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Modification of the fatty acid composition of rat heart sarcolemma with dietary cod liver oil, corn oil or butter
j Mol Cell Cardio120, 141-147 (1988)
M o d i f i c a t i o n o f t h e Fatty A c i d C o m p o s i t i o n o f R a t H e a r t S a r c o l e m m a w i t h D i e t a r y Cod Liver Oil, C o r n Oil or B u t t e r V. E. Benediktsdottir and S. Gudbjarnason* University of Iceland, Science Institute, Dunhaga 3, 220 Reykjavik, Iceland (Received 2 March 1987, accepted in revisedform 1 December 1987) V. E. BENEDIKTSDOTTIRAND S. (~UDBJARNASON.Modification of the Fatty Acid Composition of Rat Heart Sarcolemma with Dietary Cod Liver Oil, Corn Oil or Butter. Journal of Molecular and Cellular Cardiology (1988) 20, 141 147. T h e effect of dietary cod liver oil, corn oil or butter upon the lipid composition of cardiac sarcolemma and the activity of sarcolemmal Na +, K + ATPase was examined in male Wistar rats. The cod liver oil diet caused significant changes in the fatty acid composition of the major phospholipids of sarcolemma, phosphatidyl choline and phosphatidyl ethanolamine. In both these phospholipids arachidonic acid, 20:4 (n - 6) was reduced by about 50% compared to rats fed butter or corn oil and was replaced by the (n - 3) fatty acids eicosapentaenoic and docosahexaenoic acids. The corn oil diet caused a significant diminution in the oleic acid content of phosphatidyl choline and elevation of linoleic acid in phosphatidyl ethanolamine. T h e phospholipid class composition, total phospholipid fatty acid content and cholesterol content ofsarcolemma were not altered by the diets used. The activity of Na +, K + ATPase in the cardiac sarcolemma was not significantly changed by the different diets. KEY WORDS: Fatty acid composition; Heart sarcolemma; Arachidonic acid; Eicosapentaenoic acid; Docosahexaenoic acid ; Na +, K + ATPase.
Introduction Interest in (n -- 3) fatty acids has increased in recent years when it was observed that Greenland Eskimos and Japanese fishermen, whose diets are rich in (n -- 3) fatty acids, have a low incidence of cardiovascular diseases [11, 20]. Lower cardiovascular diseases mortality has also been observed in relation to regular consumption of fish [22]. Experimental studies have shown that the fatty acid composition of phospholipids in total membranes of the heart can be modified by diet [15, 16, 18, 19]. Increased availability of the ( n - 3) fatty acids, eicosapentaenoic acid (20:5 ( n - - 3 ) , EPA) and docosahexaenoic acid (22:6 ( n - 3), DHA) in dietary fish oils increase their level in heart muscle and decrease the level of (n -- 6) fatty acids, primarily arachidonic acid. Similar changes take place also in other tissues such as red blood cells, platelets and liver [1l, 27, 30]. This reduces the availability of arachidonic acid and the ratio of arachidonic acid to EPA and DHA. These ( n - 3) fatty acids can serve as competitive inhibitors in the conversion of arachidonic
acid to eicosanoids by cyclo-oxygenase and lipoxygenase. An increased level of ( n - 3) fatty acids can also result in formation of oxidation products which have physiological effects different from those of corresponding products ofarachidonic acid [10]. Alterations in fatty acid composition of phospholipids in the heart muscle could also affect the functions of membrane bound enzymes or other functional elements located in the different membranes of the heart muscle cell. The activity of the sarcolemmal enzyme Na +, K + ATPase has been shown to be lipid dependent in vitro. Studies on the enzyme in reconstituted liposomes show that its activity depends on the lipid composition of the liposomes [1, 8, 29, 23]. Dietary modifications of cardiac sarcolemmal phospholipids may thus be accompanied by important changes in activities of sarcolemmal enzymes. Other important functions of the cell membrane for example receptor mediated transduction of signals across the membrane and the ion transport across the sarcolemma could
* To whom correspondence should be addressed. 0022-2828/88/020141 + 0 7 $03.00/0
9 1988 Academic Press Limited
142
V.E. Benediktsdottir and S. Gudbjarnason TABLE 1. Fatty acid composition of rat feed Fatty acid
10% butter
10% corn oil
C4-Ca0 12:0 14:0 16:0 16:1 18:0 18 : 1 18:2 20:0/18:3 20: I 22 : 1 20:5(n - 3) 2 2 : 5 ( n - 3) 22:6(n - 3) unidentified
10. I 4.0 10.6 29.5 3.7 9.4 20.6 7.4
12.9 2.2 1.6 22.3 57.8
4.7
3.2
10% cod liver oil
3.8 14.1 10.5 2.4 22.2 12.2 1.7 9.0 5.3 6.9 0.9 7.2 3.8
% fatty acid of total fatty acids.
also be affected by a small change in the composition, fluidity or p e r m e a b i l i t y of the cell m e m b r a n e [9, 21]. T h e p u r p o s e of this study was to c o m p a r e the effects of (n -- 3) fatty acids from d i e t a r y cod liver oil a n d (n - 6) fatty acids from corn oil u p o n the lipid composition of c a r d i a c sarc o l e m m a a n d the activity of the s a r c o l e m m a l N a +, K + A T P a s e in the rat.
Experimental procedure
Experimental animals M a l e W i s t a r rats aged two months were d i v i d e d into three groups a n d fed the following diets for 4 months : 90% s t a n d a r d diet (rat a n d mouse m a i n t e n a n c e diet no 1, Special Diets Services Ltd., Essex) a n d (1) 10% b u t t e r ; (2) 10% corn oil or (3) 10% cod liver oil. T h e fatty acid composition of the diets is shown in T a b l e 1. T h e rats were killed b y d e c a p i t a t i o n a n d hearts r e m o v e d for sarcol e m m a l isolation a n d d e t e r m i n a t i o n of enzyme activity a n d lipid composition.
