking) and 100 heart patients (drinking four to five drinks a day) were 0.765 and 0.798, respectively. The highest frequency among the alleles was A4 and A5 in healthy controls while among the heart patients A2 and A3 had the highest frequencies. Since FAEEs in concentrations as high as 115 lmol/l were identified in hearts obtained at autopsy from patients either acutely intoxicated at the time of death or chronically exposed to alcohol (Lange and Sobel, 1983b), the identification of a polymorphic marker corresponding to a known gene provides a bridge between genetic and physical mapping information.
This study will help in the understanding of the genetic control mechanism of the synthase enzyme(s) in alcoholism and alcohol-induced cardiomyopathy. Since both synthase-I and synthase-III are members of the GST enzyme family, it was assumed that synthase-II was yet another isoenzyme of the same faily, yet our laboratories have proven otherwise. We have purified and characterized synthase-II from human myocardium and determined that it is a monomer with a molecular mass of 65 kDa. There was no cross-reactivity observed between synthaseII and antibodies specific for synthase-III leading to the conclusion that synthase-II is distinct from the GST family (Bora et al., 1992a). Furthermore, in terms of possible connections to other enzyme families, a limited amount of homology was demonstrated between synthase-II and two heme binding proteins, human hemopexin and rabbit cytochrome P-45011C1. Still, absence of cross reactivity between synthase-II and antibodies to either of these two proteins provides evidence that synthase-II, despite the homology, was not related to either protein. Therefore, this study supports the conclusion that FAEE synthase-II is a novel protein belonging to another unique class of enzymes which can metabolize ethanol to FAEEs. The elution profile of a DEAE-cellulose chromatography assay of human myocardial cytosol determined that synthase-II accounts for up to 50% of total FAEE synthesis in human heart (Bora et al., 1992a). Hence, regulation of the synthase-II enzyme could prove significant when addressing alcohol-induced heart muscle disease considering that this enzyme pathway plays a prominent role in human heart. Consequently, the purification and characterization of synthase-II by our laboratories
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M. E. Beckemeier and P. S. Bora Table 4 N-terminal sequences of human synthase/carboxylesterase and rat adipose tissue synthase/carboxylesterase Human heart 62 kDa synthase/ Carboxylesterase
1
8
17
NH2 - (Y) P S S P P V V D T V H G K V L G
Rat adipose tissue 60 kDa synthase/ carboxylesterase
NH2 - Y P S S P P V V N T V K G K V L G
Vertical bars indicate the differences in the amino acid of two enzymes. Amino acid within bracket is ambiguous.
from human myocardium serves as an important step in the determination of possible regulatory mechanisms for this FAEE synthase enzyme (Bora et al., 1992a). A fourth FAEE synthase enzyme, molecular mass of 62 kDa, has also been purified and characterized from human myocardium (Bora et al., 1996b). Antibodies for this synthase enzyme did not crossreact with synthase I, II, or III, providing no evidence of homology among GST and synthase-II. This new class of ethanol metabolizing enzyme has been found to have 88% homology to the first 17 N-terminal residues in rat liver and adipose tissue carboxlyesterase (Table 4). The carboxylesterase enzyme family works throughout the body by detoxifying harmful agents by hydrolyzing them with ester and amide bonds. Homology between carboxlyesterase and FAEE synthase was found by Tsujita and Okuda (Tsujita and Okuda, 1992), who suggested that the FAEE synthase they purified from rat adipose tissue, among other tissues, contained FAEE synthase activity along with carboxlyesterase activity. This finding was confirmed by our laboratories who have recently purified and characterizied this particular FAEE synthase from human heart. We have found it to possess both synthase and carboxylesterase activities and have therefore named it FAEE synthase/carboxylesterase (Bora et al., 1996b). In terms of whether there is up or down regulation of the FAEE synthase enzyme(s) in those tissues most affected by alcohol abuse, there have been conflicting reports. One study reported seeing elevated activity of FAEE synthase in the brains of alcoholics as compared with nonalcoholics, correlating high levels of FAEE synthase with ethanol toxicity (Laposata et al., 1987). Furthermore, Bora et al. have found a four-fold increase in FAEE synthase activity in the heart tissue of ethanol-fed rabbits who received 20% of their calories as ethanol for 1 month (unpublished data). However, a slight decrease in the level of FAEE synthase was found
in the pancreas of rats when they were administered 36% of their calories as ethanol for 7 weeks (Hamamoto et al., 1990). FAEE synthase levels in the livers of mice also showed decreased enzyme levels under the same parameters (Hamamoto et al., 1991). Furthermore, a look at blood FAEE levels in humans revealed that alcoholics have approximately 50% less white blood cell FAEE synthase activity than their nonalcoholic counterparts (Gorski et al., 1996). Gorski et al. (1996) have speculated that the down regulation of FAEE synthase in blood tissue could be due to an adaptive response to limit the production of FAEEs. Further study is necessary to explain the regulation of FAEE synthase for it may play an integral role in the management of alcohol-induced end-organ damage and in the screening for possible predispositions to this damage.
