Alcohol, Vol. 9, pp. 329-334, 1992
0741-8329/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
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Effect of Chronic Consumption of Ethanol and Vitamin E on Fatty Acid Composition and Lipid Peroxidation in Rat Heart Tissue S E R G E I V. P I R O Z H K O V * , CLEAMOND D. E S K E L S O N * t I, R O N A L D R. W A T S O N ~ : I!, G L E N C. H U N T E R § , J O S E P H J. P I O T R O W S K I § AND VICTOR BERNHARD§
*Research Institute f o r Medico-Biological Problems o f Addictions, Moscow 121921, Russia and t Departments o f Surgical Biology, ~Family and Community Medicine and §Surgery, IIN I A A A Specialized Alcohol Research Center, University o f Arizona Health Sciences Center, Tucson, A Z 85724 R e c e i v e d 23 S e p t e m b e r 1991; A c c e p t e d 27 J a n u a r y 1992 PIROZHKOV, S. V., C. D. ESKELSON, R. R. WATSON, G. C. HUNTER, J. J. PIOTROWSKI AND V. BERNHARD. Effect of chronic consumption of ethanol and vitamin E on fatty acid composition and lipid peroxidation in rat heart tissue. ALCOHOL 9(4) 329-334, 1992.--Lipid peroxidation products and the fatty acid composition of phospholipids were studied in the hearts of rats chronically consuming ethanol supplemented with large amounts of vitamin E. Ethanol representing 3607o of the total calories was ingested for 7 weeks in a modified Lieber-DeCarli liquid diet that contained vitamin E at 30 IU/L in the control or 172 IU/L in the supplemental dietary group. Ethanol and/or vitamin E did not change the absolute content (#g per mg of phospholipids) of the main fatty acids (C,8:0, Cts:z, and C2o:,) of heart phospholipids but increased the amount of the minor C20-C22 fatty acids. Cardiac phospholipid levels increased in rats chronically consuming excess vitamin E and/or alcohol. Chronic ethanol consumption caused elevations of the relative content (percent of total fatty acids) of tri-, tetra-, and hexaenoic acids and peroxidizability index (PI) of the cardiac phospholipids. Supplementation with vitamin E blocked this ethanol-induced shift in the fatty acid profile toward unsaturation and decreased the PI. Ethanol enhanced accumulation of vitamin E in heart tissue by 30070 irrespective of the vitamin E content in the diet. Enrichment of the diet with vitamin E coincided with the low levels of fluorescent products in heart lipids. A positive correlation (r = 0.36; p = 2°70) was found between vitamin E and diene conjugates in the heart cells. Thus, vitamin E has a stabilizing effect on heart phospholipids by preventing changes in their fatty acid composition and peroxidative deterioration. Ethanol
Vitamin E
Fatty acid composition
Lipid Peroxidation
E T H A N O L stimulates lipid peroxidation (LPO) in various tissues (19). In the heart, chronic ethanol consumption produced significant increases in m e m b r a n e peroxidizability (I). Lipid peroxidation in this study was initiated in vitro by addition o f F E 3 + / A D P / a s c o r b a t e and detected by enhancement of low-level chemiluminescence and accumulation o f the thiobarbituric acid (TBA)-reactive substances. Glutathione content decreased and diene conjugates were increased in the myocardium after chronic ethanol consumption (9). These effects of ethanol were attributed to the depletion of antioxidants in the membranes o f cardiomyocytes as a result of activated LPO. Changes in fatty acid composition induced by
Heart
ethanol may make heart lipids more liable to peroxidation. For example, feeding pigs or rats with a diet enriched with fish oil causes a shift in fatty acid profile toward more unsaturated species and facilitates accumulation of lipofuscin in the heart indicating an oxidative deterioration o f lipids (18,25). Chronic alcohol administration decreased hepatic arachidonic (C20:4) and docosahexaenoic (C~2:6) acids (17) and C2o:4 in brain synaptosomes (14), although it increased C~: 4 and Cts:~ and decreased C~6:o in erythrocyte membranes ( l l ) . Ethanol consumption reduced the relative content of C2o:4 and C2z:6 in the heart (24). Although the antioxidant vitamin E prevented ethanol-
Requests for reprints should be addressed to Cleamond D. Eskelson, Department of Surgical Biology, NIAAA Specialized Alcohol Research Center, University of Arizona Health Sciences Center, Tucson AZ 85724. 329
330
PIROZHKOV ET AL.
induced changes of LPO parameters in myocardial tissue (1,9), its effect on fatty acid composition of heart lipids was not elucidated. A strong positive correlation was demonstrated between alpha-tocopherol content and the levels of C22: 6 and C22:~in the heart when rats were fed diets with different n-3/n-6 ratio (5), but no correlation was found between vitamin E and C~8:2or C20:, in the membranes of pulmonary artery endothelial cells (26). Also, there exists no data concerning the combined action of ethanol and vitamin E on fatty acid patterns of heart membranes. The present study investigated changes in the fatty acids profile of the cardiac phospholipids during the consumption of alcoholic diets supplemented with vitamin E. Additionally, we studied the association between phospholipid polyunsaturated fatty acid composition, vitamin E content, and LPO in the myocardial cells.
total content): C~,:o-O; C]6:o-15.8%; CI6:1-1.0~70; C,~:o-3.2%; C,a:1-49.2%; CIa:2-29-7%; C]a:3-1.2%0; C~0:z-traces; C._o:s-0; Cz2:~-0; C22 t-0. The duration of the experiment was 7 weeks.