Isolation of sarcolemma T h e isolation p r o c e d u r e was based on the m e t h o d of F r a n k et al. [14], except t h a t the sucrose g r a d i e n t was b y Philipson et al. [26]. H e a r t s from three rats were pooled for each e x p e r i m e n t a n d the fresh ventricles were
t r i m m e d of excess fat a n d h o m o g e n i z e d in a Polytron P T A - 10-35 homogenizer for 2 • 3.5 s at setting 5.5 in 4 0 m l 250 mM sucrose, 100 mM KC1, 25 mM sodium p y r o p hosphate, 1 mM dithiothreitol a n d 20 mM T R 1 S p H 7.6 at 4~ T h e h o m o g e n a t e was centrifuged a t 177000 g for 20 min. T h e s u p e r n a t a n t was discarded a n d the pellet was suspended in 40 ml of the same m e d i u m a n d centrifuged again. T h e sides of the centrifuge tube were wiped d r y a n d the pellet, except the firm d a r k b r o w n p o r t i o n at the b o t t o m of the tube, was suspended in 20 ml 250 mM sucrose, 1 mM dithiothreitol a n d 20 mM T R I S p H 7 . 6 . 4 m g DNase 1 ( D N - 1 0 0 ) from S I G M A was a d d e d a n d the suspension was i n c u b a t e d for 45 min at 30~ a n d then further d i s r u p t e d with Polytron for 7 s at setting 5.5 and centrifuged at 177000 g for 45 min. T h e resulting supern a t a n t was discarded a n d the pellet was resuspended in 2 ml 45% sucrose (wt/wt) with a small glass-teflon homogenizer. This suspension was p u t in a 17.5 ml centrifuge tube. A discontinuous sucrose g r a d i e n t was formed by l a y e r i n g 3.1 ml each of 34, 32, 30, 27 a n d 2.6 ml of 20% (wt/wt) sucrose on top of the suspension. T h e sample was centrifuged overnight (18 h) in a B e c k m a n n 27 swinging bucket rotor. T h e s a r c o l e m m a was collected from the 30% sucrose layer, diluted with buffer a n d centrifuged at 177 000 g for
Modification o f the Fatty Acid C o m p o s i t i o n o f Rat Heart S a r c o l e m m a
55 min. T h e pellet was homogenized in 0 . 3 m l 140 mM KC1, 5 m M T R I S p H 7.4 and frozen in liquid N2.
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(80:20:1 by vol.). T h e lipids were identified by comparison with authentic standards.
Preparation and analysis of fatty acid methyl esters Determination of enzyme activities After preincubation of 0.5 m g sarcolemmal protein with 0.3 mg SDS per ml for 30 rain in 20~ [6], the N a +, K + ATPase was assayed in 100 mM NaC1, 20 mM KC1, 5 mM MgC12, 0.5 mM E D T A and 5 mM N a 2 A T P in a buffer of 20 mM glycylglycine and 20 mM imidazole p H 7.4, with about 12 #g sarcolemmal protein in 1.5 ml total volume. T h e same assay medium with 0.1 mM ouabain was used as a blank. T h e reaction was stopped and inorganic phosphate was measured with the method of Post and Sen [28] except that ethyl acetate was used instead of butyl acetate to extract the phosphomolybdate. Succinate dehydrogenase was measured according to the method of B a c h m a n n et al. [4] and N A D P H - c y t o c h r o m e c reductase was measured with the method of Masters et al. [24] to monitor the contamination of mitochondria and sarcoplasmic reticulum respectively. Protein content was measured with Hartrees modification of the L o w r y method [17], using bovine serum albumin as standard.
Lipid analysis Sarcolemmal preparations were combined and extracted twice with the method of Bligh and Dyer [5]. Whole hearts were homogenized and extracted as described previously [12]. In both cases diheptadecanoyl PC was used as internal standard to measure the recovery of the phospholipid. T h e antioxidant B H T was added to the extraction medium at 0.5 mg/100 ml.
Lipid separation Lipids were separated with two solvent systems, each used for single development on thin layer plates. (Adsorbosil-H, Alltech Associates Inc.). The first solvent system was chloroform/methanol/acetic acid/water ( 7 5 : 3 5 : 1 2 : 6 by vol.) and the second was petroleum ether/diethyl ether/acetic acid
The fatty acids were methylated with the method of Morrison and Smith [25] and heneicosanic acid methyl ester was added as an internal standard to quantify the phospholipid fatty acid content. The methyl esters were analysed in a Packard model 419 gas chromatograph, using 10% SP-2330 on 100/120 chromosorb W A W (Supelco) as column packing in a 180 cm column. The heater was p r o g r a m m e d at 3~ from 140 to 240~ Cholesterol was measured from the plate with the method of V e e r k a m p and Broekhuyse [32]. Students t-test was used to determine significances, except in the case of Na +, K + ATPase activity, where the paired t-test was used. Results Electronmicroscopy showed that the sarcolemma preparation contained m e m b r a n e vesicles at the size of 0.1-0.4/2m. T h e activity of the marker enzyme for sarcoplasmic reticulum N A D P H - c y t o c h r o m e c reductase was 0.35 + 0.03 /2mols/h mg protein (n = 27). This is comparable to what Tibbits et al. [31] found in rat sarcolemma and estimated the microsomal content < 1 0 % . Mitochondrial contamination was minimal. Succinate dehydrogenase specific activity was 1 . 0 4 _ 0.06 Bmols/h m g protein (n = 25) in the sarcolemmal fraction. T h a t was < 0 . 0 2 % of the total activity on the gradient and also similar to what Tibbits and coworkers found. Those results indicate that the sarcolemmal preparation was comparable to those of other investigators. Table 2 shows the fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) in sarcolemma and whole heart muscle from rats fed 10% butter. I n PC 16:0 was higher and 2 0 : 4 (n -- 6) was lower in sarcolemma than in whole muscle, but in PE the main difference was that 22:6 (n - 3) was lower in sarcolemma. Oleic acid was higher in sarcolemma than in whole heart muscle in both PC and PE. This difference
144
V. E. Benediktsdottir and S. Gudbjarnason TABLE 2. Fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine in rat heart sarcolemma and whole heart muscle Phosphatidyl ethanolamine
Phosphatidyl choline Fatty acid
Heart Muscle
Sarcolemma
Heart Muscle
16:0 18:0 18:1 18:2 20:4 22:5 22:6
22.3 -t- 0.2 27.7 -t- 0.4 9.6 + 0.I 10.5 • 0.5 22.2 -t- 0.7 1.7 _ 0.2 2.9 _ 0.2
26.2 _ 0.9 a 27.6 _ 0.6 11.5 +_ 0.4 a 12.0 _ 0.7 17.2 + 0.9 a 1.2 • 0.2 2.4 • 0.4
15.0 -t- 0.4 30.2 • 0.5 7.2 +_ 0.2 5.0 _+ 0.3 18.5 + 0.2 3.3 _ 0.3 17.5 _ 0.8
Sarcolemma 16.4 _ 27.3 • 9.5 + 5.7 • 18.9 _ 3.4 • 11.7 •
0.8 0.8 0.4~ 0.3 0.8 0.4 1.5b
" Significantdifferencecompared to whole heart muscleP < 0.01 b Significantdifferencecompared to whole heart muscleP < 0.02 % Patty acid __+s.~., n = 5 Diet: 10% butter
further illustrates that the sarcolemma preparation is distinct from the whole heart muscle preparation. T h e cholesterol content was 0.341 _ 0.013 a n d the total phospholipid content of the sarcolemma of the b u t t e r fed rats was 0.736 ___0.023 (#mol lipid/mg protein ___ S.E., n = 6). T h e phospholipid class composition was as follows : Phosphatidyl choline: 51.8 ___ 1.0, phosphatidyl ethanolamine: 23.1 __ 0.8, phosphatidyl serine -t- phosphatidyl inositol: 11.5 ___0.4 a n d sphingomyelin: 13.0 • 0.4 (mol % phospholipid ___ s.E., n = 4). No significant differences were seen between the three diet groups neither in phospholipid fatty acid a n d cholesterol content nor in the phospholipid class composition in sarcolemma. T h e fatty acid composition of PC in sarcol e m m a from the three diet groups is shown in T a b l e 3. F a t t y acids a n d Cx6 and ClS aldehydes present in small a m o u n t s in these phospholipids are not listed in Tables 2 a n d 3. Rats fed corn oil had significantly lower levels ofoleic acid t h a n rats fed butter, the reference group. I n the cod liver oil fed group there were differences in four i m p o r t a n t fatty acids. T h e linoleic acid was higher t h a n in the butter group a n d arachidonic acid was less t h a n half of what was found in the other groups. Eicosapentaenoic acid which was not detectable in the other groups was 1.7%. Docosahexaenoic acid was four times higher in the cod liver oil group compared to the corn oil group a n d also significantly higher than in the butter
group. T a b l e 3 also shows the fatty acid composition of PE in the three diet groups. Linoleic acid was significantly higher in the corn oil t h a n in the reference group. I n the cod liver oil group arachidonic acid was replaced by the ( n - 3) fatty acids docosahexaenoic acid a n d eicosapentaenoic acid as in PC. N a +, K § A T P a s e activity was 84-t-4.1 #mols/h mg protein (n = 9) in rats fed butter. T h e activity of this sarcolemmal enzyme was not significantly different in the rats fed corn oil or cod liver oil, despite markedly different fatty acid composition of sarcolemmal phospholipids.