Conclusion Alcohol abuse has long proven detrimental to the body, resulting in damage to such organs as heart, brain, pancreas and liver. In terms of a molecular explanation for this end-organ damage researchers have discovered the nonoxidative metabolism of ethanol in those organs damaged by ethanol abuse. The products of this nonoxidative pathway, FAEEs, have been shown to bind to the mitochondrion of the cell and to uncouple oxidative phosphorylation. It has been suggested that alcohol metabolism may be under genetic control and related to the presence of different amounts or types of ethanolmetabolizing enzymes (Cloninger et al., 1981; Goodwin et al., 1987). The FAEE synthase enzymes could be likely gene product candidates for the underlying genetic vulnerability to the effects of alcohol since no other significant pathway for the metabolism of alcohol is known to exist in the heart or other extrahepatic organs. Genetic studies using peripheral human leukocytes have shown that high synthase
Fatty Acid Ethyl Esters
activity is inheritable in an autosomal recessive pattern (Gilligan et al., 1989). This pattern is expected for a gene controlling the product of a toxic agent, such as FAEE synthase enzymes and their potentially toxic FAEE product. With this in mind, the cloning and mapping of the synthase enzymes to specific chromosomes, as has been done with synthase-III, opens up a new possibility to map a gene for alcoholism or alcohol-induced myocardial damage. Because FAEEs are important metabolites of ethanol and are found in highest concentrations in the heart and other target organs that are damaged by alcohol abuse, establishing a genetic link between alcohol abuse and end-organ damage may now be feasible.
Acknowledgements This work was supported by grants from Lichtenstein Foundation and V.A. Merit Review to P.S. Bora. The authors thank Drs Kelly Ludwig and Bryan Charles Doonan for their critical review of the manuscript.
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N GR, S NA, E S, M FL, A RW, 1983. Alcohol-induced congestive cardiomyopathy: an animal model. Cardiovasc Res 17: 81–87. P OM, T H, M JC, F JM, B RJ, 1973. The effect of prolonged administration of ethanol on cardiac metabolism and performance in the dog. J Clin Invest 52: 2690–2696. P MR, D SW, 1994. Alcoholic heart disease: a review. Heart Lung 23: 3–17. R TJ, L GE, O HA, F MJ, W AB, M CB 1969. Ventricular function in noncardiacs with alcoholic fatty liver: role of ethanol in the production of cardiomyopathy. J Clin Invest 48: 397–407. R WC, S RJ, MM BM, 1987. Idiopathic dilated cardiomyopathy: analysis of 152 necropsy patients. Am J Cardiol 60: 1340–1355. S JSM, I S, F R, M Y, W R, B RJ, 1976. Biochemical and contractile properties of heart muscle after prolonged alcohol administration. J Mol Cell Cardiol 8: 951–972. S LD, R SV, C Y, C K, A EA, M DT, 1975. Effects of chronic graded ethanol consumption on the metabolism ultrastructure and mechanical function of the rat heart. Cardiovasc Res 9: 649–663. S LD, R SV, M DT, 1981. Alcohol-induced hemodynamic and Ca++ flux dysfunctions are reversible. J Mol Cell Cardiol 13: 443–455. S CM, B PS, B NS, J C, G DS, 1995. Genetic and radiation-reduced somatic cell hybrid sublocation of the human GSTP1 gene. Cyto Cell Genet 71: 235–239. S ZM, D RG, L M, 1995. Fatty acid ethyl ester decreases human hepatoblastoma cell proliferation and protein synthesis. Gastroenterology 108: 515–522. T T, O H, 1992. Fatty acid ethyl ester synthase in rat adipose tissue and its relationship to carboxylesterase. J Biol Chem 267: 23489–23494. U-M A, E R, N-L F, G JM, M L, R E, 1989. The effects of alcoholism on skeletal and cardiac muscle. N Engl J Med 320: 409–416. V AM, T VG, C NM, 1986. Alcoholic cardiomyopathy and sudden cardiac death. J Am Coll Cardiol 8: 3A–11A. W JW, T M, K AM, R E, 1975. Effect of ethanol and acetaldehyde on the (Na++K+)-activated adenosine triphosphatase activity of cardiac plasm membranes. Biochem Pharmacol 24: 27–32.