Lipid Analysis Lipids were extracted from the heart samples using a Folch reagent, chloroform:methanol = 2 : 1. To aliquots of the extract an internal standard (heptadecanoyl phosphatidylcholine, Sigma Chem. Co) was added before they were evaporated under nitrogen, dissolved in 0.1 ml of the Foleh reagent and layered on Whatman Silicagel 150A, LKSD plates. Separation of the phospholipids was performed as described (21) using a solvent system hexane:diethyl ether:glacial acetic acid = 82: 18 : 2. Regions of the chromatogram-containing phospholipids were visualized using a 0.2% dichlorofluorescein spray and were scraped into tubes. 0.1 ml of chloroform and then 2.0 ml of a 14% boron trifluoride-methanol solution (Sigma Chem. Co, St. Louis, MO) were added and the transesterification allowed to proceed for 30 rain at 70°C. The fatty acid methyl esters were extracted twice with hexane from the reaction mixture following the addition of 2 ml of water. The hexane was evaporated under nitrogen at 550C and the sediment was redissoived in 0. I mi of chloroform. One to 10 t~l of this solution was injected on the gas chromatograph. Fatty acid analysis was performed as described (27) using Hewlett Packard 5890 Gas Chromatograph and a 0.53 mm capillary column, 30 m long, coated with I t~m of 50% cyanpropylmethyl-50% methylphenylsilicone. The content of fatty acids was expressed as/~g per mg of phospholipids or as a percentage of the total amount of fatty acids. Peroxidizability Index (PI) was calculated according to the formula (31): PI = (percent of monoenoic acids x 0,025 + (percent of dienoic acids × 1) + (percent of trienoic acids × 2) + (percent of tetraenoic acids x 4) + (percent of pentaenoic acids × 6) + (percent of hexaenoic acids × 8). Vitamin E was determined fluorometrically according to
METHOD
Dietary Treatment Forty male Sprague-Dawley rats, weighing 240-260 g (Harlan Lab., Indianapolis, IN) were house-paired in a cage in a temperature controlled room maintained at 22"-240C, with a 12-h diurnal light cycle. After the animals had acclimated for 1 week to the basal purified liquid diet designed by Lieber and DeCarli (12) they were randomly assigned to one of four dietary groups with or without ethanol and/or supplemented with vitamin E. The Lieber-DeCarli diets were supplemented as follows: (a) no additions, (lo) vitamin E, (c) ethanol, and (d) vitamin E plus ethanol. The composition of the basal liquid diet was previously described (20). Ethanol diets were made isocaloric by replacing 94.75 g of maltose-dextrin with 67.29 mi of 95% ethanol. The vitamin E supplemented diets received an additional 142 IU of d-alpha-tocopherol/L of diet (total = 172 IU/L). Fatty acid composition of the vegetable oils used for the basal liquid diet was (percent of
150:
120 ¢
_o
90:
o
60
0.
0
30~
t
O~ I
I
1
5
10
---I
15
I
I
I
1
I
20
25
30
35
40
45
Day
FIG. 1. Time-course of the diet consumption in the pair-feeding experiments using the control and ethanol-fed rats. Consumption of food by rats fed ethanol-containing Lieber-DeCarli diets (36% of total calories), supplied with 30 IU/L or 172 IU/L of vitamin E, was constantly registered, and the control rats were given the same amount of fond as their ethanol-fed counterparts. Each point represents mean (of 10 rats) ± SE quantity of the diet in ml consumed by one rat during a day.
ETHANOL,
V I T A M I N E, A N D R A T H E A R T T I S S U E
331
TABLE ! EFFECT OF DIFFERENT DIETARY TREATMENTS ON FATTY ACID COMPOSITION OF HEART PHOSPHOLIPIDS Fatty acid (ug/mg of phospholipids) C-14:0 C-14:1 C-16:0 C-16:(n = 7) C-18:0 C-18:1,n9 C-18:2,n6 C-18:3 C-20:0 C-20:1 C-20:02,n9 C-20:03.n9 C-20:01 C-20.4,n6 C-20:5,n3 C-22:0 C-22:1 C-22:4,n6 C-22:6,n3 o70ofmonoenoic fattyacids °7o of dienoic fatty acids °70 of trienoic fatty acids o70oftetraenic fattyacids % ofhexaenic fattyacids Peroxidizability index
Control 1.95 0.85 123 1.29 304 84.6 184 0 1.91 1.51 2.22 2.97 1.91 218 0 1.14 0.78 14.9 61.6 8.96 18.7 0.30 23.5 6.2 162.
+_ 0.16 + 0.21 ± 5.64 + 0.10 ± 10.6 + 2.52 ± 6.5 ± ± ± ± + ±
0.151. 0.111. 0.15 0.331 0.15¢ 8.1
± 0.13§ ± 0.21 ± 0.531. ± 2.6 ± 0.30 ± 0.441. +_ 0.031. _+ 0.85:1: ± 0.191. ± 4.41.~
Vitamin E 1.79 1.30 127 1.33 324 90.7 178 0 3.46 3.02 1.98 2.81 3.46 212 0 1.34 101 13.9 63/0 8.98 18.2 0.28 21.9 6.3 157.2
+ 0.35 + 0.11 ± 5.09 ± 0.29 ± 15.8 ± 4.41 _+ 7.9 ± ± ± ± ± ±
0.54"t" 0.381.1" 0.30 0.261. 0.541 10.2
± 0.19~. _+ 0.23 ± 0.99'I ± 1.5 +_ 0.32 + 0.421. ± 0.021. ± 0.65I ± 0.15"I,+ ± 3.51.
Ethanol 145 I.I1 104 0.83 319 88.2 172 0 2.55 1.93 2.48 5.60 2.55 258 0 1.39 0.59 20.3 77.8 8.61 16.4 0.53 26.3 7.2 180.3
Ethanol + Vitamin E
_+ 0.27 ± 0.10 _+ 7.63 + 0.20 ± 24.7 ± 4.77 ± 11.3 ± 0.371. ± 0.231. ± 0.24 ± 0.651. _+ 0.371. ± 23.7 ± 0.181. ± 0.05 ± 2.681. ± 10.0 ± 0.25 ± 0.52, ± 0.05~ ± 1.01§ ± 0.39§ _+ 6.9§
1.82 0.91 185 0.65 283 86.3 164 0 2.03 1.43 2.63 4.71 2.03 2264 0 1.22 15.7 64.2 9.14 17.7 0.47 24.6 6.5 169.2
+ ± ± ± ± ± ±
0.46 0.21 58 0.02 21.4 8.83 b 10.6
± 0.221.1 ± 0.21¢ _+ 0.21~: ± 0.30** ± 0.221 ± 12.4 ± ± ± ± + ± ± ± ±
0.251.§ 01.351. 3.5 0.40 0.851.,+ 0.03** 0.90§ 0.29.~§ 5/3~:§
*Data are given as the mean + SE of 7-10 rats. Symbols t,*+, §, II, and ¶, refer to significant difference between one group and all the rest in Duncan 5°70 level multiple range analysis. If one group shares the same symbol with any other it means that the difference between the man values for these groups is not significant.
the published procedure (7). P h o s p h o l i p i d content was analyzed by the m e t h o d o f R a h e j a et al. (23).