Discussion This study shows that the dietary availability of (n -- 3) fatty acids, earlier demonstrated to influence the fatty acid composition of phospholipids in the whole heart [15], significantly influences the fatty acid composition of the sarcolemma or cell m e m b r a n e in the heart. Cardiac phospholipids consist primarily of phospholipids from m i t o c h o n d r i a a n d sarcoplasmic reticulum with sarcolemma as a m i n o r component. T h e sarcolemma has a fatty acid profile that is significantly different from the whole heart. It should be noted that the sarcolemmal p r e p a r a t i o n contains also plasma m e m b r a n e s from other cells in the heart such as smooth muscle cells a n d endothelial cells. It is, however, reasonable to assume that most of the m e m b r a n e material is derived from the myocytes.
Modification o f the Fatty Acid C o m p o s i t i o n o f Rat Heart S a r c o l e m m a
145
TABLE 3. Fatty acid composition ofphosphatidyl choline and phosphatidyl ethanolamine Phosphatidyl choline Fatty acid 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6
(n -(n -(n-(n -(n --
6) 6) 3) 3) 3)
10% butter
10% corn oil
26.2 + 0.9 27.6 -t- 0.6 11.5__+0.4 12.0 -+ 0.7 17.2 -+ 0.9
23.1 -+ 0.9 29.1 -+ 1.1 9.3-t-0.2 *a 14.2 _ 0.5 19.7 + 0.7
1.2 -+ 0.2 2.4 -+ 0.4
0.6 -+ 0.1 1.5 -+ 0.2
Phosphatidyl ethanolamine
10% cod liver oil
10% butter
10% corn oil
10% cod liver oil
24.9 -+ 0.8 26.6 -+ 0.8 12.5-+0.1 15.4 _ 0.4 *a 8.3 -+ 0.1 **b 1.7 -+ 0.1 1.0 -+ 0.1 6.5 -+ 0.6 *b
16.4 + 0.8 27.3 -+ 0.8 9.5-+0.4 5.7 _ 0.3 18.9 -+ 0.8
14.0 -+ 0.6 25.2 _ 1.1 9.7-+0.4 9.4 -+ 0.1 **a 19.9 -+ 0.5
14.5 + 1.2 24.6 + 1.9 11.0 _+ 0.3 6.1 -t-0.3 8.9 ___0.2 **b 2.2--+0.1 3.1 -+ 0.1 19.1 -+ 0.9 *b
3.4 -+ 0.4 11.7 -+ 1.5
2.2 -+ 0.2 7.8 -+ 0.4
* P<0.01. ** P < 0.001. a Significant difference compared to rats fed 10% butter. b Significant difference compared to rats fed 10% butter or 10% corn oil. T h e m a i n differences between the fatty acid profile o f phospholipids from whole h e a r t muscle a n d those from isolated s a r c o l e m m a are significantly lower levels of a r a c h i d o n i c acid in PC a n d docosahexaenoic acid in PE of sarcolemma. M o r e s a t u r a t e d fatty acids, i.e. 16:0 a n d 18:1, substitute for a r a c h i d o n i c acid in PC a n d 18:1 replaces docosahexaenoic acid in PE. Changes i n d u c e d b y d i e t a r y fat are, however, similar in s a r c o l e m m a a n d in the whole h e a r t ['16]. R a t s fed diets containing either 10% butter, corn oil or cod liver oil showed different fatty acid composition of i n d i v i d u a l phospholipids in sarcolemma. D i e t a r y cod liver oil lowered the a r a c h i d o n i c acid level in sarcol e m m a l PC a n d PE by 50% c o m p a r e d to rats fed b u t t e r or corn oil, replacing a r a c h i d o n i c acid with ( n - - 3 ) fatty acids, both 2 2 : 6 ( n - 3) a n d 2 0 : 5 ( n - 3). T h e d a t a suggest that ( n - 3) fatty acids are stronger competitors t h a n 20 : 4 (n - 6), a n d 22 : 5 (n -- 3) is preferred over 2 0 : 5 ( n - - 3). These ( n - - 3) fatty acids are available in equal a m o u n t s in the diet, T a b l e 1. T h e fatty acid composition of s a r c o l e m m a l phospholipids does not seem to influence the activity of N a +, K + A T P a s e , as characteristic sarcolemmal enzyme. A l t h o u g h the N a + , K + A T P a s e is d e p e n d e n t on a specific lipid composition in vitro, it seems to be p r o t e c t e d from alterations in fatty acid composition in vivo. O u r results are consistent with those of A b e y w a a r d e n a et al. [2] a n d A w a d a n d C h a t t o p a d h y a y [3] a l t h o u g h they modified the fatty
acid composition of s a r c o l e m m a in a different way. I n o u r e x p e r i m e n t the fatty acid composition of the diet did not influence the cholesterol or p h o s p h o l i p i d content or the lipid class composition of the h e a r t muscle cell m e m b r a n e a n d that is in a g r e e m e n t with the other authors [2, 3]. T h e total p h o s p h o l i p i d specific content was 0.736-t-0.023 # m o l / m g protein which is similar to t h a t observed b y T i b b i t s et al. [3/] using the same isolation procedure. T h e r e a p p e a r s to be a species v a r i a t i o n in sarcol e m m a l p h o s p h o l i p i d content. Philipson et al. [26] using an isolation p r o c e d u r e almost identical to t h a t used in our study observed 1.6 times greater p h o s p h o l i p i d content in the r a b b i t heart. Weglicki et al. [34] a n d Colvin et al. [7] observed 2.7-4.2 times greater phosp h o l i p i d content in canine ventricular myocytes or h e a r t muscle SL preparations. These species variations in p h o s p h o l i p i d content were confirmed b y T i b b i t s et al. ['3/] using the same isolation p r o c e d u r e on rat a n d r a b b i t hearts. These authors suggest t h a t the species variations m a y be a reflection of the differences in the r a t m y o c a r d i a l E - C coupling c o m p a r e d to other m a m m a l i a n species
[z~.