Fatty Acid Ethyl Esters: Potentially Toxic Products of Myocardial Ethanol Metabolism Mary E. Beckemeier∗ and Puran S. Bora∗† ∗Veterans Affairs Medical Center, GRECC Service, St. Louis, Missouri, 63108 and †Department of Internal Medicine, Division of Cardiology, Saint Louis University Medical Center, St. Louis, Missouri, 63110, USA. (Received 7 May 1998, accepted in revised form 25 August 1998) M. E. B and P. S. B. Fatty Acid Ethyl Esters: Potentially Toxic Products of Myocardial Ethanol Metabolism. Journal of Molecular and Cellular Cardiology (1998) 30, 2487–2494. The chronic consumption of alcohol has proven detrimental to heart tissue and can lead to alcohol-induced heart muscle disease, a condition which may result in arrhythmias, cardiomegaly, and congestive heart failure. A search for the molecular mechanism underlying observed alcohol-induced end-organ damage, such as that seen in heart, has lead to the discovery of a nonoxidative pathway for the metabolism of alcohol in several human tissues including heart, brain, pancreas, and liver. It has been revealed that nonesterified fatty acids are esterified with ethanol to produce fatty acid ethyl esters (FAEE), neutral molecules which can accumulate in mitochondria and impair cell function. The observation that FAEEs are synthesized at high rates in the heart, and other organs that lack oxidative ethanol metabolism, provides a plausible link between the observed tissue damage, the ingestion of alcohol, and the subsequent development of alcohol-induced heart muscle disease. The synthesis of FAEEs are catalyzed by FAEE synthase enzyme, four of which have been characterized and purified to homogeneity from the human myocardium. Further analysis of these FAEE synthase enzymes opens up a new possibility to characterize and map a gene for alcohol-induced end-organ damage, such as that observed in heart and other organs. FAEEs have been found to be important metabolites of alcohol and are most commonly accumulated in those organs which are damaged by alcohol abuse, i.e. heart. It may now be important to establish a genetic link between alcohol abuse and alcohol-induced heart muscle disease in order to understand the mechanism of alcohol-induced 1998 Academic Press cardiomyopathy. K W: Metabolism; Alcohol; Mitochondria; Nonoxidative; Cardiomyopathy; Fatty Acids.
Introduction The chronic abuse of alcohol has been known to adversely affect the health of many individuals, causing life-threatening conditions in numerous organs including heart, brain, pancreas, and liver. With respect to the heart, alcohol abuse has proven to be a major cause of non-ischemic cardiomyopathy in Western society (Anderson and Waagstein, 1993; Roberts et al., 1987). These cases have come to be collectively known as alcoholinduced heart muscle disease, a condition in which
heart muscle is plagued with abnormal contractile function and energy metabolism that sometimes results in arrhythmias, cardiomegaly, and congestive heart failure (Asokan et al., 1972). On a cellular level, alcohol-induced myocardial damage includes loss and/or disruption of myocytes, myofibrils, muscle fibers, sarcolemma, sarcoplasmic reticulum, the Na+–K+–ATPase pump, and mitochondria (Alexander et al., 1967; Burch et al., 1971; Hibbs et al., 1965; Kupari et al., 1991; Noren et al., 1983; Urbano-Marquez et al., 1989; Vikhert et al., 1986). In terms of a cellular mechanism for the
Please address all correspondence to: Dr Puran S. Bora, Department of Medicine, Division of Cardiology, Saint Louis Medical Center, 3635 Vista at Grand, USA.