Analysis of Lipid Peroxidation Products Diene conjugates were analyzed by e v a p o r a t i n g 2.0 mi o f the Folch extract of the heart tissue u n d e r nitrogen at 55°C, redissolving the lipid residue in methylene chloride a n d washing it twice with water. The emulsion thus f o r m e d in the m e t h ylene chloride after the last wash was clarified by adding 0.5 ml o f m e t h a n o l . The c o n t e n t o f diene conjugates was determined by measuring a b s o r b a n c e at 232 n m (28). Fluorescent products were m e a s u r e d in the Folch extracts o f heart tissue as described previously (29). The content o f fluorescent products is expressed in relative units o f fluorescence at excitation wavelength 365 n m a n d emission wavelength in the range o f 430-450 n m per mg o f heart p h o s p h o l i p i d s . The exact position o f the emission m a x i m a was observed in each case by recording the emission s p e c t r u m o f the sample at excitation wavelength 365 n m using Hitachi F2000 s p e c t r o f l u o r o m e t e r .
Statistical Analysis The significance o f differences between the rat groups was tested using D u n c a n 5 % level multiple range analysis (8) or Student's t test. Data are expressed as the m e a n _+ SE.
RESULTS M e a n ( + SE) final body weights o f rats receiving the control (345 +_ 5.8 g) or vitamin E s u p p l e m e n t e d (345 + 5.8 g) diets were significantly higher (p < 0.05) t h a n those o f rats fed ethanol (325 _ 7.8 g) or ethanol plus vitamin E (294 +_ 15.5 g). The pattern o f diets c o n s u m p t i o n is s h o w n on Fig. I. A f t e r 3 weeks o f pair-feeding food c o n s u m p t i o n stabilized. At that time rats receiving the vitamin E s u p p l e m e n t e d diets c o n s u m e d approximately 15.7 mg daily a n d those fed the basic diets 2.7 mg o f alpha-tocopheryl acetate. M e a n daily intake o f ethanol stabilized at the level o f a p p r o x i m a t e l y 15 g / k g body weight. The average levels of e t h a n o l in b l o o d serum o b t a i n e d during sacrifice were very variable a n d did not show significant differences in rats c o n s u m i n g e t h a n o l with the basic (5.2, 3.0 m g / d l ) or vitamin E s u p p l e m e n t e d (1.5, 0.6 m g / dl) diets. Stearic (C~8:0), linoleic (C~s:z) a n d arachidonic (C2o:4) acids constitute a b o u t 70°70 o f the total fatty acid content o f phospholipids in the heart tissue (Table 1). C o n s u m p t i o n o f ethanol plus supplemental vitamin E did not change the profile o f the principal fatty acids a n d p r o d u c e d a significant effect only on the less a b u n d a n t ones. E t h a n o l feeding caused a n increase in C2o:2, C2o:3, a n d C22:4 whereas supplemental vitamin E augm e n t e d the q u a n t i t y o f C20:0, (C2o:j), a n d (Ca:0). C o m b i n e d a d m i n i s t r a t i o n o f ethanol and vitamin E annihilates all these
332
PIROZHKOV ET AL.
changes, leaving only significantly elevated levels of C20:3compared to controls. Although vitamin E has no influence on the percentage distribution of fatty acid species with different amounts of double bonds, ethanol evokes a decrease in a relative content of dienoic acids and an increase of trienoic, tetraenoic, and hexaenoic acids. As a result, the P! of the heart phospholipids in the ethanol group is significantly higher than in untreated and vitamin E supplemental groups. Addition of supplemental vitamin E to the ethanol diet attenuated the ethanol-induced shift toward unsaturation and decreased the peroxidizability index to control values. Ethanol alone and especially in conjunction with vitamin E promoted accumulation of vitamin E in the myocardial cells not associated with retention of lipids (Table 2). The cardiac phospholipid levels are increased in rats consuming ethanol and/or supplemental vitamin E. Elevation in the myocardial vitamin E content corresponded to an increase of diene conjugates in the heart lipids and to a decrease of fluorescent products of LPO. A significant positive correlation was found between vitamin E concentration and diene conjugates content in the heart (correlation coefficient = 0.36; p = 2070), whereas a similar correlation with the fluorescent products was insignificant. DISCUSSION
Effects of ethanol on fatty acid composition of cardiac lipids may depend on several variables, such as dose, route, and duration of ethanol administration, type and composition of the dietary lipids etc. In our studies, we used a pair-feeding approach and nutritionally balanced liquid Lieber-DeCarli diet to prevent differences between groups in consumption of calories. Concentrations of fatty acids were expressed in absolute quantities per mg of phospholipids. For the minor fatty acids, constituting less than 5070 of the total fatty acid amount, we consider this presentation as more accurate than usually given data in percent of total fatty acids. Slight changes in the total amount of major fatty acids due to alterations in the spectrum of phospholipids (e.g., increase in lysophospholipids) may affect the relative content of minor fatty acids without changes in their absolute concentrations.