T h e b a l a n c e between ( n - 6) a n d ( n - 3) fatty acids in m e m b r a n e phospholipids influences eicosanoid m e t a b o l i s m differently in different cells. I n platelets increased levels of 20:5 (n -- 3) reduce p r o d u c t i o n of t r o m b o x ane A 2 a n d m a y t h e r e b y reduce the risk of thrombosis a n d vasoconstriction [11-]. I n
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V . E . Benediktsdottir and S. Gudbjarnason
e n d o t h e l i a l cells i n c r e a s e d a v a i l a b i l i t y of ( n - 3) fatty acids reduces p r o s t a c y c l i n p r o d u c t i o n [ 1 0 ] . I n l e u c o c y t e s (n -- 3) f a t t y acids r e d u c e l e u c o t r i e n e p r o d u c t i o n [23]. T h e effects o f (n -- 3) f a t t y acids on m e t a b o lism a n d f u n c t i o n o f m y o c y t e s are l a r g e l y unknown b u t e i c o s a n o i d m e t a b o l i s m of m y o c y t e s is p o o r l y u n d e r s t o o d . S i g n i f i c a n t r e p l a c e m e n t o f 2 0 : 4 ( n - 6) by 2 2 : 6 ( n - 3) in s a r c o l e m m a m a y i n f l u e n c e the response o f the h e a r t muscle cell to i n c r e a s e d f u n c t i o n a l d e m a n d s . I t is c o n c e i v a b l e t h a t 2 0 : 4 (n -- 6) a n d 2 2 : 6 ( n - 3) p l a y a n i m p o r t a n t role in m e m b r a n e f u n c t i o n t h a t is n o t r e l a t e d to eicosanoid f o r m a t i o n . I n c o n c l u s i o n it c a n be stated t h a t the fatty acid c o m p o s i t i o n of d i e t a r y fat m a y signifi-
l 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20
c a n t l y m o d i f y the fatty acid c o m p o s i t i o n of major sarcolemmal phospholipids. Increased a v a i l a b i l i t y of (n - 3) fatty acids f r o m d i e t a r y cod l i v e r oil leads to r e p l a c e m e n t of a r a c h i d o n i c a c i d b y d o c o s a h e x a e n o i c a c i d a n d eicos a p e n t a e n o i c acid in s a r c o l e m m a l P C a n d PE. I n c r e a s e d a v a i l a b i l i t y o f linoleic acid f r o m c o r n oil leads to i n c r e a s e d linoleic acid level in PE. A l t e r a t i o n s in d i e t a r y a v a i l a b i l i t y o f (n - 6) or (n -- 3) f a t t y acids did n o t result in m a r k e d c h a n g e s in c h o l e s t e r o l c o n t e n t or p h o s p h o l i p i d class c o m p o s i t i o n of s a r c o l e m m a . T h e a c t i v i t y of s a r c o l e m m a l N a +, K + A T P a s e r e m a i n e d u n a f f e c t e d b y the c h a n g e s in fatty acid c o m position of m e m b r a n e p h o s p h o l i p i d s i n d u c e d b y d i e t a r y cod l i v e r oil or c o r n oil.
References ABEYWARDENA,M. Y., ALLEN, T. M., CHARNOCK~J. S. Lipid-protein interactions of reconstituted membraneassociated adenosinetriphosphates. Use of gel filtration procedure to examine the phospholipid-activity. Biochim Biophys Acta 729, 62 74 (1984). ABEYWARDENA,M. Y., AC MURCHIE,E.J., RUSSEL,G.J., SAWYER,W. H., CnARNOCK,J. S. Response of rat heart membranes and associated ion-transporting ATPases to dietary lipids. Biochim Biophys Acta 776, 48 59 (1984). AWAD,A. B., CHATTOPADHYAY,J. P. Effect of dietary fat on the lipid composition and enzyme activities of rat cardiac sarcolemma. J Nutr 113, 1878-1884 (1983). BACHMANN,A. B., ALLMAN,D. W., GREEN,D. E. The membrane system of the mitochondrion. 1. The S fraction of the outer membrane of beef heart mitochondria. Arch Biochem Biophys 115, 153-164 (1966). BLIGH,E. G., DYER, W. K. A rapid method of total lipid extraction and purification. Can J Biochim Physiol 37, 811 917 (1959). CARON1,P., REINLIB, a., CARAFOLI,E. Charge movements during the Na-Ca exchange in heart sarcolemma vesicles. Proc Natl Acad Sci USA 77, 6354~6358 (1980). COLVIN,R. A., ASHAVAID,T. F., HERBETTE, L. G. Structure-function studies of canine cardiac sarcolemmal membranes. I. Estimation ofreceptor site densities. Biochim Biophys Acta 812, 601 608 (1985). CORNELIUS,F., SKOU,J. C. Reconstitution of (Na + + K +) ATPase into phospholipid vesicles with full recovery of its specific activity. Biochim Biophys Acta 772, 357 373 (1984). DE GIER, J., MANDERSLOOT,J. G., VAN DEENEN, L. L. M. Lipid composition and permeability of liposomes. Biochim Biophys Acta 150, 666 675 (1968). DYERBERG,J., JORGENSEN,K. A., ARNFRED,T. Human umbilical blood vessel converts all cis- 5, 8, ll, 14, 17 eicosapentaenoic acid to prostaglandin 13 . Prostaglandins 22, 857 862 (1981). DYERBERG,J., BANG,H. O. A hypothesis on the development of acute myocardial infarct in Greenlanders. Scand J Clin Invest 42, [Suppl. 161], 7 13 (1983). EMILSSON,A., GUDBJARNASON,S. Reversible alterations in fatty acid profile of glycerophospholipids in rat heart muscle induced by repeated norepinephrine administrations. Biochim Biophys Acta 750, 1-6 (1983). FABIATO,A., FAmATO,F. Calcium and cardiac excitation-contraction coupling. Ann Rev Physiol 41, 473 484 (1979). FRANK,J. S., PHILIPSON,K. D., BEYDLER,S. Uhrastructure of isolated sarcolemma from dog and rabbit myocardium. Circ Res 54, 414-423 (1984). GUDBJAItNASON,S., OSKARSDOTTIR,G. Modification of fatty acid composition of rat heart lipids by feeding cod liver oil. Biochim Biophys Acta ttl7, 1~15 (1977). GUDBJARNASON,S., EMILSSON,A., GUDMUNDSDOTTIR,A. In Arterial Pollution : An integrated view on atherosclerosis, Peeters, H., Gresham, G. A., Paoletti, R. (Eds). pp. t 15-124. New York: Plenum Press (1984). HARTREE,E. F. Determination of protein : A modification of the Lowry method that gives a linear response. Anal Biochem'ill, 122 127 (1972). HOLMAN,R. T. Nutritional and metabolic interrelationship between fatty acids. Fed Proc 23, 1062 1067 (1972). INNIS,S. M., CLANIDXN,M. T. Dynamic modulation of mitochondrial inner membrane lipids in rat heart by dietary fat. BiochemJ 193, 155 167 (1981). KAGAWA,Y., NISHIZAWA,M., SUZUKI,M. Eicosapolyenoic acid of serum of Japanese Islanders with low cardiovascular disease. J Nutr Sci Vit 24, 441-453 (1982).
