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Effects of chronic ethanol exposure on fatty acids of rat brain glycerophospholipids
Alcohol, Vol. 6, pp. 139-146. ©PergamonPress plc, 1989.Printed in the U.S.A.
0741-8329/89$3.00 + .00
Effects of Chronic Ethanol Exposure on Fatty Acids of Rat Brain Glycerophospholipids LENA GUSTAVSSON AND CHRISTER ALLING
Department of Psychiatry and Neurochemistry, University of Lund P.O. Box 638, S-220 06 Lund, Sweden. R e c e i v e d 22 A u g u s t 1988; A c c e p t e d 1 D e c e m b e r 1988 GUSTAVSSON, L. AND C. ALLING. Effects of chronic ethanol exposure on fatty acids of rat brain glycerophospholipids. ALCOHOL 6(2) 139-146, 1989.--The lipid composition was analysed in forebrain subcellular fractions from rats treated with ethanol for three weeks and control rats. Increased proportions of oleic acid and a decrease in palmitic acid were consistently found in total glycerophospholipid fractions after ethanol exposure. The fatty acid compositions of individual phospholipids were also significantly changed. The proportion of docosahexaenoic acid was decreased in brain phosphatidylserine. In contrast to the decrease in the degree of unsaturation in phosphatidylserine, there was an opposite change in phosphatidylcholine wherein the degree of unsaturation was increased. No changes were produced in total cholesterol or phospholipid concentrations. These results point to a high degree of complexity of the mechanisms behind ethanol-induced changes in membrane lipid composition, The decrease in unsaturation in phosphatidylserine is probably an adaptive effect in order to counteract the fluidizing effect of ethanol. There are two possible explanations for the increase in unsaturation in brain phosphatidylcholine. The change may be due to adaptation to other biophysical effects, e.g., expansion of the membrane surface or be secondary to a change in liver lipid metabolism. Brain
Cell membranes
Ethanol
Fatty acids
Glycerophospholipids
ever, no conclusive results supporting this hypothesis have emerged. Inconsistent results have been reported on concentrations of cholesterol. Both increased concentrations (7,8) as well as unaltered (5) and decreased amounts (12) have been reported from studies on brain membranes after chronic ethanol exposure. Decreased proportions of polyunsaturated fatty acids in phospholipids have been reported to occur in synaptosomes from ethanol tolerant mice (19,20). Another study has indicated results in the same direction in liver and erythrocyte membranes, but no changes were found in synaptosomal plasma membranes (31). In contrast, chronic ethanol treatment of guinea pigs resulted in increased proportions of polyunsaturated fatty acids in phospholipids from synaptosomal membrane fraction (26). Other investigations have not found any differences in brain membranes after chronic ethanol exposure (8,25). Thus, our knowledge about the effect of ethanol on cell membrane lipids is incomplete. It is possible that different membrane structures as well as different lipids respond to ethanol in different ways. Even if changes in lipid composition in brain membranes are small they could have considerable consequences for membrane function. Brain lipid composition is relatively inert to changes in the environment compared to other organs, a fact that has been shown in studies of dietary effects (2) and toxic influences of organic solvents (15). It has been suggested that the vulnerability of
IT has been suggested that cell membranes adapt to the perturbing effect of ethanol during chronic exposure of the drug. Fluidization of membranes by ethanol and tolerance to this disordering effect have been measured by EPR and fluorescerise polarisation techniques (12, 17, 21). Recent NMRstudies have demonstrated that ethanol at low concentrations ordered the polar head group region of phosphatidylcholine in synaptosomal plasma membranes (14). The main effect at higher concentrations was a disordering of the membrane core. In addition to the effect on membrane fluidity, ethanol changed the polymorphic phase behaviour of membrane lipids (9,27). This phenomenon may be related to expansion of the membrane in the polar head group region, and consequently to a change in the molecular form ofphospholipids (27,29). This variety of effects points to the high complexity of ethanol-membrane interactions. Much of the attention in alcohol research on membrane lipids has been focused on ethanol-induced chronic changes in the lipid composition that could explain an adaptation to the presence of ethanol. The glycerophospholipids in particular, have been in focus due to their properties which make them important for regulation of membrane fluidity and other biophysical characteristics. Examples of such properties are high proportions of polyunsaturated fatty acids and fast turnover. Adaptation to a fluidizing effect includes a decrease in the degree of unsaturation in fatty acids of phospholipids and increased concentrations of cholesterol. How-
139
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GUSTAVSSON AND ALLING
brain for dietary changes is increased by ethanol (1). The aim of this study was to elucidate the effect of three weeks of ethanol intoxication on membrane glycerophospholipid fatty acid composition of rat brain in an experiment where the dietary intake of nutrients including essential fatty acids was well controlled. METHOD
Experimental Procedure Male Sprague-Dawley rats (n= 12) were given ethanol in the form of a liquid diet (Liquidiet ®, Rat Diet Lieber DeCarli Formula, BioServ Inc., N J) for three weeks. Control rats (n= 12) were pair-fed with an isocaloric diet. The procedure was performed as described by Lieber and DeCarli (10,18). The liquid diet was the only source of food and water. The amount of ethanol in the diet was increased from 1.5% (w/v) to a final concentration of 5% (w/v) at day 8. The rats weighed 136-+21 g at the beginning of the experiment (range 114-161). The weight gain after three weeks was 64___7.8 g in the ethanol-treated group and 81_+13 in the control group. The blood ethanol concentration was 67.2-+12.5 mmol/1 at the day of decapitation. The daily ethanol intake increased constantly from 9.3_+1.63 g ethanol/kg body weight at the first day of administration to 14.2_+2.09 at day 21. While intoxicated by ethanol, the animals were decapitated under ether anesthesia. Forebrain and liver were dissected immediately after decapitation. Subcellular fractions were prepared from forebrain. The tissues were stored at -70°C until analysis.
Isolation of Subcellular Fractions Synaptosomes, myelin and mitochondria were isolated from forebrains (28). The tissue was homogenized in 0.32 M sucrose in an all-glass homogenizer. Two ml of this total homogenate were taken for lipid analyses. The rest was used for isolation of subcellular fractions. This homogenate was centrifuged for 10 minutes at 1000xg. The pellet was resuspended in 0.32 M sucrose and centrifuged as above. The combined supernatants were centrifuged at 17500×g for 1 hour. The resulting pellet was suspended in 0.32 M sucrose and this homogenate was layered on the top of a sucrose density gradient. The discontinuous gradient consisted of 1.2 M sucrose, 0.8 M sucrose and 0.32 M sucrose. The gradient was centrifuged at 50000×g for 2 hours. The fractions enriched in myelin (interface between 0.32 M and 0.8 M sucrose) and synaptosomes (interface between 0.8 M and 1.2 M sucrose) were collected using a Pasteur pipette. The collected volumes were equal in all samples. The mitochondrial fraction (pellet) was suspended in 0.156 M KC1. All subcellular fractions, as" well as the total homogenate, were purified from the sucrose by suspension in 40 ml 0.156 M KCI and centrifugation at 17500×g for 30 minutes. This washing procedure was performed three times. The fractions were finally suspended in 0.156 M KCI. A portion was taken from the samples for protein analyses. All samples were kept at -70°C until analyses. The purity of subcellular fractions was evaluated by electron microscopy.