0022–2828/98/112487+08 $30.00/0
1998 Academic Press
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observed ethanol-induced tissue daage, past attempts have been hampered due to a lack of evidence of a metabolic, or biochemical, alcohol metabolizing pathway in the heart. Ethanol has been shown to be metabolized oxidatively creating a toxic product known as acetaldehyde, yet it was revealed that only the liver substantially metabolizes alcohol in this manner (Goldstein, 1983). However, a nonoxidative pathway for ethanol metabolism has been discovered in heart, brain, pancreas, and several tissues including blood (Bora et al., 1989a, b, c, 1991, 1992a, 1996a; Bora and Lange, 1990, 1991a, b; Lange et al., 1981; Gorski et al., 1996). This metabolic ethanol pathway has a potentially toxic product known as fatty acid ethyl ester (FAEE), a neutral family of molecules that are thought to accumulate in the mitochondria of the cell and facilitate the uncoupling of oxidative phosphorylation. The uncoupling of the energy transduction process of the mitochondria can result in an inefficiency of energy production as well as cell damage, providing a link between ethanol ingestion and the subsequent development of alcohol-induced heart muscle disease as well as other end-organ damage (Lange and Sobel, 1983a; Bora et al., 1996a). The synthesis of FAEE in the body is catalyzed by an enzyme known as FAEE synthase. In the human heart four FAEE synthase enzymes have been characterized and purified to homogeneity. These myocardial synthase enzymes are known as synthase-I, synthase-II, synthase-III, and synthase/carboxylesterase (Bora et al., 1989a, b, c, 1991, 1992a, 1996b; Bora and Lange, 1990, 1991a, b). Activity of the FAEE synthase enzymes is highest in heart, brain, and pancreas, the extrahepatic organs most commonly damaged by alcohol abuse (Laposata and Lange, 1986; Laposata et al., 1987). Because these organs lack or have minimal oxidative ethanol metabolism and because a genetic component to selectivity of alcohol-induced end-organ damage exists for some of these syndromes, gene products that modulate ethanol-associated tissue injury must reside within these organs. Thus, FAEE synthase may be at least one candidate for the gene product producing alcoholinduced heart muscle disease. A better understanding of the FAEE synthase enzymes could provide for regulation of the toxic effects of FAEE as well as insight into a possible genetic predisposition for alcohol-induced heart muscle disease.
Fatty Acid Ethyl Esters Alcohol is easily absorbed and distributed into tissues high in water content and blood flow, i.e. heart (Goldstein, 1983). The acute and chronic
administration of alcohol in several human and animal models has demonstrated depressed cardiac function (Ettinger et al., 1976; Gimeno et al., 1962; Hastillo et al., 1980; Regan et al., 1969; Pachinger et al., 1973; Sarma et al., 1976). Furthermore, electron-microscopic and histochemical analysis have revealed shrunken, disorganized myocytes, loss of myofibrils, a dilated and disordered sarcoplasmic reticulum, the excessive accumulation of lipids, and enlarged mitochondira with chaotic cristae (Alexander et al., 1967; Burch and De Pasquale, 1971; Hibbs et al., 1965; Kupari et al., 1991; Urbano-Marquez et al., 1989; Vikhert et al., 1986). It has been found that chronic alcohol administration decreases activity of the Na+–K+–ATPase pump which functions in providing the necessary ionic concentrations for membrane potentials needed for cardiac muscle contraction (Noren et al., 1983). Also, several weeks of chronic alcohol consumption by rats demonstrated in the myocytes a leaky sarcolemma and increases in cardiac intracellular calcium (Noren et al., 1983). The observed compromising of the heart cell membrane can have deleterious effects on heart muscle contraction. Studies have also found disturbances in sarcoplasmic reticulum, responsible for Ca2+ release and uptake during contraction and relaxation (Bing et al., 1974; Williams et al., 1975; Sarma et al., 1976; Segal et al., 1975; 1981). Most notable for the purposes of this review is the observation of decreased myocardial mitochondrial function which includes, as Piano and Dorie (Piano and Dorie, 1994) have pointed out in their 1994 review of alcoholic heart disease, a drop in mitochondrial oxidative enzyme activity and a drop in levels of ATP and oxygen consumption (Pachinger et al., 1973; Sarma et al., 1976; Williams et al., 1975). The mitochondria manufactures most of the cell’s energy currency, ATP, via the tricarboxylic acid cycle (TCA cycle), b-oxidation of fatty acids, and oxidative phosphorylation. The very high energy requirements of the heart muscle are mostly met by b-oxidation of fatty acids (Lopaschuk et al., 1994). Fatty acids bound to albumin, or from chylomicrons and very-low-density lipoproteins, enter the myocyte where they are converted, after a series of steps, to acetyl CoA through b-oxidation in the mitochondrial matrix. Acetyl CoA then enters the TCA cycle, the products of which will enter the electron transport chain of the inner mitochondrial membrane where most of the cell’s ATP is produced (Lopaschuk et al., 1994). It has been established, through the work of Lange and Sobel (Lange and Sobel, 1983a, b), that esters formed in the heart after alcohol consumption