Modification o f the Fatty Acid C o m p o s i t i o n o f Rat Heart S a r c o l e m m a 21 22 23
24 25 26 27 28
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KIRILOVSKY,J., SCHRAMM,M. Delipidation of a b-adrenergic receptor preparation and reconstitution by specific lipids.J Biol Chem 258, 6841 6849 (1983). KROmfOUT,D., BOSSCmETER,E. B., COULANDER,C. L. The inverse relation between fish consumption and 20-year mortality from coronary heart disease. New EnglJ Med 312, 1205 1209 (1985). LEE, T. H. HOOVER, R. L., WILLIAMS,J. D., SPERLING,R. I., RAVALESE,J., SPUR, B. W., ROBINSON,D. R , CORKY, E.J., LEwis, R. A , AUSTEN, K. F. Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrofil and monocyte leucotriene generation and neutrofil function. New EnglJ Med 312, 1217 1223 (1985), MASTERS,B. S. S., WXLL~AMS,C. H., KAMIN,H. The preparation and properties ofmicrosomal TPNH-cytochrome c reductase from pig liver. Methods Enzymol 10, 565-573 (1967). MORmSON,W. R., SMITH, L. M. Preparation of fatty acid methyl esters and dimethylacetate from lipids with boron trifluoride-methanol.J Lipid Res5, 600 608 (1964). PHmlPSON,K. D., BERS, D. M., NISHIMOTO, A. Y. The role of phospholipids in Ca binding of isolated cardiac sarcolemma.J Mol Cell Cardio112, 1159 1173 (1980). POPP-SNIJDERS,C., SCHOUTEN,J. A., DE JONG, A. P., VAN DER VEEN, E. A. Effect of dietary cod liver oil on the lipid composition of human erythrocyte membranes. ScandJ Clin Lab Invest 44, 3 ~ 4 6 (1984). POST,R. L,: SEN,A. K. Sodium and potassium stimulated ATPase. Methods Enzymol 10, 762-768 (1967).
29 SIENSKY,M., PINKERTON,F., SUTVIERLAND,E., SIMON, F. R. Rate limitation of (Na+K+)-stimulated adenosinetriphosphatase by membrane acyl chain ordering. Proc Natl Acad Sci USA 76, 4893 4897 (1979). 30 TAHIN,Q. S., BLUM,M., CARAFOLI,E. The fatty acid composition ofsubcellular membranes of rat liver, heart and brain: Diat induced modifications. EurJ Biochem 121, 5-13 (1981). 31 TIBBITS,G. F., SASAKI, M., IKEDA, M., SHIMADA, K., TSURUHARA,T., NAGATOMO,T.J. Characterisation of rat myocardial sarcolemma.J Mol Cell Cardiol 13, 1051-1061 (1981). 32 VEERKAMP,J. H., BROEKHUYSE,R. M. In Biochemical analysis of membranes: Techniques for analysis of membrane lipids. Maddy, D. H. (Ed.) pp. 252 282. London: Chapman and Hall (1976). 33 WALKER,J. A., W~EELER, K. P. Polar head group and acyl side chain requirements for phospholipid dependent (Na + + K +) ATPase. Biochim Biophys Acta 394, 135-144 (1979). 34 WEGLICKI,W. B., OWENS, K., KE~NETT, F. F., KESSNER,A., HARRIS, L., WISE, R. M. Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes. J Biol Chem 255, 3605-3609 (1980).
M o d i f i c a t i o n o f t h e Fatty A c i d C o m p o s i t i o n o f R a t H e a r t S a r c o l e m m a w i t h D i e t a r y Cod Liver Oil, C o r n Oil or B u t t e r V. E. Benediktsdottir and S. Gudbjarnason* University of Iceland, Science Institute, Dunhaga 3, 220 Reykjavik, Iceland (Received 2 March 1987, accepted in revisedform 1 December 1987) V. E. BENEDIKTSDOTTIRAND S. (~UDBJARNASON.Modification of the Fatty Acid Composition of Rat Heart Sarcolemma with Dietary Cod Liver Oil, Corn Oil or Butter. Journal of Molecular and Cellular Cardiology (1988) 20, 141 147. T h e effect of dietary cod liver oil, corn oil or butter upon the lipid composition of cardiac sarcolemma and the activity of sarcolemmal Na +, K + ATPase was examined in male Wistar rats. The cod liver oil diet caused significant changes in the fatty acid composition of the major phospholipids of sarcolemma, phosphatidyl choline and phosphatidyl ethanolamine. In both these phospholipids arachidonic acid, 20:4 (n - 6) was reduced by about 50% compared to rats fed butter or corn oil and was replaced by the (n - 3) fatty acids eicosapentaenoic and docosahexaenoic acids. The corn oil diet caused a significant diminution in the oleic acid content of phosphatidyl choline and elevation of linoleic acid in phosphatidyl ethanolamine. T h e phospholipid class composition, total phospholipid fatty acid content and cholesterol content ofsarcolemma were not altered by the diets used. The activity of Na +, K + ATPase in the cardiac sarcolemma was not significantly changed by the different diets. KEY WORDS: Fatty acid composition; Heart sarcolemma; Arachidonic acid; Eicosapentaenoic acid; Docosahexaenoic acid ; Na +, K + ATPase.