Biochemical Analyses Lipid extraction. Tissues from different organs were homogenized with all-glass homogenizers to make a 12.5% homogenate in water, except for the subcellular fractions
which were homogenized in one ml of 0.156 M KCI. Samples from the homogenate were taken for protein analyses. The tissue homogenates were extracted as described previously (4). The total lipid extract were separated into a neutral and an acidic fraction by ion-exchange chromatography (23). Quantitative analyses. Lipid phosphorus (6) and cholesterol (11) concentrations in lipid extracts were quantitated by colorimetric methods. The amounts of protein were determined on samples from tissue homogenates (13).
Fatty acid composition of total glycerophospholipids. Fatty acid methyl esters were prepared by alkaline methanolysis of the total lipid fraction. The methanolysis was performed in 0.2 M sodium methoxide in methanol at 37°C for 1 hour. The reaction was stopped by addition of 0.1 M acetic acid. The fatty acid methyl esters were extracted by light petroleum and purified by preparative thin layer chromatography. Dichloromethane was used as the solvent system. The fatty acid methyl esters were detected by bromphenolblue reagent and scraped into methanol. They were extracted by light petroleum. The composition of fatty acid methyl esters was determined by GLC on a packed column (2 m, 2 mm ID) with 10% SP-2330 on 100/120 Chromosorb WAW (Supelco Inc., PA). A temperature gradient from 160-200°C was used.
Fatty acid composition of individual glycerophospholipids. Phosphatidylinositol (PI) and phosphatidylserine (PS) in the acidic fraction and phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the neutral fraction were isolated by thin-layer chromatography (silica gel 60 thinlayer plates, Merck, Darmstadt, F.R.G.). Chloroform:methanol:acetic acid:formic acid 50:30:4.5:6.5 was used as solvent system for acidic fractions and chloroform:methanol: water 65:25:4 for neutral fractions. The lipids were visualized by bromphenolblue reagent and the gel spots scraped. Fatty acid methyl esters from individual phospholipids were prepared by alkaline methanolysis as described above after drying in a vacuum desiccator. The fatty acid methyl esters were separated by GLC either by the method described above or on a capillary column (DB 225, 30 m, 0.25 mm ID from J&W Scientific Inc., CA). The temperature for capillary column separation was 225°C. The column used was not changed within a series of runs of ethanol-control pairs; furthermore, the two methods gave the same results.
Statistics The statistical significances of differences in values between ethanol and control groups were determined by the Student's two-tailed t-test. The nonparametric Wilcoxon test was used when the standard deviations in two compared groups differed significantly (p <0.01 determined by F-test). RESULTS
Concentrations of Lipids and Protein The concentrations of total phospholipids and cholesterol are presented in relation to the protein amount (Table 1). There were no significant differences in these lipid concentrations or in the phospholipid/cholesterol ratio between ethanol and control groups in different brain subcellular fractions or in liver.
Fatty Acid Composition of Total Glycerophospholipids The proportion of palmitic acid (16:0) was decreased and oleic acid (18:1) increased in the total glycerophospholipid
G L Y C E R O P H O S P H O L I P I D F ATTY ACIDS IN BRA I N
141
TABLE 1 CONCENTRATIONS OF LIPIDS IN DIFFERENT BRAIN SUBCELLULAR FRACTIONS AND LIVER TOTAL HOMOGENATE (nmol/mg PROTEIN) Phospholipids
Cholesterol
Phospholipids Cholesterol
Ethanol Synaptosomes Myelin Mitochondria Liver
572 ~ 33 1035 ~ 112 5 0 0 ± 77 235 ~ 13
Control 570 1025 492 234
± ± ± ±
41 82 72 17
Ethanol 275 1204 150 67
Control
± 32 ± 174 ± 28 ± 11
264± 1130 ± 154 ± 72 ±
Ethanol
22 83 34 6
2.10 ± 0.87 ± 3.402 3.59 ±
Control
0.23 0.07 0.58 0.45
2.17 0.92 3.24 3.25
± ± ± ±
0.16 0.08 0.32 0.23
The values are expressed as mean ± SD. None of the differences between ethanol and control groups were statistically significant when analysed by Student's t-test. (n= 12 in each group).
TABLE 2 FATTY ACID COMPOSITIONS OF TOTAL GLYCEROPHOSPHOLIPIDS IN DIFFERENT BRAIN SUBCELLULAR FRACTIONS FROM ETHANOL-TREATED RATS AND CONTROL RATS Synaptosomes Ethanol
Control
Myelin Ethanol
16:0 16:1 18:0 18:1 18:2 (n-6) 18:3 (n-3) 20:1 20:3 (n-6) 20:4 (n-6) 20:5 (n-3) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3)
27.2 0.9 21.5 17.1 0.7 0.1 0.5 0.4 11.7 0.1 3.1 0.5 0.1 16.0
± 1.40" ± 0.14 ___ 0.60t --- 0.51t _ 0.05 -+ 0.03 ± 0.10 _+ 0.10" --- 0.42 ___0.04 -+ 0.23 _+ 0.06 _+ 0.03 -+ 1.54
28.6 ± 1.18 0.8 ___ 0.06 20.8 --- 0.63 16.6 ± 0.25 0.7 --- 0.05 0.1 -+ 0.04 0.5 -+ 0.04 0.3 ± 0.06 11.7 _ 0.57 0.1 ± 0.09 3.1 _+ 0.19 0.6 ± 0.06 0.I -+ 0.03 15.9 --+ 0.75
19.7 0.8 21.7 30.9 0.7 0.6 2.9 1.0 8.7 0.4 3.8 0.5 0.2 7.9
± 1.49" -+ 0.10 ± 0.48 _ 0.85~t ± 0.06 _+ 0.07 ± 0.29* _+ 0.11t -+ 0.26 _ 0.17" ± 0.34 ± 0.06 _+ 0.03 _+ 0.66
% saturated % monounsaturated % polyunsaturated DBI/% saturated
48.7 _+ 1.69 18.5 _ 0.59t 33.9 -+ 2.70 3.71
49.3 _+ 1.22 17.9 ± 0.26 32.7 -+ 1.42 3.63
41.3 _+ 1.65" 34.7 ± 1.06~t 24.1 _+ 1.27 3.49
Mitochondria Control
21.7 0.8 21.9 29.3 0.8 0.5 2.6 0.8 8.9 0.2 3.6 0.5 0.1 7.9
_+ 1.97 ± 0.08 _+ 0.44 --- 1.12 ___0.06 -+ 0.08 _+ 0.29 ± 0.11 -+ 0.42 ___ 0.09 -+ 0.40 --- 0.09 _+ 0.03 ± 0.97
43.7 _+ 1.92 32.7 __+ 1.14 23.7 ± 1.73 3.23
Ethanol 22.2 1.2 20.7 19.1 1.6 0.1 0.4 0.5 14.1 2.4 0.5 0.1 16.7
-+ 1.30" _+ 0.09* --- 0.50 ± 0.81" ± 0.13 _+ 0.02 _ 0.03 -+ 0.0St _+ 0.64 -_+ 0.15 -+ 0.03 _+ 0.03 _+ 0.22*
42.9 _+ 1.50# 20.6 _+ 0.85 36.5 -+ 1.71" 4.56
Control 24.4 1.3 21.1 18.4 1.6 0.1 0.4 0.4 13.8
_ 2.55 ___0.18 ± 0.88 _+ 0.58 ___0.10 ± 0.01 ± 0.06 _+ 0.05 ± 0.71 _ 2.3 _+ 0.27 0.5 _+ 0.07 0.1 _+ 0.03 15.2 _+ 1.91
45.5 _+ 2.46 20.1 _+ 0.68 34.4 _+ 2.87 4.04
The values are expressed as mean _ SD (n=12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05 tp<0.01 *p<0.001. DBI: double bond index, which is the sum of the products of molar percentage and number of double bonds of the unsaturated fatty acids present.
f r a c t i o n a f t e r 3 w e e k s o f e t h a n o l e x p o s u r e (Table 2). T h e s e c h a n g e s w e r e c o n s i s t e n t l y f o u n d in all subcellular f r a c t i o n s f r o m forebrain. The s a t u r a t e d , m o n o u n s a t u r a t e d and p o l y u n s a t u r a t e d fatty acid totals w e r e c a l c u l a t e d in o r d e r to f o r m an overall v i e w o f the effect o f e t h a n o l o n p h o s p h o l i p i d fatty acid p r o p e r t i e s . T h e r e w a s a t r e n d in all the t i s s u e s t o w a r d s an i n c r e a s e in the d e g r e e o f u n s a t u r a t i o n after e t h a n o l e x p o s u r e (Table 2).