Our results confirm a recently published work reporting no effects of chronic inhalation of ethanol vapors on the main fatty acid profile of aortic lipids but increase of eicosatrienoic (C2o:~) acid (10). We also observed an increase of C20:3 in the heart phospholipids. However in our case C20:.~belongs to n-9 series of fatty acids and this effect can not be explained by the inhibitory influence of alcohol on A'-desaturase. In earlier reports the relative amount of C ~ and C22:6in total myocardial lipids or phospholipids was decreased in rats or mice chronically consuming ethanol (15,2,1). Our data suggest that ethanol can produce a significant shift of fatty acid profile toward unsaturation. We found a marked relative enrichment of the heart phospholipids with tri-, tetra-, and hexaenoic fatty acids, though absolute amounts of these fatty acids in the heart were not significantly changed. The reason for the discrepancies may lie in the diets and routes of ethanol administration. In one of these studies, mice were treated by ethanol vapors in the inhalation chamber (15). As a result, blood ethanol levels reached 300-600 mg/dl. These levels are 100 times higher than those observed in our experiments. It has been demonstrated that perfusion of isolated heart with 1 g/dl solution of ethanol leads to activation of phospholipase A2 and 60°70 increase in cardiac lysophospholipids (6). Because C2o:, is mainly located at C2 position of glycerol moiety of phospholipids, it may be lost after ethanol-induced activation of phospholipase A2 in mice treated with ethanol vapors. In addition, consumption of food by the control and ethanol-breathing mice was not equated. In the second work (24) rats were fed Purina Chow diet and ethanol was added to drinking water. In Chow food fat contributes 12070 of total calories whereas in Lieber-DeCarli liquid diet, specially developed to mimic hepatic changes associated with fatty liver fat, constitutes 35 070 of total calories (13). This great difference in dietary fat consumption in our studies and in the earlier work may have determined the opposite response of C~0:4of cardiac phospholipids to ethanol. Thus, our study suggests that myocardial tissue may be resistant to a polyunsaturated fatty acid lowering effect of ethanol seen in liver (17) or brain (14) of ethanolconsuming animals. As a result of the relative increase in fatty acid unsaturation in cardiac phospholipids, the PI, a parameter based on the maximal rates of oxidation in vitro of the specific fatty acid,
TABLE 2 EFFECT OF ETHANOL AND VITAMIN E DIETARY TREATMENTON VITAMIN E AND LIPID PEROXIDATIONPRODUCTS CONTENT IN THE HEART Parameter* Phospholipids (mg/g of heart issue) Vitamin E ug/g of heart tissue ug/mg of heart phospholipids Diene conjugates (O.D. units/mg of phospholipids) + Fluorescent products (units/rag of phospholipids)*
Control
VitaminE
Ethanol
Ethanol + VitaminE
11.5 -± 0.36*
12.2 ± 0.285
12.5 ± 0.34J;
13.1 ± 0.5,
23.8 :l: 0.68t 2.09 ± 0.09* 1.16 ± 0.09t
31.0 ± 1.65 2.56 ± 0.16$ 1.26 ± 0.095
37.3 ± 1.7§ 2.96 ± 0.14{ 1.36 ± 0.125
49.6 ± 2.011 3.96 ± 0.2311 1.62 +_ 0.105
830 ± 43t
712 ± 87~
805 ± 225t
642 ± 94t§
*Data are given as the mean ± SE of 7 10 rats; symbols (t, t;. §, II, ¶) refer to significant difference between one group and all the rest in Duncan 5% level multiple range analysis. +Diene conjugates are expressed as units of optical density at 232 rum per mg of heart phospholipids. mFluorescent products are expressed as units of fluorescence per mg of heart phospholipids as described in the Methods section.tAnd use the insert one pica para indent before each new footnote. End the last footnote with.
E T H A N O L , V I T A M I N E, A N D R A T H E A R T T I S S U E was increased by 11070 compared to controls. That may be one cause of higher peroxidizability of the myocardial membranes from ethanol-treated rats found in vitro (1). In lung tissue, PI significantly correlated (r = 0.85) with the level of TBAreactive substances (2). Ethanol consumption yielded more pronounced increase o f vitamin E in the heart than vitamin E supplemented diet. When rats were fed a diet containing both ethanol and supplemental vitamin E, the increase o f vitamin E in the heart was additive, suggesting different mechanisms whereby it accumulates in the myocardial membranes. In a similar study, using rats fed Lieber-DeCarli alcohol diet and a pair-fed control group, changes in the myocardial vitamin E content were not found (4). It should be emphasized that we were detecting vitamin E, which represents a mixture o f tocopherols and tocotrienols, whereas in the previous study, only aipha-tocopherol was measured. Recently it has been demonstrated that as a result of chronic ethanol consumption concentration of alpha-tocopherol in lipids increases in rat testes, decreases in liver, and remains unchanged in lungs (16). In addition, concentration of gamma-tocopherol doubles in tests, where it reaches almost 5007o of alpha-tocopherol concentration. Thus, ethanol may affect distribution of alpha-tocopherol between various organs or preferentially influence accumulation of other types of tocopherols, such as gamma-tocopherol. Also, it has been suggested that membrane structure is a critical factor determining how much vitamin E may be incorporated in a particular m e m b r a n e when an excess of vitamin is available (22). Because ethanol exerts a disordering effect on various types of biological membrane (32) and vitamin E decreases
333 fluidity of the lipid bilayer (22) incorporation of vitamin E in the cardiac membrane may be adaptive. It is noteworthy that vitamin E opposed the ethanolinduced increase in polyunsaturated fatty acids content in the myocardium bringing down the PI to the normal level. In spite of this, higher levels of diene conjugates were found in cardiac lipids from the rats, consuming ethanol and large quantities o f vitamin E, than in the control rats. Vitamin E possesses high affinity for polyunsaturated fatty acids (30). Due to its capacity to scavenge free radicals vitamin E may reduce carbon-centered radical of fatty acid molecule that has undergone rearrangement to form diene conjugate. Thus, conjugated diene structure may be trapped in the fatty acid moiety of phospholipids. Alternatively, free radicals of fatty acids can give rise to hydroperoxides and then decompose to LPO products. This suggestion is indirectly supported by lower levels of fluorescent products in the hearts of rats consuming high quantities o f vitamin E. According to the established view aldehydes and ketones formed during decomposition of fatty acid hydroperoxides are one of the sources of lipofuscine-like fluorescent products (3,29). Therefore, in our experimental model, diene conjugates reflect increased rate of fatty acid free radicals formation rather than activated LPO. Thus the results o f this work provide evidence of a protective potency of vitamin E against ethanol-induced changes in fatty acid composition, PI, and accumulation of fluorescent products formed from L P O in the heart. ACKNOWLEDGEMENT This work was supported in part by NIAAA Grant No. 08034.
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31. 32.
ET AL.