Introduction Interest in (n -- 3) fatty acids has increased in recent years when it was observed that Greenland Eskimos and Japanese fishermen, whose diets are rich in (n -- 3) fatty acids, have a low incidence of cardiovascular diseases [11, 20]. Lower cardiovascular diseases mortality has also been observed in relation to regular consumption of fish [22]. Experimental studies have shown that the fatty acid composition of phospholipids in total membranes of the heart can be modified by diet [15, 16, 18, 19]. Increased availability of the ( n - 3) fatty acids, eicosapentaenoic acid (20:5 ( n - - 3 ) , EPA) and docosahexaenoic acid (22:6 ( n - 3), DHA) in dietary fish oils increase their level in heart muscle and decrease the level of (n -- 6) fatty acids, primarily arachidonic acid. Similar changes take place also in other tissues such as red blood cells, platelets and liver [1l, 27, 30]. This reduces the availability of arachidonic acid and the ratio of arachidonic acid to EPA and DHA. These ( n - 3) fatty acids can serve as competitive inhibitors in the conversion of arachidonic
acid to eicosanoids by cyclo-oxygenase and lipoxygenase. An increased level of ( n - 3) fatty acids can also result in formation of oxidation products which have physiological effects different from those of corresponding products ofarachidonic acid [10]. Alterations in fatty acid composition of phospholipids in the heart muscle could also affect the functions of membrane bound enzymes or other functional elements located in the different membranes of the heart muscle cell. The activity of the sarcolemmal enzyme Na +, K + ATPase has been shown to be lipid dependent in vitro. Studies on the enzyme in reconstituted liposomes show that its activity depends on the lipid composition of the liposomes [1, 8, 29, 23]. Dietary modifications of cardiac sarcolemmal phospholipids may thus be accompanied by important changes in activities of sarcolemmal enzymes. Other important functions of the cell membrane for example receptor mediated transduction of signals across the membrane and the ion transport across the sarcolemma could
* To whom correspondence should be addressed. 0022-2828/88/020141 + 0 7 $03.00/0
9 1988 Academic Press Limited
142
V.E. Benediktsdottir and S. Gudbjarnason TABLE 1. Fatty acid composition of rat feed Fatty acid
10% butter
10% corn oil
C4-Ca0 12:0 14:0 16:0 16:1 18:0 18 : 1 18:2 20:0/18:3 20: I 22 : 1 20:5(n - 3) 2 2 : 5 ( n - 3) 22:6(n - 3) unidentified
10. I 4.0 10.6 29.5 3.7 9.4 20.6 7.4
12.9 2.2 1.6 22.3 57.8
4.7
3.2
10% cod liver oil
3.8 14.1 10.5 2.4 22.2 12.2 1.7 9.0 5.3 6.9 0.9 7.2 3.8
% fatty acid of total fatty acids.
also be affected by a small change in the composition, fluidity or p e r m e a b i l i t y of the cell m e m b r a n e [9, 21]. T h e p u r p o s e of this study was to c o m p a r e the effects of (n -- 3) fatty acids from d i e t a r y cod liver oil a n d (n - 6) fatty acids from corn oil u p o n the lipid composition of c a r d i a c sarc o l e m m a a n d the activity of the s a r c o l e m m a l N a +, K + A T P a s e in the rat.
Experimental procedure
Experimental animals M a l e W i s t a r rats aged two months were d i v i d e d into three groups a n d fed the following diets for 4 months : 90% s t a n d a r d diet (rat a n d mouse m a i n t e n a n c e diet no 1, Special Diets Services Ltd., Essex) a n d (1) 10% b u t t e r ; (2) 10% corn oil or (3) 10% cod liver oil. T h e fatty acid composition of the diets is shown in T a b l e 1. T h e rats were killed b y d e c a p i t a t i o n a n d hearts r e m o v e d for sarcol e m m a l isolation a n d d e t e r m i n a t i o n of enzyme activity a n d lipid composition.
Isolation of sarcolemma T h e isolation p r o c e d u r e was based on the m e t h o d of F r a n k et al. [14], except t h a t the sucrose g r a d i e n t was b y Philipson et al. [26]. H e a r t s from three rats were pooled for each e x p e r i m e n t a n d the fresh ventricles were
t r i m m e d of excess fat a n d h o m o g e n i z e d in a Polytron P T A - 10-35 homogenizer for 2 • 3.5 s at setting 5.5 in 4 0 m l 250 mM sucrose, 100 mM KC1, 25 mM sodium p y r o p hosphate, 1 mM dithiothreitol a n d 20 mM T R 1 S p H 7.6 at 4~ T h e h o m o g e n a t e was centrifuged a t 177000 g for 20 min. T h e s u p e r n a t a n t was discarded a n d the pellet was suspended in 40 ml of the same m e d i u m a n d centrifuged again. T h e sides of the centrifuge tube were wiped d r y a n d the pellet, except the firm d a r k b r o w n p o r t i o n at the b o t t o m of the tube, was suspended in 20 ml 250 mM sucrose, 1 mM dithiothreitol a n d 20 mM T R I S p H 7 . 6 . 4 m g DNase 1 ( D N - 1 0 0 ) from S I G M A was a d d e d a n d the suspension was i n c u b a t e d for 45 min at 30~ a n d then further d i s r u p t e d with Polytron for 7 s at setting 5.5 and centrifuged at 177000 g for 45 min. T h e resulting supern a t a n t was discarded a n d the pellet was resuspended in 2 ml 45% sucrose (wt/wt) with a small glass-teflon homogenizer. This suspension was p u t in a 17.5 ml centrifuge tube. A discontinuous sucrose g r a d i e n t was formed by l a y e r i n g 3.1 ml each of 34, 32, 30, 27 a n d 2.6 ml of 20% (wt/wt) sucrose on top of the suspension. T h e sample was centrifuged overnight (18 h) in a B e c k m a n n 27 swinging bucket rotor. T h e s a r c o l e m m a was collected from the 30% sucrose layer, diluted with buffer a n d centrifuged at 177 000 g for
Modification o f the Fatty Acid C o m p o s i t i o n o f Rat Heart S a r c o l e m m a
55 min. T h e pellet was homogenized in 0 . 3 m l 140 mM KC1, 5 m M T R I S p H 7.4 and frozen in liquid N2.
143
(80:20:1 by vol.). T h e lipids were identified by comparison with authentic standards.
Preparation and analysis of fatty acid methyl esters Determination of enzyme activities After preincubation of 0.5 m g sarcolemmal protein with 0.3 mg SDS per ml for 30 rain in 20~ [6], the N a +, K + ATPase was assayed in 100 mM NaC1, 20 mM KC1, 5 mM MgC12, 0.5 mM E D T A and 5 mM N a 2 A T P in a buffer of 20 mM glycylglycine and 20 mM imidazole p H 7.4, with about 12 #g sarcolemmal protein in 1.5 ml total volume. T h e same assay medium with 0.1 mM ouabain was used as a blank. T h e reaction was stopped and inorganic phosphate was measured with the method of Post and Sen [28] except that ethyl acetate was used instead of butyl acetate to extract the phosphomolybdate. Succinate dehydrogenase was measured according to the method of B a c h m a n n et al. [4] and N A D P H - c y t o c h r o m e c reductase was measured with the method of Masters et al. [24] to monitor the contamination of mitochondria and sarcoplasmic reticulum respectively. Protein content was measured with Hartrees modification of the L o w r y method [17], using bovine serum albumin as standard.
Lipid analysis Sarcolemmal preparations were combined and extracted twice with the method of Bligh and Dyer [5]. Whole hearts were homogenized and extracted as described previously [12]. In both cases diheptadecanoyl PC was used as internal standard to measure the recovery of the phospholipid. T h e antioxidant B H T was added to the extraction medium at 0.5 mg/100 ml.