Fatty Acid Composition of Phosphatidylinositol (PI) Stearic (18:0) a n d a r a c h i d o n i c [20:4(n-6)] acids w e r e the d o m i n a t i n g fatty acids in p h o s p h a t i d y l i n o s i t o l (Fig. 1). T h r e e w e e k s o f e t h a n o l e x p o s u r e did not induce any significant
fatty acid c h a n g e s in PI in brain subcellular fractions (Fig. 1). O n the o t h e r h a n d , oleic (18:1) a n d linoleic acids [18:2(n-6)] w e r e i n c r e a s e d and palmitic (16:0) and a r a c h i d o n i c acids dec r e a s e d in liver (Fig. 1).
Fatty Acid Composition of Phosphatidylserine (PS) P h o s p h a t i d y l s e r i n e c o n t a i n e d high a m o u n t s o f stearic and d o c o s a h e x a e n o i c acids [22:6(n-3)] (Fig. 2). It is n o t e w o r t h y t h a t this p a t t e r n exists in b r a i n tissue but n o t in liver. T h e p r o p o r t i o n o f d o c o s a h e x a e n o i c acid w a s d e c r e a s e d in brain mitochondria and myelin after chronic ethanol exposure (Fig. 2). This finding w a s r e f l e c t e d in a significant d e c r e a s e in t h e d e g r e e o f p o l y u n s a t u r a t i o n in PS in t h e s e fractions. O n
142
GUSTAVSSON AND ALLING
,d O 50
SYNAPTOSOMES
P~
MITOCHONDRIA ]
Ethanol
]
Control
30'
_
16:0
O
50
MYELIN
180
18 1
18 2
2O 4
226
PI
160
18 0
16:0
18 0
18 1
18 2
20 4
22 6
5O
3O
30 •
• 16:0
18:0
18:1
18:2
20:4
22:6
181
182
204
226
FIG. 1. Fatty acid composition of phosphatidylinositol in different brain subceUular fractions and liver from ethanol-treated rats and controls. Each bar represents mean-S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
the other hand, docosahexaenoic acid was increased in liver PS (Fig. 2).
Fatty Acid Composition of Phosphatidylethanolamine (PE) Phosphatidylethanolarnine contained a high amount of polyunsaturated fatty acids including both arachidonic and docosahexaenoic acids. Few changes were seen in the fatty acid composition of PE in synaptosomes and mitochondria after ethanol exposure (Fig. 3). Arachidonic acid was decreased in synaptosomes and myelin after chronic ethanol exposure. In myelin, the major change was an increase in the proportion of oleic acid (18:1). In liver, oleic and docosahexaenoic acids were increased while palmitic and arachidonic acids were decreased.
Fatty Acid Composition of Phosphatidylcholine (PC) Phosphatidylcholine contained the highest proportion of
saturated fatty acids, and was especially enriched in palmitic acid compared to the other phospholipids (Fig. 4). The degree of unsaturation in PC was increased in synaptosomes and mitochondria after chronic ethanol exposure. Palmitic acid (16:0) was decreased and docosahexaenoic acid was increased in synaptosomes, mitochondria and liver (Fig. 4). DISCUSSION
Total Glycerophospholipids in Brain The fatty acid composition of total glycerophospholipid fraction was analysed in order to achieve an overall view. The major findings in this fraction were an increase in the monoene, oleic acid and a decrease in the saturate, palmitic acid after ethanol exposure. There was an increase in the degree of unsaturation in all the tissues analysed. The changes in fatty acid proportions of glycerophospholipids after chronic ethanol exposure did not differ essentially in
GLYCEROPHOSPHOLIPID FATTY ACIDS IN BRAIN
SYNAPTOSOMES
50
PS
]
Et.aoo,
]
Control
143
MITOCHONORIA
60
PS
30'
I0
10
16:0
18:0
18:1
18:2
MYELIN
50
20:4
16:0
22 6
PS
180
tS: 1
182
LIVER
50
20:4
22:6
PS
30
3O
10
r,r~,,~ -- I 16:0
18:0
l 18:1
10
18:2
20:4
22:6
***
tS:0
18:0
18:1
18:2
20:4
22:6
FIG. 2. Fatty acid compositionof phosphatidylserine in different brain subcellular fractions and liver from ethanoltreated rats and controls. Each bar represents mean_+S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
character between different subcellnlar fractions. It has been suggested that different cell membranes could respond in varying ways to ethanol treatment due to their different original lipid composition (31). This was obviously not the case in the present study. Myelin and synaptosomes which greatly differ from each other in their lipid compositions, reacted in the same way to the ethanol exposure. Increased proportions of oleic acid have been found in several studies on ethanol effects, although this phenomenon has not received much attention. Chronic ethanol treatment of rodents has been reported to induce increased proportions of oleic acid in synaptosomes, both in the total phospholipid fraction (16) and in phosphatidylcholine (5). In the latter study (5), oleic acid also tended to be increased in different phospholipids from myelin and mitochondria although the changes were not statistically significant. Enhanced proportions of oleic acid have also been reported to occur in peripheral tissues such as heart (20), liver (20,31), erythrocytes
(16,31) as well as in liver mitochondrial membranes (22,24) after ethanol exposure of rodents. Furthermore, an increase in oleic acid has been a consistent f'mding in different phospholipids in erythrocytes and platelets from chronic alcoholics (3,4). Only three studies have resulted in decreases (26) or no changes (8,25) in oleic acid proportions after chronic ethanol exposure. An increase in oleic acid is obviously one of the most general effects of chronic ethanol treatment on different membrane structures.
Individual Glycerophospholipids Only a few studies have been published where the fatty acid composition of PI and PS in rat brain subeellnlar fractions have been analysed separately after chronic ethanol exposure. Harris and co-workers (12) demonstrated decreased proportions of docosahexaenoic acid and an increase in palmitic acid in synaptosomal membrane PS after
144
GUSTAVSSON AND ALLING
5O
SYNAPTOSOMES
PE
]
El..ool
]
Control
5oI
MITOCHONDRIA
I J
! 30
18;0
18:0
181
162
204
22 6
i
I 16:0
18 0
~81
204
226
.,J 0 ~50
MYELIN
t IVER
PE
PE
30
16:0
18:0
18:1
18:2
20:4
22:6
16:0
18:O
18:1
18:2
i 204
22:6
FIG. 3. Fatty acid composition of phosphatidylethanolamine in different brain subcellular fractions and liver from ethanol-treated rats and controls. Each bar represents mean-+S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
chronic ethanol exposure to mice. The fmdings in the present study extend these changes to include also brain mitochondria and myelin. In the present study ethanol induced opposite changes in the fatty acid composition of PS and PC. Decreased proportions of polyunsaturated fatty acids were found in PS. In brain this was most obvious for docosahexaenoic acid. On the other hand, the degree of unsaturation in PC was increased. The observation that fatty acid changes after ethanol exposure have opposite character in different phospholipids have previously been reported from studies on erythrocyte membranes (30). The decreased amount of polyunsaturated fatty acids found in that study was mainly located to phospholipids with high degree of unsaturation, namely PI+PS and PE.