Fatty Acid Ethyl Esters
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Figure 1 Nonesterified fatty acids (FAs) bound in the cell cytosol react with ethanol (EtOH) to form FAEEs by FAEE synthase. The neutral FAEEs accumulate and bind to the inner mitochondrial membrane where they cause damage by uncoupling oxidative phosphorylation. Additionally, some of the FAEEs are hydrolyzed to FAs. Since FAs are a known uncoupler of oxidative phosphorylation, the FAs generated after FAEE hydrolysis can also cause cell damage (Lange and Sobel, 1983a).
can attach to the mitochondria and disrupt the energy process. This family of molecules, created by the nonoxidative metabolism of ethanol, are known as fatty acid ethyl esters (FAEEs) and have been found in those tissues damaged by ethanol abuse (Laposata and Lange, 1986). These potentially dangerous molecules are synthesized at high rates in myocardium, as well as in brain, pancreas, and blood, as a response to the ingestion of alcohol (Bora et al., 1989a, b, c, 1991, 1992a, 1996; Bora and Lange, 1990, 1991a, b; Lange et al., 1981; Gorski et al., 1996). In terms of molecular mechanism for FAEEs, it has been shown that fatty acids bound in the cytosol of the myocyte react with ethanol to form neutral FAEE molecules which bind to and accumulate in the mitochondrial inner membrane (Fig. 1) (Lange et al., 1981; Lange and Sobel, 1983b). The mitochondria then hydrolyze some of the FAEEs, by way of carboxylesterase enzyme (Bora et al., 1996b), to fatty acids (Lange et al., 1981; Lange and Sobel, 1983a; Bora et al., 1996b). Free fatty acids are a known uncoupler of oxidative phosphorylation in concentrations as low as 5 l (Lange and Sobel, 1983a; Borst et al., 1962; Jezek and Freisleben, 1994). Hence, the free fatty acids, as well as the esters themselves, cause uncoupling of oxidative phosphorylation, damaging the ATP synthesis activity of the mitochondria, providing for cellular dysfunction and possible cell death (Lange and Sobel, 1983a). Thus, a mechanism for extrahepatic damage due to alcohol abuse has been provided via the nonoxidative metabolism of ethanol and its products, FAEEs. The biological half-life of FAEE spans from 20–24 h, with FAEEs accumulating in the tissues of those individuals who chronically abuse alcohol
(Lange and Sobel, 1983a). This exposes the heart and other tissues to the potentially toxic effects of FAEEs for long periods of time, increasing the risk for possible end-organ damage (Bora et al., 1996a; Lange and Sobel, 1983a). The normal concentration of FAEEs in the heart muscle is less than 0.001 lmol/l, yet in human hearts obtained at autopsy of persons who were either acutely intoxicated or where chronic ethanol abusers concentrations reached as high as 115 lmol/l (Lange and Sobel, 1983a, b). Moreover, to illustrate the damage caused by FAEEs, rat pancreatic lysosomes incubated with the esters became unstable and fragile (Haber et al., 1993) and the incubation of mouse brain synaptosomes with FAEEs caused a disordering of the membrane lipid bilayer (Hungund et al., 1988). Additionally, our laboratory has shown that by injecting small amounts of FAEE into rat myocardium the histopathological changes, noted through light microscopy, were significant and dose dependent (Bora et al., 1996a). The damage was evident in the myocardial tissue, four and 30 days after injection of the FAEE, as swollen and deformed cells (Table 1). The results coincide with a study done by Szczepiorkowski et al., 1995, in which intact HepG2 liver cells exposed to FAEE showed marked histological differences when compared to a control. However, more studies are needed to determine the damage caused by FAEEs, including the development of transgenic and knockout animal models. We have also shown that FAEEs have binding capabilities to the mitochondria (Bora et al., 1996a). Our results demonstrate that the binding of ethyl oleate to the mitochondria increases linearly with time, providing the evidence that FAEEs can bind to
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Table 1 Myocardial cell damage analysis by a computer software program with digital microscopic planimetry Days
Injection amount (lmol)
Damage area (lm2 )
30 30 30 50 50 50
270±2.1 932±0.9 3692±0.4 418±1.9 1298±1.5 5263±0.8
Table 2 GST Activity of synthase I, II, III and synthase/ carboxylesterase enzymes#
Enzyme 2 4 30 2 4 30
Values are mean±...