Lipid separation Lipids were separated with two solvent systems, each used for single development on thin layer plates. (Adsorbosil-H, Alltech Associates Inc.). The first solvent system was chloroform/methanol/acetic acid/water ( 7 5 : 3 5 : 1 2 : 6 by vol.) and the second was petroleum ether/diethyl ether/acetic acid
The fatty acids were methylated with the method of Morrison and Smith [25] and heneicosanic acid methyl ester was added as an internal standard to quantify the phospholipid fatty acid content. The methyl esters were analysed in a Packard model 419 gas chromatograph, using 10% SP-2330 on 100/120 chromosorb W A W (Supelco) as column packing in a 180 cm column. The heater was p r o g r a m m e d at 3~ from 140 to 240~ Cholesterol was measured from the plate with the method of V e e r k a m p and Broekhuyse [32]. Students t-test was used to determine significances, except in the case of Na +, K + ATPase activity, where the paired t-test was used. Results Electronmicroscopy showed that the sarcolemma preparation contained m e m b r a n e vesicles at the size of 0.1-0.4/2m. T h e activity of the marker enzyme for sarcoplasmic reticulum N A D P H - c y t o c h r o m e c reductase was 0.35 + 0.03 /2mols/h mg protein (n = 27). This is comparable to what Tibbits et al. [31] found in rat sarcolemma and estimated the microsomal content < 1 0 % . Mitochondrial contamination was minimal. Succinate dehydrogenase specific activity was 1 . 0 4 _ 0.06 Bmols/h m g protein (n = 25) in the sarcolemmal fraction. T h a t was < 0 . 0 2 % of the total activity on the gradient and also similar to what Tibbits and coworkers found. Those results indicate that the sarcolemmal preparation was comparable to those of other investigators. Table 2 shows the fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) in sarcolemma and whole heart muscle from rats fed 10% butter. I n PC 16:0 was higher and 2 0 : 4 (n -- 6) was lower in sarcolemma than in whole muscle, but in PE the main difference was that 22:6 (n - 3) was lower in sarcolemma. Oleic acid was higher in sarcolemma than in whole heart muscle in both PC and PE. This difference
144
V. E. Benediktsdottir and S. Gudbjarnason TABLE 2. Fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine in rat heart sarcolemma and whole heart muscle Phosphatidyl ethanolamine
Phosphatidyl choline Fatty acid
Heart Muscle
Sarcolemma
Heart Muscle
16:0 18:0 18:1 18:2 20:4 22:5 22:6
22.3 -t- 0.2 27.7 -t- 0.4 9.6 + 0.I 10.5 • 0.5 22.2 -t- 0.7 1.7 _ 0.2 2.9 _ 0.2
26.2 _ 0.9 a 27.6 _ 0.6 11.5 +_ 0.4 a 12.0 _ 0.7 17.2 + 0.9 a 1.2 • 0.2 2.4 • 0.4
15.0 -t- 0.4 30.2 • 0.5 7.2 +_ 0.2 5.0 _+ 0.3 18.5 + 0.2 3.3 _ 0.3 17.5 _ 0.8
Sarcolemma 16.4 _ 27.3 • 9.5 + 5.7 • 18.9 _ 3.4 • 11.7 •
0.8 0.8 0.4~ 0.3 0.8 0.4 1.5b
" Significantdifferencecompared to whole heart muscleP < 0.01 b Significantdifferencecompared to whole heart muscleP < 0.02 % Patty acid __+s.~., n = 5 Diet: 10% butter
further illustrates that the sarcolemma preparation is distinct from the whole heart muscle preparation. T h e cholesterol content was 0.341 _ 0.013 a n d the total phospholipid content of the sarcolemma of the b u t t e r fed rats was 0.736 ___0.023 (#mol lipid/mg protein ___ S.E., n = 6). T h e phospholipid class composition was as follows : Phosphatidyl choline: 51.8 ___ 1.0, phosphatidyl ethanolamine: 23.1 __ 0.8, phosphatidyl serine -t- phosphatidyl inositol: 11.5 ___0.4 a n d sphingomyelin: 13.0 • 0.4 (mol % phospholipid ___ s.E., n = 4). No significant differences were seen between the three diet groups neither in phospholipid fatty acid a n d cholesterol content nor in the phospholipid class composition in sarcolemma. T h e fatty acid composition of PC in sarcol e m m a from the three diet groups is shown in T a b l e 3. F a t t y acids a n d Cx6 and ClS aldehydes present in small a m o u n t s in these phospholipids are not listed in Tables 2 a n d 3. Rats fed corn oil had significantly lower levels ofoleic acid t h a n rats fed butter, the reference group. I n the cod liver oil fed group there were differences in four i m p o r t a n t fatty acids. T h e linoleic acid was higher t h a n in the butter group a n d arachidonic acid was less t h a n half of what was found in the other groups. Eicosapentaenoic acid which was not detectable in the other groups was 1.7%. Docosahexaenoic acid was four times higher in the cod liver oil group compared to the corn oil group a n d also significantly higher than in the butter
group. T a b l e 3 also shows the fatty acid composition of PE in the three diet groups. Linoleic acid was significantly higher in the corn oil t h a n in the reference group. I n the cod liver oil group arachidonic acid was replaced by the ( n - 3) fatty acids docosahexaenoic acid a n d eicosapentaenoic acid as in PC. N a +, K § A T P a s e activity was 84-t-4.1 #mols/h mg protein (n = 9) in rats fed butter. T h e activity of this sarcolemmal enzyme was not significantly different in the rats fed corn oil or cod liver oil, despite markedly different fatty acid composition of sarcolemmal phospholipids.
Discussion This study shows that the dietary availability of (n -- 3) fatty acids, earlier demonstrated to influence the fatty acid composition of phospholipids in the whole heart [15], significantly influences the fatty acid composition of the sarcolemma or cell m e m b r a n e in the heart. Cardiac phospholipids consist primarily of phospholipids from m i t o c h o n d r i a a n d sarcoplasmic reticulum with sarcolemma as a m i n o r component. T h e sarcolemma has a fatty acid profile that is significantly different from the whole heart. It should be noted that the sarcolemmal p r e p a r a t i o n contains also plasma m e m b r a n e s from other cells in the heart such as smooth muscle cells a n d endothelial cells. It is, however, reasonable to assume that most of the m e m b r a n e material is derived from the myocytes.