Pathophysiological Considerations The different effects of ethanol exposure on PS fatty acids
compared to PC point to the high complexity of ethanol interactions both with the membrane itself as well as effects on metabolic processes. The final pattern of changes is probably produced by different mechanisms. In the present study rats were pair-fed with an isocaloric diet in order to exclude changes due to differences in dietary intake of essential fatty acids. Contradictory results from different studies on ethanol effects on brain membrane lipids might partly del~nd on different dietary regimens. Thus, studies reporting a decrease in polyunsaturated fatty acid content in total phospholipids (20,31) or phosphatidylcholine and phosphatidylethanolamine (5) did not include pairfeeding of control rats or isocaloric compensation for ethanol intake. In contrast to dietary effects, a change in fatty acid supply to the brain due to changes in liver lipid metabolism and subsequent transport to brain cannot be excluded as a cause of the changes in the total glycerophospholipid pool or PC in the present study. However, the ethanol-induced de-
G L Y C E R O P H O S P H O L I P I D FATTY ACIDS IN BRAIN
145
4 SYNAPTOSOMES
50"
PC
]
Ethanol
]
Control
MITOCHONDRIA
50"
PC
30
3O
10 -
10 -
I 16:0
18:0
18:1
18:2
20:4
MYELIN
50
22:6
PC
16:0
180
181
18:2
LIVER
50
m 204
22:6
PC
30
30
101
16:0
18:0
18:1
18:2
20:4
22:6
ili 16:0
18:0
18:1
18:2
20:4
22:6
FIG. 4. Fatty acid composition of phosphatidylcholine in different brain subceUular fractions and liver from ethanol-treated rats and controls. Each bar represents mean_+S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
crease in brain PS docosahexaenoic acid could not have been caused by this mechanism since liver fatty acids showed no decrease in docosahexaenoic acid. This change in PS must therefore have been formed by another mechanism. It has been claimed that cell membranes adapt their lipid composition in order to counteract the fluidizing effect of ethanol. The decrease in polyunsaturated fatty acids in PS could be explained by such a hypothesis. Other compositional changes in the present study point in an opposite direction. The PC fatty acid compositions were changed in the direction of a higher degree of unsaturation and cholesterol concentrations were normal after ethanol exposure. This is contrary to what could be assumed if an antifluidization hypothesis would be valid for bulk lipids. However, the interaction between ethanol and membrane lipids is more complex than just a fluidization (9, 14, 27). The increased degree of unsaturation found in PC in the present study could be an adaptation to restore the molecular form and membrane packing properties of phospholipids after surface ex-
pansion by ethanol (29). These possibilities need to be further studied before a conclusion about etiologic mechanisms can be made. Taken together, the present results indicate that chronic ethanol treatment induces changes in the fatty acid composition of rat brain subcellular fractions. Oleic acid was increased and palmitic acid was decreased in total glycerophospholipid fraction. The fatty acid compositions of PC and PS were changed in different directions. This points to the high complexity of the action of ethanol on cell membranes. The observed deviations in lipids from their normal composition are probably of importance for membrane function. ACKNOWLEDGEMENTS This study was supported by grants from The Swedish Medical Research Council (project No. 05249), The Bank of Sweden Tercentenary Foundation and The Medical Faculty of Lund University. Mrs. Berit F~rjh is gratefully acknowledged for her excellent technical assistance.
146
GUSTAVSSON AND ALLING REFERENCES
1. Alling, C.; Becker, W.; Jones, A. W.; Angg~trd, E. Effects of chronic ethanol treatment on lipid composition and prostaglandins in rats fed essential fatty acid deficient diets. Alcohol. Clin. Exp. Res. 8:238-242; 1984. 2. Ailing, C.; Bruce, A.; Karlsson, I.; Sapia, O.; Svennerholm, L. Effect of maternal essential fatty acid composition of brain, liver, muscles and serum in 21-day-old rats. J. Nutr. 102:773782; 1972. 3. Alling, C.; Gustavsson, L.; Kristensson-Aas, A.; Wallerstedt, S. Changes in fatty acid composition of major glycerophospholipids in erythrocyte membranes from chronic alcoholics during withdrawal. Scand. J. Clin. Lab. Invest. 44:283-289; 1984. 4. Ailing, C.; J6nsson, G.; Gustavsson, L.; Jensen, L.; Simonsson, P. Anionic glycerophospholipids in platelets from alcoholics. Drug Alcohol Depend. 16:309-320; 1986. 5. Ailing, C.; Liljequist, S.; Engel, J. The effect of chronic ethanol administration on lipids and fatty acids in subcellular fractions of rat brain. Med. Biol. 60:149-154; 1982. 6. Bartlett, G. R. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466--468; 1959. 7. Chin, J. H.; Parsons, L. M.; Goldstein, D. B. Increased cholesterol content of erythrocyte and brain membranes in ethanoltolerant mice. Biochim. Biophys. Acta 513:358-363; 1978. 8. Crews, F. T.; Majchrowicz, E.; Meeks, R. Changes in cortical synaptosomal plasma membrane fluidity and composition in ethanol dependent rats. Psychopharmacoiogy 81:208-213; 1983. 9. Cullis, P. R.; Hornby, A. P.; Hope, M. J. Effects of anesthetics on lipid polymorphism. In: Fink, B. R., ed. Molecular mechanisms of anesthesia. Progress in anesthesiology, vol 2. New York: Raven Press; 1980:397-403. 10. DeCarli, L. M.; Lieber, C. S. Fatty liver in the rat after prolonged intake of ethanol with a nutritionally adequate new liquid diet. J. Nutr. 91:331-336; 1967. 11. Franey, R. J.; Amador, E. Serum cholesterol measurement based on ethanol extraction and ferric chloride-sulfuric acid. Clin. Chim. Acta 21:255-263; 1968. 12. Harris, R. A.; Baxter, D. M.; Mitchell, M. A.; Hitzemann, R. J. Physical properties and lipid composition of brain membranes from ethanol tolerant-dependent mice. Mol. Pharmacol. 25:401--409; 1984. 13. Hartree, E. F. Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48:422.-427; 1972. 14. Hitzemann, R. J.; Schueler, H. E.; Graham-Brittain, C.; Kreishman, G. P. Ethanol-induced changes in neuronal membrane order. An NMR study. Biochim. Biophys. Acta 859:189197; 1986. 15. Kyrklund, T.; Ailing, C.; Haglid, K.; Kjellstrand, P. Chronic exposure to trichloroethylene: Lipid and acyl group composition in gerbil cerebral cortex and hippocampus. Neurutoxicology 4:35--42; 1983. 16. La Droitte, P.; Lamboeuf, Y.; De Saint Blanquat, G. Ethanol sensitivity and membrane lipid composition in three strains of mouse. Comp. Bioehem. Physiol. [C] 77:351-356; 1984.
