Alcohol and Alcoholism Advance Access originally published online on September 16, 2006
Alcohol and Alcoholism 2006 41(6):598-603; doi:10.1093/alcalc/agl069
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THE TOTAL BODY MASS OF FATTY ACID ETHYL ESTERS IN SKELETAL MUSCLES FOLLOWING ETHANOL EXPOSURE GREATLY EXCEEDS THAT FOUND IN THE LIVER AND THE HEART
1 Massachusetts General Hospital and Harvard Medical School, Division of Laboratory Medicine, Department of Pathology, 55 Fruit Street, Gray 235, Boston, MA 02114, USA and 2 King's College London, Nutritional Sciences Research Division, London SE1 9NU, UK
* Author to whom correspondence should be addressed. Tel: +1 617 726 8172; Fax: +1 617 726 3256; E-mail: mlaposata{at}partners.org
(Received 20 August 2005; first review notified 24 October 2005; in revised form 27 July 2006; accepted 28 July 2006)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aims: Skeletal muscle appears to be susceptible to chronic and acute excess alcohol intake, giving rise to alcoholic myopathy, a common disease among alcoholics. Fatty acid ethyl esters (FAEE), non-oxidative metabolites of ethanol, have been shown to be toxic to cells in vitro and in vivo. We hypothesized that accumulation of FAEE in skeletal muscle could contribute to the development of alcoholic myopathy. Methods: Male wistar rats were treated either with 75 mmol ethanol/kg body weight or saline, in the fed state or starved for 1 or 2 days before administration. Rats were thus divided into the following groups: fed-saline (n = 8); fed-ethanol (n = 8); starved 1 day, saline (n = 8); starved 1 day, ethanol (n = 9); starved 2 days, saline (n = 7); and starved 2 days, ethanol (n = 8). At the end of the incubation, skeletal muscles (abdominal and gastrocnemius), liver, and heart were isolated and processed for FAEE isolation and analysis by gas chromatography-mass spectrometry (GC-MS). Results: Total mass of FAEE in the muscles was much greater than that found in the liver and the heart. In general, the animals that were fasted for 1 day and received ethanol had the highest FAEE levels among the three groups of animals. The major ethyl ester species in all cases were ethyl 16:0, ethyl 18:0, ethyl 18:1 n–9, and ethyl 18:2 n–6. Ethyl 20:4 n–6 and ethyl 22:6 n–3 were also present, except in the fasted 1-day group, where ethyl 22:6 disappeared, though it reappeared in the fasted 2-day group. Conclusion: These findings demonstrate that skeletal muscles contain high levels of FAEE that are synthesized in the body after ethanol exposure. The concentration of FAEE in skeletal muscle in this study was very similar to FAEE concentration in the liver. This differs from previous studies suggesting a low concentration of skeletal muscle FAEE with ethanol exposure.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Upon ingestion of ethanol, the production of the non-oxidative metabolites of ethanol known as fatty acid ethyl esters (FAEE) is initiated (Doyle et al., 1994
, 1996). There have been a number of reports both in vitro and in vivo indicating that FAEE are toxic to the cells. It has been proposed that FAEE mediate at least in part some of the toxic effects of ethanol (Lange and Sobel, 1983; Haber et al., 1993; Ponnappa et al., 1994; Szczepiorkowski et al., 1995; Laposata, 1998). The appealing connection that brought FAEE into consideration as a toxic mediator is that they have been found in highest concentrations in the organs most commonly damaged by ethanol abuse, notably pancreas, liver, heart, and brain (Laposata and Lange, 1986). FAEE have also been detected in significant quantities in adipose tissue where the FAEE may be stored after production (Laposata and Lange, 1986; Laposata et al., 1989; Salem et al., 2001). Much attention has been focused on the production of FAEE by the pancreas, the liver, the heart, the brain, and adipose tissue, because these are the organs and tissues in which the production is the highest on a per weight basis (Laposata and Lange, 1986).
Between 40 and 60% of patients who drink ethanol to excess can develop skeletal muscle abnormalities giving rise to the disease entity alcoholic myopathy (Martin et al., 1985). This myopathy, which affects skeletal muscle, is a clinically observed toxic effect of ethanol, but the mechanism to produce this toxic effect has not been established. A previous report showed that skeletal muscle contains smaller amounts of FAEE, per fixed weight of organ or tissue, in individuals who have ingested ethanol compared with pancreas, liver, heart, and brain (Laposata and Lange, 1986). Because the skeletal muscle mass accounts for 
40% of the total mammalian body mass, a large percentage of total FAEE in the body may be present in the muscles (Preedy et al., 2001b). We hypothesized that skeletal muscle may harbour more of total body FAEE than any other organ or tissue.