J.; Dormandy, T.; Watson, R. R. Plasma fatty acid pattern including dicalc-conjugated linoleic acid in ethanol users and patients with ethanol-related liver disease. Alcohol Clin. Exp. Res. 10:647-650; 1986. Tappel, A. L. Lipid peroxide damage to cell components. Fed. Proc. 32: ! 870-1874; 1973. Tappel, A. L. Measurement and protection from m vivo lipid peroxidation. In: Pryor, W. A., ed. Free radicals in biology, vol. 4, New York:Academic Press; 1980:1-47. Urano, S.; Iida, M.; Otani, I.; Matsuo, M. Membrane stabilization of vitamin E: Interaction of cc-tocopherol with phospholipids in bilayer liposomes. Biochim. Biophys. Acta 146:1413-1418; 1987. Witting, L . . A . ; Horwitt, M. K. Effect of degree of fatty acid unsaturation in tocopherol deficiency induced creatinuria..I. Nutr. 82:!9-25; 1964. Wood, W. G.; Schroeder, F. Membrane effects o! ethanol: Bulk lipid versus lipid domains. Life Sci. 43:467-475; 1988.
0741-8329/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
Effect of Chronic Consumption of Ethanol and Vitamin E on Fatty Acid Composition and Lipid Peroxidation in Rat Heart Tissue S E R G E I V. P I R O Z H K O V * , CLEAMOND D. E S K E L S O N * t I, R O N A L D R. W A T S O N ~ : I!, G L E N C. H U N T E R § , J O S E P H J. P I O T R O W S K I § AND VICTOR BERNHARD§
*Research Institute f o r Medico-Biological Problems o f Addictions, Moscow 121921, Russia and t Departments o f Surgical Biology, ~Family and Community Medicine and §Surgery, IIN I A A A Specialized Alcohol Research Center, University o f Arizona Health Sciences Center, Tucson, A Z 85724 R e c e i v e d 23 S e p t e m b e r 1991; A c c e p t e d 27 J a n u a r y 1992 PIROZHKOV, S. V., C. D. ESKELSON, R. R. WATSON, G. C. HUNTER, J. J. PIOTROWSKI AND V. BERNHARD. Effect of chronic consumption of ethanol and vitamin E on fatty acid composition and lipid peroxidation in rat heart tissue. ALCOHOL 9(4) 329-334, 1992.--Lipid peroxidation products and the fatty acid composition of phospholipids were studied in the hearts of rats chronically consuming ethanol supplemented with large amounts of vitamin E. Ethanol representing 3607o of the total calories was ingested for 7 weeks in a modified Lieber-DeCarli liquid diet that contained vitamin E at 30 IU/L in the control or 172 IU/L in the supplemental dietary group. Ethanol and/or vitamin E did not change the absolute content (#g per mg of phospholipids) of the main fatty acids (C,8:0, Cts:z, and C2o:,) of heart phospholipids but increased the amount of the minor C20-C22 fatty acids. Cardiac phospholipid levels increased in rats chronically consuming excess vitamin E and/or alcohol. Chronic ethanol consumption caused elevations of the relative content (percent of total fatty acids) of tri-, tetra-, and hexaenoic acids and peroxidizability index (PI) of the cardiac phospholipids. Supplementation with vitamin E blocked this ethanol-induced shift in the fatty acid profile toward unsaturation and decreased the PI. Ethanol enhanced accumulation of vitamin E in heart tissue by 30070 irrespective of the vitamin E content in the diet. Enrichment of the diet with vitamin E coincided with the low levels of fluorescent products in heart lipids. A positive correlation (r = 0.36; p = 2°70) was found between vitamin E and diene conjugates in the heart cells. Thus, vitamin E has a stabilizing effect on heart phospholipids by preventing changes in their fatty acid composition and peroxidative deterioration. Ethanol
Vitamin E
Fatty acid composition
Lipid Peroxidation
E T H A N O L stimulates lipid peroxidation (LPO) in various tissues (19). In the heart, chronic ethanol consumption produced significant increases in m e m b r a n e peroxidizability (I). Lipid peroxidation in this study was initiated in vitro by addition o f F E 3 + / A D P / a s c o r b a t e and detected by enhancement of low-level chemiluminescence and accumulation o f the thiobarbituric acid (TBA)-reactive substances. Glutathione content decreased and diene conjugates were increased in the myocardium after chronic ethanol consumption (9). These effects of ethanol were attributed to the depletion of antioxidants in the membranes o f cardiomyocytes as a result of activated LPO. Changes in fatty acid composition induced by
Heart
ethanol may make heart lipids more liable to peroxidation. For example, feeding pigs or rats with a diet enriched with fish oil causes a shift in fatty acid profile toward more unsaturated species and facilitates accumulation of lipofuscin in the heart indicating an oxidative deterioration o f lipids (18,25). Chronic alcohol administration decreased hepatic arachidonic (C20:4) and docosahexaenoic (C~2:6) acids (17) and C2o:4 in brain synaptosomes (14), although it increased C~: 4 and Cts:~ and decreased C~6:o in erythrocyte membranes ( l l ) . Ethanol consumption reduced the relative content of C2o:4 and C2z:6 in the heart (24). Although the antioxidant vitamin E prevented ethanol-
Requests for reprints should be addressed to Cleamond D. Eskelson, Department of Surgical Biology, NIAAA Specialized Alcohol Research Center, University of Arizona Health Sciences Center, Tucson AZ 85724. 329
330
PIROZHKOV ET AL.
induced changes of LPO parameters in myocardial tissue (1,9), its effect on fatty acid composition of heart lipids was not elucidated. A strong positive correlation was demonstrated between alpha-tocopherol content and the levels of C22: 6 and C22:~in the heart when rats were fed diets with different n-3/n-6 ratio (5), but no correlation was found between vitamin E and C~8:2or C20:, in the membranes of pulmonary artery endothelial cells (26). Also, there exists no data concerning the combined action of ethanol and vitamin E on fatty acid patterns of heart membranes. The present study investigated changes in the fatty acids profile of the cardiac phospholipids during the consumption of alcoholic diets supplemented with vitamin E. Additionally, we studied the association between phospholipid polyunsaturated fatty acid composition, vitamin E content, and LPO in the myocardial cells.
total content): C~,:o-O; C]6:o-15.8%; CI6:1-1.0~70; C,~:o-3.2%; C,a:1-49.2%; CIa:2-29-7%; C]a:3-1.2%0; C~0:z-traces; C._o:s-0; Cz2:~-0; C22 t-0. The duration of the experiment was 7 weeks.