17. Le Bourhis, B.; Beaug6, F.; Aufr~re, G.; Nordmann, R. Membrane fluidity and alcohol dependence. Alcohol.: Clin. Exp. Res. 10:337-342; 1986. 18. Lieber, C. S.; DeCadi, L. M. The feeding of alcohol in liquid diets: Two decades of applications and 1982 update. Alcohol.: Clin. Exp. Res. 6:523-531; 1982. 19. Littleton, J. M.; John, G. R. Synaptosomal membrane lipids of mice during continuous exposure to ethanol. J. Pharm. Pharmacol. 29:579--580; 1977. 20. Littleton, J. M.; John, G. R.; Grieve, S. J. Alterations in phospholipid composition in ethanol tolerance and dependence. Alcohol.: Clin. Exp. Res. 3:50-56; 1979. 21. Lyon, R. C.; Goldstein, D. B. Changes in synaptic membrane order associated with chronic ethanol treatment in mice. Mol. Pharmacol. 23:86-91; 1983. 22. Marco, C.; Ceacero, F.; Garcia-Peregrin, E.; Segovia, J. L. Ethanol sensitivity and fatty acid composition of chick liver mitochondrial and microsomal membranes. Biochem. Int. 11:291-299; 1985. 23. Nilsson, O.; Fredman, P.; Klinghardt, G. W.; Dreyfus, H.; Svennerholm, L. Chioroquine-induced accumulation of gangliosides and phospholipids in skeletal muscles. Eur. J. Biochem. 116:565-571; 1981. 24. Rouach, H.; Clement, M.; Orfanelli, M.-T.; Janvier, B.; Nordmann, R. Fatty acid composition of rat liver mitochondrial phospholipids during ethanol inhalation. Biochim. Biophys. Acta 795:125-129; 1984. 25. Smith, T. L.; Gerhart, M. J. Alterations in brain lipid composition of mice made physically dependent to ethanol. Life Sci. 31:1419-1425; 1982. 26. Sun, G. Y.; Sun, A. Y. Effect of chronic ethanol administration on phospholipid acyl groups of synaptic plasma membrane fraction isolated from guinea pig brain. Res. Commun. Chem. Pathol. Pharmacol. 24:405-408; 1979. 27. Tilcock, C. P. S.; Cullis, P. R. Lipid polymorphism. In: Rubin, E., ed. Alcohol and the cell. New York: The New York Academy of Sciences; 1987:88-101. (Ann. NY Acad. Sci., vol. 492.) 28. Whittaker, V. P.; Barker, L. A. The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In: Fried, R., ed. Methods in neurochemistry, vol. 2. New York: Marcel Dekker. 1972:1-52. 29. Wieslander, A.; Rilfors, L.; Lindblom, G. Metabolic changes of membrane lipid composition in Acholeplasma laidlawii by hydrocarbons, alcohols and detergents: Arguments for effects on lipid packing. Biochemistry 25:7511-7517; 1986. 30. Wing, D. R.; Harvey, D. J.; Belcher, S. J.; Paton, W. D. M. Changes in membrane lipid content after chronic ethanol administration with respect to fatty acyl compositions and phospholipid type. Biochem. Pharmacol. 33:1625-1632; 1984. 31. Wing, D. R.; Harvey, D. J.; Hughes, J.; Dunbar, P. G. McPherson, K. A.; Paton, W. D. M. Effects of chronic ethanol administration on the composition of membrane lipids in the mouse. Biochem. Pharmacoi. 31:3431-3139; 1982.
0741-8329/89$3.00 + .00
Effects of Chronic Ethanol Exposure on Fatty Acids of Rat Brain Glycerophospholipids LENA GUSTAVSSON AND CHRISTER ALLING
Department of Psychiatry and Neurochemistry, University of Lund P.O. Box 638, S-220 06 Lund, Sweden. R e c e i v e d 22 A u g u s t 1988; A c c e p t e d 1 D e c e m b e r 1988 GUSTAVSSON, L. AND C. ALLING. Effects of chronic ethanol exposure on fatty acids of rat brain glycerophospholipids. ALCOHOL 6(2) 139-146, 1989.--The lipid composition was analysed in forebrain subcellular fractions from rats treated with ethanol for three weeks and control rats. Increased proportions of oleic acid and a decrease in palmitic acid were consistently found in total glycerophospholipid fractions after ethanol exposure. The fatty acid compositions of individual phospholipids were also significantly changed. The proportion of docosahexaenoic acid was decreased in brain phosphatidylserine. In contrast to the decrease in the degree of unsaturation in phosphatidylserine, there was an opposite change in phosphatidylcholine wherein the degree of unsaturation was increased. No changes were produced in total cholesterol or phospholipid concentrations. These results point to a high degree of complexity of the mechanisms behind ethanol-induced changes in membrane lipid composition, The decrease in unsaturation in phosphatidylserine is probably an adaptive effect in order to counteract the fluidizing effect of ethanol. There are two possible explanations for the increase in unsaturation in brain phosphatidylcholine. The change may be due to adaptation to other biophysical effects, e.g., expansion of the membrane surface or be secondary to a change in liver lipid metabolism. Brain
Cell membranes
Ethanol
Fatty acids
Glycerophospholipids
ever, no conclusive results supporting this hypothesis have emerged. Inconsistent results have been reported on concentrations of cholesterol. Both increased concentrations (7,8) as well as unaltered (5) and decreased amounts (12) have been reported from studies on brain membranes after chronic ethanol exposure. Decreased proportions of polyunsaturated fatty acids in phospholipids have been reported to occur in synaptosomes from ethanol tolerant mice (19,20). Another study has indicated results in the same direction in liver and erythrocyte membranes, but no changes were found in synaptosomal plasma membranes (31). In contrast, chronic ethanol treatment of guinea pigs resulted in increased proportions of polyunsaturated fatty acids in phospholipids from synaptosomal membrane fraction (26). Other investigations have not found any differences in brain membranes after chronic ethanol exposure (8,25). Thus, our knowledge about the effect of ethanol on cell membrane lipids is incomplete. It is possible that different membrane structures as well as different lipids respond to ethanol in different ways. Even if changes in lipid composition in brain membranes are small they could have considerable consequences for membrane function. Brain lipid composition is relatively inert to changes in the environment compared to other organs, a fact that has been shown in studies of dietary effects (2) and toxic influences of organic solvents (15). It has been suggested that the vulnerability of
IT has been suggested that cell membranes adapt to the perturbing effect of ethanol during chronic exposure of the drug. Fluidization of membranes by ethanol and tolerance to this disordering effect have been measured by EPR and fluorescerise polarisation techniques (12, 17, 21). Recent NMRstudies have demonstrated that ethanol at low concentrations ordered the polar head group region of phosphatidylcholine in synaptosomal plasma membranes (14). The main effect at higher concentrations was a disordering of the membrane core. In addition to the effect on membrane fluidity, ethanol changed the polymorphic phase behaviour of membrane lipids (9,27). This phenomenon may be related to expansion of the membrane in the polar head group region, and consequently to a change in the molecular form ofphospholipids (27,29). This variety of effects points to the high complexity of ethanol-membrane interactions. Much of the attention in alcohol research on membrane lipids has been focused on ethanol-induced chronic changes in the lipid composition that could explain an adaptation to the presence of ethanol. The glycerophospholipids in particular, have been in focus due to their properties which make them important for regulation of membrane fluidity and other biophysical characteristics. Examples of such properties are high proportions of polyunsaturated fatty acids and fast turnover. Adaptation to a fluidizing effect includes a decrease in the degree of unsaturation in fatty acids of phospholipids and increased concentrations of cholesterol. How-
139
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GUSTAVSSON AND ALLING
brain for dietary changes is increased by ethanol (1). The aim of this study was to elucidate the effect of three weeks of ethanol intoxication on membrane glycerophospholipid fatty acid composition of rat brain in an experiment where the dietary intake of nutrients including essential fatty acids was well controlled. METHOD
Experimental Procedure Male Sprague-Dawley rats (n= 12) were given ethanol in the form of a liquid diet (Liquidiet ®, Rat Diet Lieber DeCarli Formula, BioServ Inc., N J) for three weeks. Control rats (n= 12) were pair-fed with an isocaloric diet. The procedure was performed as described by Lieber and DeCarli (10,18). The liquid diet was the only source of food and water. The amount of ethanol in the diet was increased from 1.5% (w/v) to a final concentration of 5% (w/v) at day 8. The rats weighed 136-+21 g at the beginning of the experiment (range 114-161). The weight gain after three weeks was 64___7.8 g in the ethanol-treated group and 81_+13 in the control group. The blood ethanol concentration was 67.2-+12.5 mmol/1 at the day of decapitation. The daily ethanol intake increased constantly from 9.3_+1.63 g ethanol/kg body weight at the first day of administration to 14.2_+2.09 at day 21. While intoxicated by ethanol, the animals were decapitated under ether anesthesia. Forebrain and liver were dissected immediately after decapitation. Subcellular fractions were prepared from forebrain. The tissues were stored at -70°C until analysis.