In this study, rats were given 75 mmol ethanol/kg. The animals were sacrificed 2.5 h after intraperitoneal injection with ethanol. At that time, samples of skeletal muscle, as well as liver and heart, were removed for analysis. The FAEE were quantitated on a per gram basis for all of these organs and tissues, In addition, the relative mass of skeletal muscle, heart, and liver relative to total body weight was also considered in the calculation to determine the total body mass of FAEE in these two organs and tissues. The findings are that (i) FAEE were present in skeletal muscle in animals exposed to ethanol; (ii) on a total FAEE mass basis within the body, the skeletal muscle FAEE accounted for much more of the total body FAEE than that found in liver and in heart; and (iii) the concentration of FAEE in skeletal muscle was similar to the FAEE concentration in liver, which differs from previous observations (Laposata and Lange, 1986). Therefore, we conclude that skeletal muscle FAEE represents a major component of total FAEE in the body.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal mode and ethanol treatment
Male Wistar rats were obtained from commercial suppliers at
60 g body weight. Rats were maintained and studied according to Home Office Guidelines and a specific project license. Rats were maintained in a temperature and humidity-controlled animal house for 1 week until they weighed about 100–150 g. The rats were 6–7 weeks old when treatment with alcohol commenced. Rats were either treated in the fed state or starved 1 or 2 days before use (i.e. total food deprivation 24 and 48 h with free access to water at all times). Rats were thus divided into the following groups: fed-saline (n = 8); fed-ethanol (n = 8); starved 1 day, saline (n = 8); starved 1 day, ethanol (n = 9); starved 2 days, saline (n = 7); and starved 2 days, ethanol (n = 8). The dosage of ethanol used was 75 mmol ethanol/kg body weight for treated rats (alcohol in 0.15 mM NaCl to a volume of 1 ml/100 g body wt); controls were given an identical volume of 0.15 mM NaCl. Ethanol or saline was administrated by intraperitoneal injection (1 ml/100 g body weight).
Collection of tissues
After 2.5 h of exposure to alcohol or saline, rats were sacrificed by decapitation and livers rapidly removed, blotted onto paper, and then weighed. The average weight of liver was 4.707 ± 0.139 g. After weighing, the livers were immediately frozen in liquid nitrogen. The hearts were trimmed of fat and atria, which were discarded. The ventricles of the heart were then exposed to release the blood, rinsed in an ice-water slurry, weighed, and frozen in liquid nitrogen. The average weight of heart was 0.457 ± 0.007 g. The hind limbs were stripped of skin and placed in ice-water slurry to cool the muscles. The gastrocnemius muscles were then dissected out, blotted, weighed, and frozen. The average weight of muscles was 0.618 ± 0.009 g. A portion of the abdominal muscles was also dissected, blotted, weighed, and frozen. At least three individuals performed these tasks simultaneously to ensure rapid dissection and cooling of all tissues. After freezing all samples in liquid nitrogen, all tissues were stored at –70°C.
Tissue extraction and analysis
Frozen tissue, weighing between 0.1 and 0.3 g, was thawed while on ice and this wet weight recorded to the nearest 0.1 mg. Tissue was then placed in 1.0 ml of homogenization buffer [phosphate buffered saline containing 20 mg/l phenylmethylsulfonyl fluoride, 1 mmol/l benzamidine and 0.1 trypsin inhibitor (pH 7.34)] and homogenized using a Fisher PowerGen 125 Homogenizer equipped with a 10 x 195 mm sawtooth generator (Fisher Scientific, Pittsburgh, PA). Ethyl heptadecanoate (2000 pmol) was added as an internal standard (Nu Check Prep, Elysian, MN). The samples were extracted using acetone–hexane (2/8, v/v), then dried under nitrogen to 300 µl (Bernhardt et al., 1996). FAEE were isolated by solid phase extraction using a Bond Elut-LRC aminopropyl column (Analytichem International, Varian Diagnostics, Palo Alto, CA) as described earlier (Kaluzny et al., 1985; Bernhardt et al., 1996). The aminopropyl-silica columns were placed on a Vac-Elut vacuum apparatus set at 10 kPa. The columns were first conditioned with 4 ml of dichloromethane, followed by 4 ml of hexane. Then 200 µl of sample suspended in hexane was applied to the column immediately after the solvent reservoir became empty. The sample was followed by 4 ml hexane and 4 ml dichloromethane. The two organic fractions were then combined and dried completely under stream of N2, then resuspended in 35 µl hexane for FAEE quantitation using gas chromatography-mass spectrometry (GC-MS) (Dan et al., 1998).