Lipid Analysis Lipids were extracted from the heart samples using a Folch reagent, chloroform:methanol = 2 : 1. To aliquots of the extract an internal standard (heptadecanoyl phosphatidylcholine, Sigma Chem. Co) was added before they were evaporated under nitrogen, dissolved in 0.1 ml of the Foleh reagent and layered on Whatman Silicagel 150A, LKSD plates. Separation of the phospholipids was performed as described (21) using a solvent system hexane:diethyl ether:glacial acetic acid = 82: 18 : 2. Regions of the chromatogram-containing phospholipids were visualized using a 0.2% dichlorofluorescein spray and were scraped into tubes. 0.1 ml of chloroform and then 2.0 ml of a 14% boron trifluoride-methanol solution (Sigma Chem. Co, St. Louis, MO) were added and the transesterification allowed to proceed for 30 rain at 70°C. The fatty acid methyl esters were extracted twice with hexane from the reaction mixture following the addition of 2 ml of water. The hexane was evaporated under nitrogen at 550C and the sediment was redissoived in 0. I mi of chloroform. One to 10 t~l of this solution was injected on the gas chromatograph. Fatty acid analysis was performed as described (27) using Hewlett Packard 5890 Gas Chromatograph and a 0.53 mm capillary column, 30 m long, coated with I t~m of 50% cyanpropylmethyl-50% methylphenylsilicone. The content of fatty acids was expressed as/~g per mg of phospholipids or as a percentage of the total amount of fatty acids. Peroxidizability Index (PI) was calculated according to the formula (31): PI = (percent of monoenoic acids x 0,025 + (percent of dienoic acids × 1) + (percent of trienoic acids × 2) + (percent of tetraenoic acids x 4) + (percent of pentaenoic acids × 6) + (percent of hexaenoic acids × 8). Vitamin E was determined fluorometrically according to
METHOD
Dietary Treatment Forty male Sprague-Dawley rats, weighing 240-260 g (Harlan Lab., Indianapolis, IN) were house-paired in a cage in a temperature controlled room maintained at 22"-240C, with a 12-h diurnal light cycle. After the animals had acclimated for 1 week to the basal purified liquid diet designed by Lieber and DeCarli (12) they were randomly assigned to one of four dietary groups with or without ethanol and/or supplemented with vitamin E. The Lieber-DeCarli diets were supplemented as follows: (a) no additions, (lo) vitamin E, (c) ethanol, and (d) vitamin E plus ethanol. The composition of the basal liquid diet was previously described (20). Ethanol diets were made isocaloric by replacing 94.75 g of maltose-dextrin with 67.29 mi of 95% ethanol. The vitamin E supplemented diets received an additional 142 IU of d-alpha-tocopherol/L of diet (total = 172 IU/L). Fatty acid composition of the vegetable oils used for the basal liquid diet was (percent of
150:
120 ¢
_o
90:
o
60
0.
0
30~
t
O~ I
I
1
5
10
---I
15
I
I
I
1
I
20
25
30
35
40
45
Day
FIG. 1. Time-course of the diet consumption in the pair-feeding experiments using the control and ethanol-fed rats. Consumption of food by rats fed ethanol-containing Lieber-DeCarli diets (36% of total calories), supplied with 30 IU/L or 172 IU/L of vitamin E, was constantly registered, and the control rats were given the same amount of fond as their ethanol-fed counterparts. Each point represents mean (of 10 rats) ± SE quantity of the diet in ml consumed by one rat during a day.
ETHANOL,
V I T A M I N E, A N D R A T H E A R T T I S S U E
331
TABLE ! EFFECT OF DIFFERENT DIETARY TREATMENTS ON FATTY ACID COMPOSITION OF HEART PHOSPHOLIPIDS Fatty acid (ug/mg of phospholipids) C-14:0 C-14:1 C-16:0 C-16:(n = 7) C-18:0 C-18:1,n9 C-18:2,n6 C-18:3 C-20:0 C-20:1 C-20:02,n9 C-20:03.n9 C-20:01 C-20.4,n6 C-20:5,n3 C-22:0 C-22:1 C-22:4,n6 C-22:6,n3 o70ofmonoenoic fattyacids °7o of dienoic fatty acids °70 of trienoic fatty acids o70oftetraenic fattyacids % ofhexaenic fattyacids Peroxidizability index
Control 1.95 0.85 123 1.29 304 84.6 184 0 1.91 1.51 2.22 2.97 1.91 218 0 1.14 0.78 14.9 61.6 8.96 18.7 0.30 23.5 6.2 162.
+_ 0.16 + 0.21 ± 5.64 + 0.10 ± 10.6 + 2.52 ± 6.5 ± ± ± ± + ±
0.151. 0.111. 0.15 0.331 0.15¢ 8.1
± 0.13§ ± 0.21 ± 0.531. ± 2.6 ± 0.30 ± 0.441. +_ 0.031. _+ 0.85:1: ± 0.191. ± 4.41.~
Vitamin E 1.79 1.30 127 1.33 324 90.7 178 0 3.46 3.02 1.98 2.81 3.46 212 0 1.34 101 13.9 63/0 8.98 18.2 0.28 21.9 6.3 157.2
+ 0.35 + 0.11 ± 5.09 ± 0.29 ± 15.8 ± 4.41 _+ 7.9 ± ± ± ± ± ±
0.54"t" 0.381.1" 0.30 0.261. 0.541 10.2
± 0.19~. _+ 0.23 ± 0.99'I ± 1.5 +_ 0.32 + 0.421. ± 0.021. ± 0.65I ± 0.15"I,+ ± 3.51.
Ethanol 145 I.I1 104 0.83 319 88.2 172 0 2.55 1.93 2.48 5.60 2.55 258 0 1.39 0.59 20.3 77.8 8.61 16.4 0.53 26.3 7.2 180.3
Ethanol + Vitamin E
_+ 0.27 ± 0.10 _+ 7.63 + 0.20 ± 24.7 ± 4.77 ± 11.3 ± 0.371. ± 0.231. ± 0.24 ± 0.651. _+ 0.371. ± 23.7 ± 0.181. ± 0.05 ± 2.681. ± 10.0 ± 0.25 ± 0.52, ± 0.05~ ± 1.01§ ± 0.39§ _+ 6.9§
1.82 0.91 185 0.65 283 86.3 164 0 2.03 1.43 2.63 4.71 2.03 2264 0 1.22 15.7 64.2 9.14 17.7 0.47 24.6 6.5 169.2
+ ± ± ± ± ± ±
0.46 0.21 58 0.02 21.4 8.83 b 10.6
± 0.221.1 ± 0.21¢ _+ 0.21~: ± 0.30** ± 0.221 ± 12.4 ± ± ± ± + ± ± ± ±
0.251.§ 01.351. 3.5 0.40 0.851.,+ 0.03** 0.90§ 0.29.~§ 5/3~:§
*Data are given as the mean + SE of 7-10 rats. Symbols t,*+, §, II, and ¶, refer to significant difference between one group and all the rest in Duncan 5°70 level multiple range analysis. If one group shares the same symbol with any other it means that the difference between the man values for these groups is not significant.
the published procedure (7). P h o s p h o l i p i d content was analyzed by the m e t h o d o f R a h e j a et al. (23).