Isolation of Subcellular Fractions Synaptosomes, myelin and mitochondria were isolated from forebrains (28). The tissue was homogenized in 0.32 M sucrose in an all-glass homogenizer. Two ml of this total homogenate were taken for lipid analyses. The rest was used for isolation of subcellular fractions. This homogenate was centrifuged for 10 minutes at 1000xg. The pellet was resuspended in 0.32 M sucrose and centrifuged as above. The combined supernatants were centrifuged at 17500×g for 1 hour. The resulting pellet was suspended in 0.32 M sucrose and this homogenate was layered on the top of a sucrose density gradient. The discontinuous gradient consisted of 1.2 M sucrose, 0.8 M sucrose and 0.32 M sucrose. The gradient was centrifuged at 50000×g for 2 hours. The fractions enriched in myelin (interface between 0.32 M and 0.8 M sucrose) and synaptosomes (interface between 0.8 M and 1.2 M sucrose) were collected using a Pasteur pipette. The collected volumes were equal in all samples. The mitochondrial fraction (pellet) was suspended in 0.156 M KC1. All subcellular fractions, as" well as the total homogenate, were purified from the sucrose by suspension in 40 ml 0.156 M KCI and centrifugation at 17500×g for 30 minutes. This washing procedure was performed three times. The fractions were finally suspended in 0.156 M KCI. A portion was taken from the samples for protein analyses. All samples were kept at -70°C until analyses. The purity of subcellular fractions was evaluated by electron microscopy.
Biochemical Analyses Lipid extraction. Tissues from different organs were homogenized with all-glass homogenizers to make a 12.5% homogenate in water, except for the subcellular fractions
which were homogenized in one ml of 0.156 M KCI. Samples from the homogenate were taken for protein analyses. The tissue homogenates were extracted as described previously (4). The total lipid extract were separated into a neutral and an acidic fraction by ion-exchange chromatography (23). Quantitative analyses. Lipid phosphorus (6) and cholesterol (11) concentrations in lipid extracts were quantitated by colorimetric methods. The amounts of protein were determined on samples from tissue homogenates (13).
Fatty acid composition of total glycerophospholipids. Fatty acid methyl esters were prepared by alkaline methanolysis of the total lipid fraction. The methanolysis was performed in 0.2 M sodium methoxide in methanol at 37°C for 1 hour. The reaction was stopped by addition of 0.1 M acetic acid. The fatty acid methyl esters were extracted by light petroleum and purified by preparative thin layer chromatography. Dichloromethane was used as the solvent system. The fatty acid methyl esters were detected by bromphenolblue reagent and scraped into methanol. They were extracted by light petroleum. The composition of fatty acid methyl esters was determined by GLC on a packed column (2 m, 2 mm ID) with 10% SP-2330 on 100/120 Chromosorb WAW (Supelco Inc., PA). A temperature gradient from 160-200°C was used.
Fatty acid composition of individual glycerophospholipids. Phosphatidylinositol (PI) and phosphatidylserine (PS) in the acidic fraction and phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the neutral fraction were isolated by thin-layer chromatography (silica gel 60 thinlayer plates, Merck, Darmstadt, F.R.G.). Chloroform:methanol:acetic acid:formic acid 50:30:4.5:6.5 was used as solvent system for acidic fractions and chloroform:methanol: water 65:25:4 for neutral fractions. The lipids were visualized by bromphenolblue reagent and the gel spots scraped. Fatty acid methyl esters from individual phospholipids were prepared by alkaline methanolysis as described above after drying in a vacuum desiccator. The fatty acid methyl esters were separated by GLC either by the method described above or on a capillary column (DB 225, 30 m, 0.25 mm ID from J&W Scientific Inc., CA). The temperature for capillary column separation was 225°C. The column used was not changed within a series of runs of ethanol-control pairs; furthermore, the two methods gave the same results.
Statistics The statistical significances of differences in values between ethanol and control groups were determined by the Student's two-tailed t-test. The nonparametric Wilcoxon test was used when the standard deviations in two compared groups differed significantly (p <0.01 determined by F-test). RESULTS
Concentrations of Lipids and Protein The concentrations of total phospholipids and cholesterol are presented in relation to the protein amount (Table 1). There were no significant differences in these lipid concentrations or in the phospholipid/cholesterol ratio between ethanol and control groups in different brain subcellular fractions or in liver.
Fatty Acid Composition of Total Glycerophospholipids The proportion of palmitic acid (16:0) was decreased and oleic acid (18:1) increased in the total glycerophospholipid
G L Y C E R O P H O S P H O L I P I D F ATTY ACIDS IN BRA I N
141
TABLE 1 CONCENTRATIONS OF LIPIDS IN DIFFERENT BRAIN SUBCELLULAR FRACTIONS AND LIVER TOTAL HOMOGENATE (nmol/mg PROTEIN) Phospholipids
Cholesterol
Phospholipids Cholesterol
Ethanol Synaptosomes Myelin Mitochondria Liver
572 ~ 33 1035 ~ 112 5 0 0 ± 77 235 ~ 13
Control 570 1025 492 234
± ± ± ±
41 82 72 17
Ethanol 275 1204 150 67
Control
± 32 ± 174 ± 28 ± 11
264± 1130 ± 154 ± 72 ±
Ethanol
22 83 34 6
2.10 ± 0.87 ± 3.402 3.59 ±
Control
0.23 0.07 0.58 0.45
2.17 0.92 3.24 3.25
± ± ± ±
0.16 0.08 0.32 0.23
The values are expressed as mean ± SD. None of the differences between ethanol and control groups were statistically significant when analysed by Student's t-test. (n= 12 in each group).