the mitochondria (Bora et al., 1996a). Furthermore, we have shown that of the total ethyl oleate injected into the myocardium, 8 lmol oleate and 1 lmol ethyl oleate were bound to the mitochondria. The 8 lmol labeled oleate bound to the mitochondria suggests that the labeled ethyl loeate was broken down in the mitochondria to form fatty acid. This data is corroborated with a liver study in which 90% of the FAEE taken in by intact HepG2 cells was hydrolyzed to fatty acids (Szczepiorkowski et al., 1995). Taken together, this data provides evidence that the cell, possibly the mitochondria, breaks down FAEEs to fatty acids, a known uncoupler of oxidative phosphorylation (Lange and Sobel, 1983a; Borst et al., 1962; Jezek and Freisleben, 1994). Investigators have found FAEEs in human blood serum after the ingestion of ethanol (Doyle et al., 1994). Furthermore, FAEEs have been shown to be delivered to the tissues by way of low density lipoproteins (LDLs) in the bloodstream, affecting such organs as the liver (Szczepiorkowski et al., 1995). This study has revealed that LDLs are passing their FAEE component into human hepatoblastoma (Hep G2) cells and causing a marked decrease in protein synthesis and cell formation in the liver. The assertion that most of the extracted FAEE was hydrolyzed by the liver cells to ethanol and fatty acid has left undetermined whether the liver damage was directly caused by the FAEE, or by the suspected released fatty acid component. Nevertheless, this data supports FAEE as perhaps a new culprit in alcohol-mediated liver damage and provides general evidence of a role for FAEE in endorgan damage.
The Role of Myocardial Fatty Acid Ethyl Ester Synthase Enzymes The formation of FAEEs in the body is catalyzed by an enzyme known as FAEE synthase (Lange et al.,
Synthase I Synthase III Synthase/ Carboxylesterase Synthase II
FAEE synthase activity (nmol/mg/h)
∗GST Activity
200 100 370
225 315 0.03
140
0.02
(nmol/mg/h)
∗ GST activity was assayed according to Bora et al., 1989a. # Bora et al., 1996b J Mol Cell Cardiol 28: 2027–2032.
1982; Mogelson and Lange, 1984; Mogelson et al., 1984). FAEE synthase has been found in the liver and pancreas of rats (Hamamoto et al., 1991), as well as in the brain of mice (Hungund et al., 1998), and has been purified and characterized from rabbit myocardium (Mogelson and Lange, 1984; Mogelson et al., 1984). Additionally, four FAEE synthase enzymes have been purified and characterized from human myocardium (Bora et al., 1989a; b, c, 1990, 1991, 1992a, 1996b; Bora and Lange, 1989a, b). As mentioned earlier, these myocardial FAEE synthase enzymes are known as synthase-I, synthase-II, synthase-III and synthase/carboxylesterase. Synthase-I and synthase-III are dimers with a subunit molecular mass of 26 kDa, both possessing glutathione S-transferase (GST) enzyme activity (Bora et al., 1989a, b, c) (Table 2). The GSTs are a family of enzymes involved in detoxification and drug resistance (Mannervik et al., 1985). SynthaseIII, whose main function is FAEE synthesis, exhibits 98% homology to an isoenzyme of GST known as GSTP1, whose main function is detoxification (Smith et al., 1995). Sequence analysis revealed a six amino acid difference between the genes. This small differential region most likely confers the functional variations between synthase-III and GSTP1 (Bora et al., 1991). Yet, even with this small differential region in mind, evidence of homology and GST enzyme activity suggests that synthaseIII and synthase-I are likely members of the GST enzyme family. An amino acid residue in synthase-III (purified in human myocardium), histidine 72, has shown to be important for GST but not synthase activities (Bora et al., 1992b). This data offers significant information in designing selective inhibitors of FAEE synthase-III. Additionally, the gene structure for synthase-III has been isolated, determined, and localized to human chromosome 11q13 (Bora et al., 1997). The complete nucleotide sequence of synthase-III has also been determined. The promoter
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Table 3 Allelic frequencies of ATAAA repeat region of FAEE synthase-III gene in different groups∗
Type
Alleles size (bp)
Frequency healthy control
Frequency heart patient
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11
409 410 415 418 420 425 427 431 433 435 437
0.110 0.309 0.410 0.710 0.818 0.050 0.135 0.115 0.095 0.080 0.075
0.095 0.805 0.790 0.115 0.250 0.325 0.110 0.107 0.090 0.085 0.065
∗ Unpublished data (Bora et al., 1998).