Analysis of Lipid Peroxidation Products Diene conjugates were analyzed by e v a p o r a t i n g 2.0 mi o f the Folch extract of the heart tissue u n d e r nitrogen at 55°C, redissolving the lipid residue in methylene chloride a n d washing it twice with water. The emulsion thus f o r m e d in the m e t h ylene chloride after the last wash was clarified by adding 0.5 ml o f m e t h a n o l . The c o n t e n t o f diene conjugates was determined by measuring a b s o r b a n c e at 232 n m (28). Fluorescent products were m e a s u r e d in the Folch extracts o f heart tissue as described previously (29). The content o f fluorescent products is expressed in relative units o f fluorescence at excitation wavelength 365 n m a n d emission wavelength in the range o f 430-450 n m per mg o f heart p h o s p h o l i p i d s . The exact position o f the emission m a x i m a was observed in each case by recording the emission s p e c t r u m o f the sample at excitation wavelength 365 n m using Hitachi F2000 s p e c t r o f l u o r o m e t e r .
Statistical Analysis The significance o f differences between the rat groups was tested using D u n c a n 5 % level multiple range analysis (8) or Student's t test. Data are expressed as the m e a n _+ SE.
RESULTS M e a n ( + SE) final body weights o f rats receiving the control (345 +_ 5.8 g) or vitamin E s u p p l e m e n t e d (345 + 5.8 g) diets were significantly higher (p < 0.05) t h a n those o f rats fed ethanol (325 _ 7.8 g) or ethanol plus vitamin E (294 +_ 15.5 g). The pattern o f diets c o n s u m p t i o n is s h o w n on Fig. I. A f t e r 3 weeks o f pair-feeding food c o n s u m p t i o n stabilized. At that time rats receiving the vitamin E s u p p l e m e n t e d diets c o n s u m e d approximately 15.7 mg daily a n d those fed the basic diets 2.7 mg o f alpha-tocopheryl acetate. M e a n daily intake o f ethanol stabilized at the level o f a p p r o x i m a t e l y 15 g / k g body weight. The average levels of e t h a n o l in b l o o d serum o b t a i n e d during sacrifice were very variable a n d did not show significant differences in rats c o n s u m i n g e t h a n o l with the basic (5.2, 3.0 m g / d l ) or vitamin E s u p p l e m e n t e d (1.5, 0.6 m g / dl) diets. Stearic (C~8:0), linoleic (C~s:z) a n d arachidonic (C2o:4) acids constitute a b o u t 70°70 o f the total fatty acid content o f phospholipids in the heart tissue (Table 1). C o n s u m p t i o n o f ethanol plus supplemental vitamin E did not change the profile o f the principal fatty acids a n d p r o d u c e d a significant effect only on the less a b u n d a n t ones. E t h a n o l feeding caused a n increase in C2o:2, C2o:3, a n d C22:4 whereas supplemental vitamin E augm e n t e d the q u a n t i t y o f C20:0, (C2o:j), a n d (Ca:0). C o m b i n e d a d m i n i s t r a t i o n o f ethanol and vitamin E annihilates all these
332
PIROZHKOV ET AL.
changes, leaving only significantly elevated levels of C20:3compared to controls. Although vitamin E has no influence on the percentage distribution of fatty acid species with different amounts of double bonds, ethanol evokes a decrease in a relative content of dienoic acids and an increase of trienoic, tetraenoic, and hexaenoic acids. As a result, the P! of the heart phospholipids in the ethanol group is significantly higher than in untreated and vitamin E supplemental groups. Addition of supplemental vitamin E to the ethanol diet attenuated the ethanol-induced shift toward unsaturation and decreased the peroxidizability index to control values. Ethanol alone and especially in conjunction with vitamin E promoted accumulation of vitamin E in the myocardial cells not associated with retention of lipids (Table 2). The cardiac phospholipid levels are increased in rats consuming ethanol and/or supplemental vitamin E. Elevation in the myocardial vitamin E content corresponded to an increase of diene conjugates in the heart lipids and to a decrease of fluorescent products of LPO. A significant positive correlation was found between vitamin E concentration and diene conjugates content in the heart (correlation coefficient = 0.36; p = 2070), whereas a similar correlation with the fluorescent products was insignificant. DISCUSSION
Effects of ethanol on fatty acid composition of cardiac lipids may depend on several variables, such as dose, route, and duration of ethanol administration, type and composition of the dietary lipids etc. In our studies, we used a pair-feeding approach and nutritionally balanced liquid Lieber-DeCarli diet to prevent differences between groups in consumption of calories. Concentrations of fatty acids were expressed in absolute quantities per mg of phospholipids. For the minor fatty acids, constituting less than 5070 of the total fatty acid amount, we consider this presentation as more accurate than usually given data in percent of total fatty acids. Slight changes in the total amount of major fatty acids due to alterations in the spectrum of phospholipids (e.g., increase in lysophospholipids) may affect the relative content of minor fatty acids without changes in their absolute concentrations.