TABLE 2 FATTY ACID COMPOSITIONS OF TOTAL GLYCEROPHOSPHOLIPIDS IN DIFFERENT BRAIN SUBCELLULAR FRACTIONS FROM ETHANOL-TREATED RATS AND CONTROL RATS Synaptosomes Ethanol
Control
Myelin Ethanol
16:0 16:1 18:0 18:1 18:2 (n-6) 18:3 (n-3) 20:1 20:3 (n-6) 20:4 (n-6) 20:5 (n-3) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3)
27.2 0.9 21.5 17.1 0.7 0.1 0.5 0.4 11.7 0.1 3.1 0.5 0.1 16.0
± 1.40" ± 0.14 ___ 0.60t --- 0.51t _ 0.05 -+ 0.03 ± 0.10 _+ 0.10" --- 0.42 ___0.04 -+ 0.23 _+ 0.06 _+ 0.03 -+ 1.54
28.6 ± 1.18 0.8 ___ 0.06 20.8 --- 0.63 16.6 ± 0.25 0.7 --- 0.05 0.1 -+ 0.04 0.5 -+ 0.04 0.3 ± 0.06 11.7 _ 0.57 0.1 ± 0.09 3.1 _+ 0.19 0.6 ± 0.06 0.I -+ 0.03 15.9 --+ 0.75
19.7 0.8 21.7 30.9 0.7 0.6 2.9 1.0 8.7 0.4 3.8 0.5 0.2 7.9
± 1.49" -+ 0.10 ± 0.48 _ 0.85~t ± 0.06 _+ 0.07 ± 0.29* _+ 0.11t -+ 0.26 _ 0.17" ± 0.34 ± 0.06 _+ 0.03 _+ 0.66
% saturated % monounsaturated % polyunsaturated DBI/% saturated
48.7 _+ 1.69 18.5 _ 0.59t 33.9 -+ 2.70 3.71
49.3 _+ 1.22 17.9 ± 0.26 32.7 -+ 1.42 3.63
41.3 _+ 1.65" 34.7 ± 1.06~t 24.1 _+ 1.27 3.49
Mitochondria Control
21.7 0.8 21.9 29.3 0.8 0.5 2.6 0.8 8.9 0.2 3.6 0.5 0.1 7.9
_+ 1.97 ± 0.08 _+ 0.44 --- 1.12 ___0.06 -+ 0.08 _+ 0.29 ± 0.11 -+ 0.42 ___ 0.09 -+ 0.40 --- 0.09 _+ 0.03 ± 0.97
43.7 _+ 1.92 32.7 __+ 1.14 23.7 ± 1.73 3.23
Ethanol 22.2 1.2 20.7 19.1 1.6 0.1 0.4 0.5 14.1 2.4 0.5 0.1 16.7
-+ 1.30" _+ 0.09* --- 0.50 ± 0.81" ± 0.13 _+ 0.02 _ 0.03 -+ 0.0St _+ 0.64 -_+ 0.15 -+ 0.03 _+ 0.03 _+ 0.22*
42.9 _+ 1.50# 20.6 _+ 0.85 36.5 -+ 1.71" 4.56
Control 24.4 1.3 21.1 18.4 1.6 0.1 0.4 0.4 13.8
_ 2.55 ___0.18 ± 0.88 _+ 0.58 ___0.10 ± 0.01 ± 0.06 _+ 0.05 ± 0.71 _ 2.3 _+ 0.27 0.5 _+ 0.07 0.1 _+ 0.03 15.2 _+ 1.91
45.5 _+ 2.46 20.1 _+ 0.68 34.4 _+ 2.87 4.04
The values are expressed as mean _ SD (n=12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05 tp<0.01 *p<0.001. DBI: double bond index, which is the sum of the products of molar percentage and number of double bonds of the unsaturated fatty acids present.
f r a c t i o n a f t e r 3 w e e k s o f e t h a n o l e x p o s u r e (Table 2). T h e s e c h a n g e s w e r e c o n s i s t e n t l y f o u n d in all subcellular f r a c t i o n s f r o m forebrain. The s a t u r a t e d , m o n o u n s a t u r a t e d and p o l y u n s a t u r a t e d fatty acid totals w e r e c a l c u l a t e d in o r d e r to f o r m an overall v i e w o f the effect o f e t h a n o l o n p h o s p h o l i p i d fatty acid p r o p e r t i e s . T h e r e w a s a t r e n d in all the t i s s u e s t o w a r d s an i n c r e a s e in the d e g r e e o f u n s a t u r a t i o n after e t h a n o l e x p o s u r e (Table 2).
Fatty Acid Composition of Phosphatidylinositol (PI) Stearic (18:0) a n d a r a c h i d o n i c [20:4(n-6)] acids w e r e the d o m i n a t i n g fatty acids in p h o s p h a t i d y l i n o s i t o l (Fig. 1). T h r e e w e e k s o f e t h a n o l e x p o s u r e did not induce any significant
fatty acid c h a n g e s in PI in brain subcellular fractions (Fig. 1). O n the o t h e r h a n d , oleic (18:1) a n d linoleic acids [18:2(n-6)] w e r e i n c r e a s e d and palmitic (16:0) and a r a c h i d o n i c acids dec r e a s e d in liver (Fig. 1).
Fatty Acid Composition of Phosphatidylserine (PS) P h o s p h a t i d y l s e r i n e c o n t a i n e d high a m o u n t s o f stearic and d o c o s a h e x a e n o i c acids [22:6(n-3)] (Fig. 2). It is n o t e w o r t h y t h a t this p a t t e r n exists in b r a i n tissue but n o t in liver. T h e p r o p o r t i o n o f d o c o s a h e x a e n o i c acid w a s d e c r e a s e d in brain mitochondria and myelin after chronic ethanol exposure (Fig. 2). This finding w a s r e f l e c t e d in a significant d e c r e a s e in t h e d e g r e e o f p o l y u n s a t u r a t i o n in PS in t h e s e fractions. O n
142
GUSTAVSSON AND ALLING
,d O 50
SYNAPTOSOMES
P~
MITOCHONDRIA ]
Ethanol
]
Control
30'
_
16:0
O
50
MYELIN
180
18 1
18 2
2O 4
226
PI
160
18 0
16:0
18 0
18 1
18 2
20 4
22 6
5O
3O
30 •
• 16:0
18:0
18:1
18:2
20:4
22:6
181
182
204
226
FIG. 1. Fatty acid composition of phosphatidylinositol in different brain subceUular fractions and liver from ethanol-treated rats and controls. Each bar represents mean-S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
the other hand, docosahexaenoic acid was increased in liver PS (Fig. 2).
Fatty Acid Composition of Phosphatidylethanolamine (PE) Phosphatidylethanolarnine contained a high amount of polyunsaturated fatty acids including both arachidonic and docosahexaenoic acids. Few changes were seen in the fatty acid composition of PE in synaptosomes and mitochondria after ethanol exposure (Fig. 3). Arachidonic acid was decreased in synaptosomes and myelin after chronic ethanol exposure. In myelin, the major change was an increase in the proportion of oleic acid (18:1). In liver, oleic and docosahexaenoic acids were increased while palmitic and arachidonic acids were decreased.