GC-MS analysis was performed using a Hewlett-Packard 5890 Series II gas chromatograph coupled to Hewlett-Packard 5971 mass spectrometer (Hewlett-Packard, Palo Alto, CA) equipped with a Supelcowax 10 capillary column. The oven temperature was maintained at 150°C for 2 min, ramped at 10°C/min to 200°C held for 4 min, ramped again at 5°C/min to 240°C, where it was held for 3 min, then ramped at 10°C/min to 270°C and held for 5 min. The injector and mass spectrometer were maintained at 260 and 280°C, respectively. Carrier gas flow rate was maintained at a constant 0.8 ml/min throughout. Single ion monitoring was performed, quantifying appropriate base ions for individual FAEE species [i.e. ions 67, 88 and 101 for ethyl palmitate (E16:0), ethyl heptadecanoate (E17:0), ethyl stearate (E18:0), ethyl oleate (E18:1), and ethyl linoleate (E18:2); and ions 79, 91, and 117 for ethyl arachidonate (E20:4), ethyl eicosapentaenoate (E20:5), and ethyl docosahexaenoate (E22:6)]. FAEE quantification was determined by interpolation of the slope generated from individually prepared standard curves, comparing areas of varying concentration of E16:0–E22:6 to fixed concentrations of internal standard (E17:0). Mass relationships were obtained for each FAEE using its individual standard curve. Total FAEE mass was determined by addition of the masses of the individual FAEE (E16:0–E22:6).
Calculation of total body contents
In this manuscript tissue FAEE compositional data have been presented in terms of concentrations and contents. The distinction between the two is as follows: concentrations were defined as the amount of analyte relative to wet weight of the tissue, i.e. nmol/g wet weight; contents were defined as the total amount of analyte per anatomic region or organ, i.e. nmol per tissue (liver, heart, or muscle). All weights pertain to wet weights of the tissue or organ after blotting the tissue, or organ on absorbent paper. In order to calculate the total contents it is necessary to dissect out the entire organ. Whilst this is relatively straightforward for the liver and heart, it is more problematical for muscle as much of it is interspersed between organs or attached to bones: such as the intercostal muscles located between the ribs. However, various studies have shown that skeletal muscle is 40% of body weight and we have used this value in our calculations (Young, 1970; Waterlow et al., 1978; Preedy and Peters, 1988; Preedy et al., 1990). In response to starvation, the muscle shows profound reductions in weight and discordant changes in the weight/body weight ratio. To resolve practical difficulties in the dissection of the entire musculature, we used the changes in the mean weight of the gastrocnemius in our calculations. This is based on the fact that the gastrocnemius, containing mixed Type I and II fibres, is representative of the skeletal musculature as a whole (Preedy and Peters, 1988; Preedy et al., 1990).
Statistical analysis
Results are expressed as the mean ± SEM. Significant differences were determined using ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows the total FAEE per skeletal muscle, liver, or heart mass in animals that were fed before receiving ethanol (75 mmol ethanol/kg), in animals that were fasted 1 day and then received ethanol, and in animals that were fasted for 2 days and then received ethanol. The influence of fasting on FAEE level has not been previously explored. Recent observation regarding the impact of dietary fat intake on the persistence of FAEE in the circulation led to the inclusion of fed/fasting data in this report (Bisaga et al., 2005
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The results were derived from calculation that presumes that the muscle mass is 40.0% of total body weight for the fed animals (Young, 1970
; Waterlow et al., 1978; Preedy and Peters, 1988; Preedy et al., 1990). Thus mean body weights of the gastrocnemius muscle in the groups were as follows: fed-ethanol, 0.659 g; starved 1 day, ethanol, 0.627 g; and starved 2 days, ethanol 0.558 g. If control rats in the fed-saline weighed 150 g then total body muscle mass is 60 g (i.e. as 40% of body weight). If gastrocnemius weights are reduced by 5% at the end of 1 day of starvation (i.e. from 0.659 to 0.627 g) then total muscle mass will be similarly reduced, i.e. by 5% from 60 to 57 g. Using the same method of calculation, we showed that at the end of 2 days of starvation the weight of the gastrocnemius is reduced by 15% (i.e. from 0.659 to 0.558 g) so total muscle mass was correspondingly reduced from 60 to 51 g. Thus, using the muscle mass of 60, 57, and 51 g in fed, 1 day starved and 2 day starved rats, respectively, we were able to assess with some degree of confidence the total FAEE contents to complement values obtained for liver and heart. The effect of ethanol intake on this model is not known.