Figure 2 PCR products of fatty acid ethyl ester synthaseIII gene. The figure shows the number of alleles present in four different representative cardiovascular patients (unpublished data, Bora et al., 1998).
region has several transcription regulatory sequences including a TATA box and two SP1 recognition sequences. It has been also been shown that the repeat region of the synthase-III gene is polymorphic when PCR was performed using the DNA from unrelated individuals (unpublished data by Bora et al.). Figure 2 shows the electrophoretic patterns of the PCR products and the 11 alleles observed in the unrelated individuals. Table 3 shows the allele sizes and frequencies. The allelic frequencies from 100 unrelated healthy controls (no history of drinking) and 100 heart patients (drinking four to five drinks a day) were 0.765 and 0.798, respectively. The highest frequency among the alleles was A4 and A5 in healthy controls while among the heart patients A2 and A3 had the highest frequencies. Since FAEEs in concentrations as high as 115 lmol/l were identified in hearts obtained at autopsy from patients either acutely intoxicated at the time of death or chronically exposed to alcohol (Lange and Sobel, 1983b), the identification of a polymorphic marker corresponding to a known gene provides a bridge between genetic and physical mapping information.
This study will help in the understanding of the genetic control mechanism of the synthase enzyme(s) in alcoholism and alcohol-induced cardiomyopathy. Since both synthase-I and synthase-III are members of the GST enzyme family, it was assumed that synthase-II was yet another isoenzyme of the same faily, yet our laboratories have proven otherwise. We have purified and characterized synthase-II from human myocardium and determined that it is a monomer with a molecular mass of 65 kDa. There was no cross-reactivity observed between synthaseII and antibodies specific for synthase-III leading to the conclusion that synthase-II is distinct from the GST family (Bora et al., 1992a). Furthermore, in terms of possible connections to other enzyme families, a limited amount of homology was demonstrated between synthase-II and two heme binding proteins, human hemopexin and rabbit cytochrome P-45011C1. Still, absence of cross reactivity between synthase-II and antibodies to either of these two proteins provides evidence that synthase-II, despite the homology, was not related to either protein. Therefore, this study supports the conclusion that FAEE synthase-II is a novel protein belonging to another unique class of enzymes which can metabolize ethanol to FAEEs. The elution profile of a DEAE-cellulose chromatography assay of human myocardial cytosol determined that synthase-II accounts for up to 50% of total FAEE synthesis in human heart (Bora et al., 1992a). Hence, regulation of the synthase-II enzyme could prove significant when addressing alcohol-induced heart muscle disease considering that this enzyme pathway plays a prominent role in human heart. Consequently, the purification and characterization of synthase-II by our laboratories
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M. E. Beckemeier and P. S. Bora Table 4 N-terminal sequences of human synthase/carboxylesterase and rat adipose tissue synthase/carboxylesterase Human heart 62 kDa synthase/ Carboxylesterase
1
8
17
NH2 - (Y) P S S P P V V D T V H G K V L G
Rat adipose tissue 60 kDa synthase/ carboxylesterase
NH2 - Y P S S P P V V N T V K G K V L G
Vertical bars indicate the differences in the amino acid of two enzymes. Amino acid within bracket is ambiguous.