Modification o f the Fatty Acid C o m p o s i t i o n o f Rat Heart S a r c o l e m m a
145
TABLE 3. Fatty acid composition ofphosphatidyl choline and phosphatidyl ethanolamine Phosphatidyl choline Fatty acid 16:0 18:0 18:1 18:2 20:4 20:5 22:5 22:6
(n -(n -(n-(n -(n --
6) 6) 3) 3) 3)
10% butter
10% corn oil
26.2 + 0.9 27.6 -t- 0.6 11.5__+0.4 12.0 -+ 0.7 17.2 -+ 0.9
23.1 -+ 0.9 29.1 -+ 1.1 9.3-t-0.2 *a 14.2 _ 0.5 19.7 + 0.7
1.2 -+ 0.2 2.4 -+ 0.4
0.6 -+ 0.1 1.5 -+ 0.2
Phosphatidyl ethanolamine
10% cod liver oil
10% butter
10% corn oil
10% cod liver oil
24.9 -+ 0.8 26.6 -+ 0.8 12.5-+0.1 15.4 _ 0.4 *a 8.3 -+ 0.1 **b 1.7 -+ 0.1 1.0 -+ 0.1 6.5 -+ 0.6 *b
16.4 + 0.8 27.3 -+ 0.8 9.5-+0.4 5.7 _ 0.3 18.9 -+ 0.8
14.0 -+ 0.6 25.2 _ 1.1 9.7-+0.4 9.4 -+ 0.1 **a 19.9 -+ 0.5
14.5 + 1.2 24.6 + 1.9 11.0 _+ 0.3 6.1 -t-0.3 8.9 ___0.2 **b 2.2--+0.1 3.1 -+ 0.1 19.1 -+ 0.9 *b
3.4 -+ 0.4 11.7 -+ 1.5
2.2 -+ 0.2 7.8 -+ 0.4
* P<0.01. ** P < 0.001. a Significant difference compared to rats fed 10% butter. b Significant difference compared to rats fed 10% butter or 10% corn oil. T h e m a i n differences between the fatty acid profile o f phospholipids from whole h e a r t muscle a n d those from isolated s a r c o l e m m a are significantly lower levels of a r a c h i d o n i c acid in PC a n d docosahexaenoic acid in PE of sarcolemma. M o r e s a t u r a t e d fatty acids, i.e. 16:0 a n d 18:1, substitute for a r a c h i d o n i c acid in PC a n d 18:1 replaces docosahexaenoic acid in PE. Changes i n d u c e d b y d i e t a r y fat are, however, similar in s a r c o l e m m a a n d in the whole h e a r t ['16]. R a t s fed diets containing either 10% butter, corn oil or cod liver oil showed different fatty acid composition of i n d i v i d u a l phospholipids in sarcolemma. D i e t a r y cod liver oil lowered the a r a c h i d o n i c acid level in sarcol e m m a l PC a n d PE by 50% c o m p a r e d to rats fed b u t t e r or corn oil, replacing a r a c h i d o n i c acid with ( n - - 3 ) fatty acids, both 2 2 : 6 ( n - 3) a n d 2 0 : 5 ( n - 3). T h e d a t a suggest that ( n - 3) fatty acids are stronger competitors t h a n 20 : 4 (n - 6), a n d 22 : 5 (n -- 3) is preferred over 2 0 : 5 ( n - - 3). These ( n - - 3) fatty acids are available in equal a m o u n t s in the diet, T a b l e 1. T h e fatty acid composition of s a r c o l e m m a l phospholipids does not seem to influence the activity of N a +, K + A T P a s e , as characteristic sarcolemmal enzyme. A l t h o u g h the N a + , K + A T P a s e is d e p e n d e n t on a specific lipid composition in vitro, it seems to be p r o t e c t e d from alterations in fatty acid composition in vivo. O u r results are consistent with those of A b e y w a a r d e n a et al. [2] a n d A w a d a n d C h a t t o p a d h y a y [3] a l t h o u g h they modified the fatty
acid composition of s a r c o l e m m a in a different way. I n o u r e x p e r i m e n t the fatty acid composition of the diet did not influence the cholesterol or p h o s p h o l i p i d content or the lipid class composition of the h e a r t muscle cell m e m b r a n e a n d that is in a g r e e m e n t with the other authors [2, 3]. T h e total p h o s p h o l i p i d specific content was 0.736-t-0.023 # m o l / m g protein which is similar to t h a t observed b y T i b b i t s et al. [3/] using the same isolation procedure. T h e r e a p p e a r s to be a species v a r i a t i o n in sarcol e m m a l p h o s p h o l i p i d content. Philipson et al. [26] using an isolation p r o c e d u r e almost identical to t h a t used in our study observed 1.6 times greater p h o s p h o l i p i d content in the r a b b i t heart. Weglicki et al. [34] a n d Colvin et al. [7] observed 2.7-4.2 times greater phosp h o l i p i d content in canine ventricular myocytes or h e a r t muscle SL preparations. These species variations in p h o s p h o l i p i d content were confirmed b y T i b b i t s et al. ['3/] using the same isolation p r o c e d u r e on rat a n d r a b b i t hearts. These authors suggest t h a t the species variations m a y be a reflection of the differences in the r a t m y o c a r d i a l E - C coupling c o m p a r e d to other m a m m a l i a n species
[z~.
T h e b a l a n c e between ( n - 6) a n d ( n - 3) fatty acids in m e m b r a n e phospholipids influences eicosanoid m e t a b o l i s m differently in different cells. I n platelets increased levels of 20:5 (n -- 3) reduce p r o d u c t i o n of t r o m b o x ane A 2 a n d m a y t h e r e b y reduce the risk of thrombosis a n d vasoconstriction [11-]. I n
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V . E . Benediktsdottir and S. Gudbjarnason
e n d o t h e l i a l cells i n c r e a s e d a v a i l a b i l i t y of ( n - 3) fatty acids reduces p r o s t a c y c l i n p r o d u c t i o n [ 1 0 ] . I n l e u c o c y t e s (n -- 3) f a t t y acids r e d u c e l e u c o t r i e n e p r o d u c t i o n [23]. T h e effects o f (n -- 3) f a t t y acids on m e t a b o lism a n d f u n c t i o n o f m y o c y t e s are l a r g e l y unknown b u t e i c o s a n o i d m e t a b o l i s m of m y o c y t e s is p o o r l y u n d e r s t o o d . S i g n i f i c a n t r e p l a c e m e n t o f 2 0 : 4 ( n - 6) by 2 2 : 6 ( n - 3) in s a r c o l e m m a m a y i n f l u e n c e the response o f the h e a r t muscle cell to i n c r e a s e d f u n c t i o n a l d e m a n d s . I t is c o n c e i v a b l e t h a t 2 0 : 4 (n -- 6) a n d 2 2 : 6 ( n - 3) p l a y a n i m p o r t a n t role in m e m b r a n e f u n c t i o n t h a t is n o t r e l a t e d to eicosanoid f o r m a t i o n . I n c o n c l u s i o n it c a n be stated t h a t the fatty acid c o m p o s i t i o n of d i e t a r y fat m a y signifi-
l 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20
c a n t l y m o d i f y the fatty acid c o m p o s i t i o n of major sarcolemmal phospholipids. Increased a v a i l a b i l i t y of (n - 3) fatty acids f r o m d i e t a r y cod l i v e r oil leads to r e p l a c e m e n t of a r a c h i d o n i c a c i d b y d o c o s a h e x a e n o i c a c i d a n d eicos a p e n t a e n o i c acid in s a r c o l e m m a l P C a n d PE. I n c r e a s e d a v a i l a b i l i t y o f linoleic acid f r o m c o r n oil leads to i n c r e a s e d linoleic acid level in PE. A l t e r a t i o n s in d i e t a r y a v a i l a b i l i t y o f (n - 6) or (n -- 3) f a t t y acids did n o t result in m a r k e d c h a n g e s in c h o l e s t e r o l c o n t e n t or p h o s p h o l i p i d class c o m p o s i t i o n of s a r c o l e m m a . T h e a c t i v i t y of s a r c o l e m m a l N a +, K + A T P a s e r e m a i n e d u n a f f e c t e d b y the c h a n g e s in fatty acid c o m position of m e m b r a n e p h o s p h o l i p i d s i n d u c e d b y d i e t a r y cod l i v e r oil or c o r n oil.
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