As shown in Fig. 1, the total FAEE content in the muscles was much greater than that found in the liver, even though the liver FAEE concentration (per gram of wet tissue) has been shown to be much greater than in skeletal muscle after ethanol intake (Laposata and Lange, 1986). The heart, in terms of total body FAEE mass, accounted for only a very small portion of the total whole body FAEE concentration. In general, the animals that were fasted for 1 day and received ethanol had the highest FAEE concentration among the three groups of fed, fasted 1-day, and fasted 2-day animals.
Figure 2 shows the total FAEE per gram of wet tissue in abdominal muscle, gastrocnemius muscle, liver, and heart. In five groups of animals, the two bars to the far left in each cluster show the animals that were fed or fasted for 2 days and received saline rather than ethanol. There were no FAEE detected in any of these organs or tissues. The black bar in the cluster for each organ or tissue represents animals that were fed and received ethanol. These animals showed FAEE production. The FAEE concentrations per wet weight of tissue detected in the skeletal muscle were similar to that found in the heart and in the liver. For the skeletal muscles and the heart, a previous study using tissues from humans who died while intoxicated found approximately the same level of FAEE (Laposata and Lange, 1986). The slight elevation in FAEE levels seen in Fig. 1 in the group that was fasted for 1 day was found only in the gastrocnemius muscle samples. Although the abdominal and gastrocnemius muscles have similar contractile potential, they differ in subcellular and biochemical profiles. Certainly, they respond differently to metabolic perturbations (Meszaros et al., 1987). A comparison between different skeletal muscles was also necessary to address the argument that the results in the gastrocnemius (which was used to calculate total FAEE contents) were due to its anatomical location or some other physiological peculiarity. As FAEE concentrations in abdominal and hind-limb muscles were of the same order of magnitude, we can safely conclude that skeletal muscle contains a significant pool of FAEE.
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Figure 3A shows the distribution of the fatty acid within the FAEE for the abdominal muscle, the gastrocnemius muscle, the liver, and the heart. The major ethyl ester species in all cases were ethyl 16:0, ethyl 18:0, ethyl 18:1 n–9, and ethyl 18:2 n–6. It should be noted that ethyl 20:4 n–6 and ethyl 22:6 n–3 were also present and well above the limit of detection in all these tissues. From the animals that were fed and exposed to ethanol, ethyl 20:3 n–6 was present in the heart and to a lesser extent in gastrocnemius muscles.
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-linolenate; E20:3, ethyl dihomo-
-linolenate; E20:4, ethyl arachidonate; and E22:6, ethyl docosahexaenoate. ND, none detected.Figure 3B shows the results of the animals that were fasted for 1 day and then received ethanol. Similar to the fed group, these animals produced the same major FAEE species. The one major difference was that the ethyl 22:6 n–3 was not detected in any of these tissues, arguing for the possibility that this ethyl ester is more rapidly degraded than other ethyl esters when a fasting state is initiated. On the other hand, the other major polyunsaturated FAEE, ethyl 20:4 n–6, was retained in all the tissue analysed.
Figure 3C shows the data from animals fasted for 2 days and then injected with ethanol. The major fatty acid species were those found in both the fed group and animals fasted for 1 day. Surprisingly, the 22:6 ethyl ester was found in the animals that fasted for 2 days, despite its absence in the group fasted for 1 day. The ethyl 20:4 n–6 was present in these animals, as it was in the fed state and the animals fasted for 1 day. Also ethyl 20:3 n–6 was present in all tissues except the heart.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data in this report demonstrate that skeletal muscle is a site for a significant amount of FAEE synthesis. This notion is new, because on a per weight basis, the muscle has been shown to produce less FAEE than selected organs in humans, such as the liver and the pancreas. However, since the body, human and rat, is composed to a significant extent of skeletal muscle, the total amount of FAEE within the body's skeletal muscle is significant. As shown in this report with rats, the total amount of FAEE within skeletal muscle is much greater than that found in liver and heart (Fig. 1). This is likely to be true for humans as well.