from human myocardium serves as an important step in the determination of possible regulatory mechanisms for this FAEE synthase enzyme (Bora et al., 1992a). A fourth FAEE synthase enzyme, molecular mass of 62 kDa, has also been purified and characterized from human myocardium (Bora et al., 1996b). Antibodies for this synthase enzyme did not crossreact with synthase I, II, or III, providing no evidence of homology among GST and synthase-II. This new class of ethanol metabolizing enzyme has been found to have 88% homology to the first 17 N-terminal residues in rat liver and adipose tissue carboxlyesterase (Table 4). The carboxylesterase enzyme family works throughout the body by detoxifying harmful agents by hydrolyzing them with ester and amide bonds. Homology between carboxlyesterase and FAEE synthase was found by Tsujita and Okuda (Tsujita and Okuda, 1992), who suggested that the FAEE synthase they purified from rat adipose tissue, among other tissues, contained FAEE synthase activity along with carboxlyesterase activity. This finding was confirmed by our laboratories who have recently purified and characterizied this particular FAEE synthase from human heart. We have found it to possess both synthase and carboxylesterase activities and have therefore named it FAEE synthase/carboxylesterase (Bora et al., 1996b). In terms of whether there is up or down regulation of the FAEE synthase enzyme(s) in those tissues most affected by alcohol abuse, there have been conflicting reports. One study reported seeing elevated activity of FAEE synthase in the brains of alcoholics as compared with nonalcoholics, correlating high levels of FAEE synthase with ethanol toxicity (Laposata et al., 1987). Furthermore, Bora et al. have found a four-fold increase in FAEE synthase activity in the heart tissue of ethanol-fed rabbits who received 20% of their calories as ethanol for 1 month (unpublished data). However, a slight decrease in the level of FAEE synthase was found
in the pancreas of rats when they were administered 36% of their calories as ethanol for 7 weeks (Hamamoto et al., 1990). FAEE synthase levels in the livers of mice also showed decreased enzyme levels under the same parameters (Hamamoto et al., 1991). Furthermore, a look at blood FAEE levels in humans revealed that alcoholics have approximately 50% less white blood cell FAEE synthase activity than their nonalcoholic counterparts (Gorski et al., 1996). Gorski et al. (1996) have speculated that the down regulation of FAEE synthase in blood tissue could be due to an adaptive response to limit the production of FAEEs. Further study is necessary to explain the regulation of FAEE synthase for it may play an integral role in the management of alcohol-induced end-organ damage and in the screening for possible predispositions to this damage.
Conclusion Alcohol abuse has long proven detrimental to the body, resulting in damage to such organs as heart, brain, pancreas and liver. In terms of a molecular explanation for this end-organ damage researchers have discovered the nonoxidative metabolism of ethanol in those organs damaged by ethanol abuse. The products of this nonoxidative pathway, FAEEs, have been shown to bind to the mitochondrion of the cell and to uncouple oxidative phosphorylation. It has been suggested that alcohol metabolism may be under genetic control and related to the presence of different amounts or types of ethanolmetabolizing enzymes (Cloninger et al., 1981; Goodwin et al., 1987). The FAEE synthase enzymes could be likely gene product candidates for the underlying genetic vulnerability to the effects of alcohol since no other significant pathway for the metabolism of alcohol is known to exist in the heart or other extrahepatic organs. Genetic studies using peripheral human leukocytes have shown that high synthase
Fatty Acid Ethyl Esters
activity is inheritable in an autosomal recessive pattern (Gilligan et al., 1989). This pattern is expected for a gene controlling the product of a toxic agent, such as FAEE synthase enzymes and their potentially toxic FAEE product. With this in mind, the cloning and mapping of the synthase enzymes to specific chromosomes, as has been done with synthase-III, opens up a new possibility to map a gene for alcoholism or alcohol-induced myocardial damage. Because FAEEs are important metabolites of ethanol and are found in highest concentrations in the heart and other target organs that are damaged by alcohol abuse, establishing a genetic link between alcohol abuse and end-organ damage may now be feasible.
Acknowledgements This work was supported by grants from Lichtenstein Foundation and V.A. Merit Review to P.S. Bora. The authors thank Drs Kelly Ludwig and Bryan Charles Doonan for their critical review of the manuscript.
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