Fatty Acid Composition of Phosphatidylcholine (PC) Phosphatidylcholine contained the highest proportion of
saturated fatty acids, and was especially enriched in palmitic acid compared to the other phospholipids (Fig. 4). The degree of unsaturation in PC was increased in synaptosomes and mitochondria after chronic ethanol exposure. Palmitic acid (16:0) was decreased and docosahexaenoic acid was increased in synaptosomes, mitochondria and liver (Fig. 4). DISCUSSION
Total Glycerophospholipids in Brain The fatty acid composition of total glycerophospholipid fraction was analysed in order to achieve an overall view. The major findings in this fraction were an increase in the monoene, oleic acid and a decrease in the saturate, palmitic acid after ethanol exposure. There was an increase in the degree of unsaturation in all the tissues analysed. The changes in fatty acid proportions of glycerophospholipids after chronic ethanol exposure did not differ essentially in
GLYCEROPHOSPHOLIPID FATTY ACIDS IN BRAIN
SYNAPTOSOMES
50
PS
]
Et.aoo,
]
Control
143
MITOCHONORIA
60
PS
30'
I0
10
16:0
18:0
18:1
18:2
MYELIN
50
20:4
16:0
22 6
PS
180
tS: 1
182
LIVER
50
20:4
22:6
PS
30
3O
10
r,r~,,~ -- I 16:0
18:0
l 18:1
10
18:2
20:4
22:6
***
tS:0
18:0
18:1
18:2
20:4
22:6
FIG. 2. Fatty acid compositionof phosphatidylserine in different brain subcellular fractions and liver from ethanoltreated rats and controls. Each bar represents mean_+S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
character between different subcellnlar fractions. It has been suggested that different cell membranes could respond in varying ways to ethanol treatment due to their different original lipid composition (31). This was obviously not the case in the present study. Myelin and synaptosomes which greatly differ from each other in their lipid compositions, reacted in the same way to the ethanol exposure. Increased proportions of oleic acid have been found in several studies on ethanol effects, although this phenomenon has not received much attention. Chronic ethanol treatment of rodents has been reported to induce increased proportions of oleic acid in synaptosomes, both in the total phospholipid fraction (16) and in phosphatidylcholine (5). In the latter study (5), oleic acid also tended to be increased in different phospholipids from myelin and mitochondria although the changes were not statistically significant. Enhanced proportions of oleic acid have also been reported to occur in peripheral tissues such as heart (20), liver (20,31), erythrocytes
(16,31) as well as in liver mitochondrial membranes (22,24) after ethanol exposure of rodents. Furthermore, an increase in oleic acid has been a consistent f'mding in different phospholipids in erythrocytes and platelets from chronic alcoholics (3,4). Only three studies have resulted in decreases (26) or no changes (8,25) in oleic acid proportions after chronic ethanol exposure. An increase in oleic acid is obviously one of the most general effects of chronic ethanol treatment on different membrane structures.
Individual Glycerophospholipids Only a few studies have been published where the fatty acid composition of PI and PS in rat brain subeellnlar fractions have been analysed separately after chronic ethanol exposure. Harris and co-workers (12) demonstrated decreased proportions of docosahexaenoic acid and an increase in palmitic acid in synaptosomal membrane PS after
144
GUSTAVSSON AND ALLING
5O
SYNAPTOSOMES
PE
]
El..ool
]
Control
5oI
MITOCHONDRIA
I J
! 30
18;0
18:0
181
162
204
22 6
i
I 16:0
18 0
~81
204
226
.,J 0 ~50
MYELIN
t IVER
PE
PE
30
16:0
18:0
18:1
18:2
20:4
22:6
16:0
18:O
18:1
18:2
i 204
22:6
FIG. 3. Fatty acid composition of phosphatidylethanolamine in different brain subcellular fractions and liver from ethanol-treated rats and controls. Each bar represents mean-+S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
chronic ethanol exposure to mice. The fmdings in the present study extend these changes to include also brain mitochondria and myelin. In the present study ethanol induced opposite changes in the fatty acid composition of PS and PC. Decreased proportions of polyunsaturated fatty acids were found in PS. In brain this was most obvious for docosahexaenoic acid. On the other hand, the degree of unsaturation in PC was increased. The observation that fatty acid changes after ethanol exposure have opposite character in different phospholipids have previously been reported from studies on erythrocyte membranes (30). The decreased amount of polyunsaturated fatty acids found in that study was mainly located to phospholipids with high degree of unsaturation, namely PI+PS and PE.
Pathophysiological Considerations The different effects of ethanol exposure on PS fatty acids
compared to PC point to the high complexity of ethanol interactions both with the membrane itself as well as effects on metabolic processes. The final pattern of changes is probably produced by different mechanisms. In the present study rats were pair-fed with an isocaloric diet in order to exclude changes due to differences in dietary intake of essential fatty acids. Contradictory results from different studies on ethanol effects on brain membrane lipids might partly del~nd on different dietary regimens. Thus, studies reporting a decrease in polyunsaturated fatty acid content in total phospholipids (20,31) or phosphatidylcholine and phosphatidylethanolamine (5) did not include pairfeeding of control rats or isocaloric compensation for ethanol intake. In contrast to dietary effects, a change in fatty acid supply to the brain due to changes in liver lipid metabolism and subsequent transport to brain cannot be excluded as a cause of the changes in the total glycerophospholipid pool or PC in the present study. However, the ethanol-induced de-
G L Y C E R O P H O S P H O L I P I D FATTY ACIDS IN BRAIN
145
4 SYNAPTOSOMES
50"
PC
]
Ethanol
]
Control
MITOCHONDRIA
50"
PC
30
3O
10 -
10 -
I 16:0
18:0
18:1
18:2
20:4
MYELIN
50
22:6
PC
16:0
180
181
18:2
LIVER
50
m 204
22:6
PC
30
30
101
16:0
18:0
18:1
18:2
20:4
22:6
ili 16:0
18:0
18:1
18:2
20:4
22:6
FIG. 4. Fatty acid composition of phosphatidylcholine in different brain subceUular fractions and liver from ethanol-treated rats and controls. Each bar represents mean_+S.D. (n= 12 in each group). Statistical significances of differences between ethanol and control groups: *p<0.05, **p<0.01, ***p<0.001.
crease in brain PS docosahexaenoic acid could not have been caused by this mechanism since liver fatty acids showed no decrease in docosahexaenoic acid. This change in PS must therefore have been formed by another mechanism. It has been claimed that cell membranes adapt their lipid composition in order to counteract the fluidizing effect of ethanol. The decrease in polyunsaturated fatty acids in PS could be explained by such a hypothesis. Other compositional changes in the present study point in an opposite direction. The PC fatty acid compositions were changed in the direction of a higher degree of unsaturation and cholesterol concentrations were normal after ethanol exposure. This is contrary to what could be assumed if an antifluidization hypothesis would be valid for bulk lipids. However, the interaction between ethanol and membrane lipids is more complex than just a fluidization (9, 14, 27). The increased degree of unsaturation found in PC in the present study could be an adaptation to restore the molecular form and membrane packing properties of phospholipids after surface ex-
pansion by ethanol (29). These possibilities need to be further studied before a conclusion about etiologic mechanisms can be made. Taken together, the present results indicate that chronic ethanol treatment induces changes in the fatty acid composition of rat brain subcellular fractions. Oleic acid was increased and palmitic acid was decreased in total glycerophospholipid fraction. The fatty acid compositions of PC and PS were changed in different directions. This points to the high complexity of the action of ethanol on cell membranes. The observed deviations in lipids from their normal composition are probably of importance for membrane function. ACKNOWLEDGEMENTS This study was supported by grants from The Swedish Medical Research Council (project No. 05249), The Bank of Sweden Tercentenary Foundation and The Medical Faculty of Lund University. Mrs. Berit F~rjh is gratefully acknowledged for her excellent technical assistance.
146
GUSTAVSSON AND ALLING REFERENCES
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