Our results confirm a recently published work reporting no effects of chronic inhalation of ethanol vapors on the main fatty acid profile of aortic lipids but increase of eicosatrienoic (C2o:~) acid (10). We also observed an increase of C20:3 in the heart phospholipids. However in our case C20:.~belongs to n-9 series of fatty acids and this effect can not be explained by the inhibitory influence of alcohol on A'-desaturase. In earlier reports the relative amount of C ~ and C22:6in total myocardial lipids or phospholipids was decreased in rats or mice chronically consuming ethanol (15,2,1). Our data suggest that ethanol can produce a significant shift of fatty acid profile toward unsaturation. We found a marked relative enrichment of the heart phospholipids with tri-, tetra-, and hexaenoic fatty acids, though absolute amounts of these fatty acids in the heart were not significantly changed. The reason for the discrepancies may lie in the diets and routes of ethanol administration. In one of these studies, mice were treated by ethanol vapors in the inhalation chamber (15). As a result, blood ethanol levels reached 300-600 mg/dl. These levels are 100 times higher than those observed in our experiments. It has been demonstrated that perfusion of isolated heart with 1 g/dl solution of ethanol leads to activation of phospholipase A2 and 60°70 increase in cardiac lysophospholipids (6). Because C2o:, is mainly located at C2 position of glycerol moiety of phospholipids, it may be lost after ethanol-induced activation of phospholipase A2 in mice treated with ethanol vapors. In addition, consumption of food by the control and ethanol-breathing mice was not equated. In the second work (24) rats were fed Purina Chow diet and ethanol was added to drinking water. In Chow food fat contributes 12070 of total calories whereas in Lieber-DeCarli liquid diet, specially developed to mimic hepatic changes associated with fatty liver fat, constitutes 35 070 of total calories (13). This great difference in dietary fat consumption in our studies and in the earlier work may have determined the opposite response of C~0:4of cardiac phospholipids to ethanol. Thus, our study suggests that myocardial tissue may be resistant to a polyunsaturated fatty acid lowering effect of ethanol seen in liver (17) or brain (14) of ethanolconsuming animals. As a result of the relative increase in fatty acid unsaturation in cardiac phospholipids, the PI, a parameter based on the maximal rates of oxidation in vitro of the specific fatty acid,
TABLE 2 EFFECT OF ETHANOL AND VITAMIN E DIETARY TREATMENTON VITAMIN E AND LIPID PEROXIDATIONPRODUCTS CONTENT IN THE HEART Parameter* Phospholipids (mg/g of heart issue) Vitamin E ug/g of heart tissue ug/mg of heart phospholipids Diene conjugates (O.D. units/mg of phospholipids) + Fluorescent products (units/rag of phospholipids)*
Control
VitaminE
Ethanol
Ethanol + VitaminE
11.5 -± 0.36*
12.2 ± 0.285
12.5 ± 0.34J;
13.1 ± 0.5,
23.8 :l: 0.68t 2.09 ± 0.09* 1.16 ± 0.09t
31.0 ± 1.65 2.56 ± 0.16$ 1.26 ± 0.095
37.3 ± 1.7§ 2.96 ± 0.14{ 1.36 ± 0.125
49.6 ± 2.011 3.96 ± 0.2311 1.62 +_ 0.105
830 ± 43t
712 ± 87~
805 ± 225t
642 ± 94t§
*Data are given as the mean ± SE of 7 10 rats; symbols (t, t;. §, II, ¶) refer to significant difference between one group and all the rest in Duncan 5% level multiple range analysis. +Diene conjugates are expressed as units of optical density at 232 rum per mg of heart phospholipids. mFluorescent products are expressed as units of fluorescence per mg of heart phospholipids as described in the Methods section.tAnd use the insert one pica para indent before each new footnote. End the last footnote with.
E T H A N O L , V I T A M I N E, A N D R A T H E A R T T I S S U E was increased by 11070 compared to controls. That may be one cause of higher peroxidizability of the myocardial membranes from ethanol-treated rats found in vitro (1). In lung tissue, PI significantly correlated (r = 0.85) with the level of TBAreactive substances (2). Ethanol consumption yielded more pronounced increase o f vitamin E in the heart than vitamin E supplemented diet. When rats were fed a diet containing both ethanol and supplemental vitamin E, the increase o f vitamin E in the heart was additive, suggesting different mechanisms whereby it accumulates in the myocardial membranes. In a similar study, using rats fed Lieber-DeCarli alcohol diet and a pair-fed control group, changes in the myocardial vitamin E content were not found (4). It should be emphasized that we were detecting vitamin E, which represents a mixture o f tocopherols and tocotrienols, whereas in the previous study, only aipha-tocopherol was measured. Recently it has been demonstrated that as a result of chronic ethanol consumption concentration of alpha-tocopherol in lipids increases in rat testes, decreases in liver, and remains unchanged in lungs (16). In addition, concentration of gamma-tocopherol doubles in tests, where it reaches almost 5007o of alpha-tocopherol concentration. Thus, ethanol may affect distribution of alpha-tocopherol between various organs or preferentially influence accumulation of other types of tocopherols, such as gamma-tocopherol. Also, it has been suggested that membrane structure is a critical factor determining how much vitamin E may be incorporated in a particular m e m b r a n e when an excess of vitamin is available (22). Because ethanol exerts a disordering effect on various types of biological membrane (32) and vitamin E decreases
333 fluidity of the lipid bilayer (22) incorporation of vitamin E in the cardiac membrane may be adaptive. It is noteworthy that vitamin E opposed the ethanolinduced increase in polyunsaturated fatty acids content in the myocardium bringing down the PI to the normal level. In spite of this, higher levels of diene conjugates were found in cardiac lipids from the rats, consuming ethanol and large quantities o f vitamin E, than in the control rats. Vitamin E possesses high affinity for polyunsaturated fatty acids (30). Due to its capacity to scavenge free radicals vitamin E may reduce carbon-centered radical of fatty acid molecule that has undergone rearrangement to form diene conjugate. Thus, conjugated diene structure may be trapped in the fatty acid moiety of phospholipids. Alternatively, free radicals of fatty acids can give rise to hydroperoxides and then decompose to LPO products. This suggestion is indirectly supported by lower levels of fluorescent products in the hearts of rats consuming high quantities o f vitamin E. According to the established view aldehydes and ketones formed during decomposition of fatty acid hydroperoxides are one of the sources of lipofuscine-like fluorescent products (3,29). Therefore, in our experimental model, diene conjugates reflect increased rate of fatty acid free radicals formation rather than activated LPO. Thus the results o f this work provide evidence of a protective potency of vitamin E against ethanol-induced changes in fatty acid composition, PI, and accumulation of fluorescent products formed from L P O in the heart. ACKNOWLEDGEMENT This work was supported in part by NIAAA Grant No. 08034.
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