As shown in Fig. 1, skeletal muscles showed the highest FAEE levels compared with liver and heart. Laposata and Lang detected minimal levels of FAEE per gram of skeletal muscles compared with other organs, when they performed autopsy studies on individuals with detectable blood ethanol at the time of death (Laposata and Lange, 1986). The FAEE levels obtained from liver and heart in this animal model measuring intraperitoneal dosage of ethanol compares well with humans ingesting ethanol orally (Laposata and Lange, 1986). On the other hand, FAEE levels detected in skeletal muscles from this rat study was 10x higher than FAEE levels in the autopsy study. Since FAEE levels in the heart and the liver matched well the levels detected in the autopsy study, the quantitative results for this rat study are comparable with that for human. The low FAEE concentration detected in skeletal muscles of individual who ingested ethanol prior to death could be due to rapid degradation of FAEE in skeletal muscles. It is conceivable that the by the time the autopsy was performed in the 1986 study, most of FAEE in skeletal muscles were hydrolysed.
As shown in Fig. 2, animals that were fasted for 1 day and received ethanol had the highest FAEE concentration among the three groups of fed, fasted 1-day, and fasted 2-day animals. A likely basis for this is that fasting for 1 day is likely to produce higher concentration of free fatty acids, a substrate for FAEE formation, in the presence of ethanol. Several studies have shown that plasma free fatty acid concentrations are not constant under different fasting conditions (Quabbe et al., 1966; Court et al., 1971; Smith et al., 1977), so we speculate that there may be a different concentration of free fatty acids, and therefore FAEE levels, in our experimental model in the fasted 2-day group relative to the fasted 1-day group.
We investigated the responses of starvation due to the central importance of malnutrition in the aetiology of alcohol-related pathology (Preedy and Watson, 2005). Alcoholics also undergo episodic periods of fasting. Furthermore, fasting alters the metabolism of alcohol (Preedy and Watson, 2005), and so it was necessary to ascertain whether the results we obtained were peculiar only to fed rats. Nevertheless, despite the fact that we starved rats for up to 2 days there was still substantial amounts of FAEE in skeletal muscle and the premise that the skeletal muscle is a significant pool of FAEE still holds true.
The fatty acid composition of the FAEE species was also evaluated in this study (Fig. 3). The major FAEE species that have previously been identified in studies within other organs and tissues were the same ones that were most prominent in the skeletal muscle (Laposata et al., 2000; Salem et al., 2001; Refaai et al., 2002). The FAEE species in the skeletal muscle in this study were also essentially the same in the liver and in the heart of the rats injected with ethanol. The absence of ethyl 22:6 in the animals that were fasted for 1 day and its reappearance in different animals that were fasted for 2 days was unexpected. It is not clear why the ethyl 22:6 was depleted and then was restored. In this model, the animals fasted for 2 days do not appear to be a simple extension of the animals fed or fasted for 1 day. The results from several studies indicate that the mobilization of a specific fatty acid into plasma is not proportional to its content in adipose tissue (Conner et al., 1996; Hunter et al., 1970; Raclot and Groscolas, 1993). For this reason, the mobilization of fatty acids for FAEE synthesis was not assumed to be the same for the heart, the liver, and the two skeletal muscle tissues. Upon analysis, however, the fatty acid content of all four tissues was found to be very similar.
Alcoholic myopathy is a common disease with a prevalence of 2000 cases/100 000 alcoholic population (Preedy et al., 2001a, b). Malnutrition could contribute to muscle impairment in alcoholics. However, well-nourished alcoholics also suffer from muscle myopathy (Urbano-Marquez et al., 1989). Recent evidence suggests that alcohol-mediated apoptosis can occur in cardiac and skeletal muscles with high amounts of intake through an unknown mechanism (Fernandez-Sola et al., 2003). A study performed by Aydin et al. (2005) has shown that FAEE can induce apoptosis in a hepatoblastoma cell line (Aydin et al., 2005). Also FAEE have been shown to cause uncoupling of oxidative phosphorylation in heart mitochondria (Lange and Sobel, 1983; Bora et al., 1996). Based on these studies, FAEE could induce apoptosis and cause mitochondrial damage in muscles leading to alcoholic myopathy. However, one argument against this pertains to the observation that in the rat there is no evidence of apoptosis in the muscle of rats either acutely or chronically dosed with ethanol (Paice et al., 2003). Nevertheless, it is possible that FAEE may activate other pathways involved in muscle damage such as pre-apoptotic signalling or onco-expression such as c-myc (Nakahara et al., 2003). Taken together, these findings demonstrate that skeletal muscle is a large reservoir for FAEE that are synthesized in the body after ethanol consumption and that this large pool has long been overlooked.
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ACKNOWLEDGEMENTS |
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We wish to thank Mathew Baely for technical assistance.
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