The Effect of Dietary Erucic Acid on Cardiac Triglycerides and Free Fatty Acid Levels in Rats 1 JOHN K.G. K R A M E R and H O W A R D W. H U L A N , Animal Research Institute,
Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 ABSTRACT
Male Sprague-Dawley rats, 3 weeks of age, were fed semisynthetic diets containing test oils at 20% by weight for 3 days, 1 week, and 16 weeks. The test oils contained up to 22.3% erucic acid. Growth retardation was evident in rats fed rapeseed oil high in erucic acid, and soybean oil and Tower rapeseed oil diets containing about 5% erucic acid. Cardiac triglyceride accumulation was found in rats fed diets containing about 5% erucic acid but not in rats fed Tower rapeseed oil which contains 0.2% of this acid. The cardiac free fatty acid levels were low, 50-100 pg/g of wet heart tissue, and were not affected by feeding diets containing about 5% erucic acid. Feeding a diet containing a high erucic acid rapeseed oil did result in higher free fatty acid levels but only at 3 days and 1 week; the level at 16 weeks was similar to the other oils. The fatty acid analysis of cardiac triglycerides and free fatty acids showed high percentages of erucic acid at 3 days and 1 week; at 16 weeks these levels had declined significantly. The results indicate that the accumulated erucic and eicosenoic acids, at 3 days and 1 week, accounted for the increase in cardiac free fatty acids when rats were fed the high erucic acid rapeseed oil. There appears to be no evidence that the early cardiac triglyceride or free fatty acid accumulation is related to the formation of the long term myocardial lesions. INTRODUCTION
these lipid classes might lead to myocardial fibrosis will be discussed.
Rats fed diets containing rapeseed oil (RSO) high in erucic acid (22: 1), developed early m y o cardial lipidosis (1-6), characterized by pron o u n c e d intracellular lipid deposition mainly in the form of triglycerides (TG) (2,6-11) and free fatty acids ( F F A ) (2,7,9). F a t t y acid analysis showed the relative c o n c e n t r a t i o n of 22:1 to be very high in cardiac TGs (2,6-11) with lower levels r e p o r t e d in cardiac F F A s (2,7,9). On the other hand, rats fed diets containing RSO l o w in 22:1 showed no myocardial lipidosis as determined gravimetrically (4), and no TG a c c u m u l a t i o n (6,10), although, histologically a scanty fat deposition was d e m o n s t r a t e d by oil redO staining (3-5). The cause of the early m y o cardial lipidosis was a t t r i b u t e d to 22:1 (1-8,12) and to a lesser extent to eicosenoic acid (12). It was suggested that the TG a n d / o r the F F A a c c u m u l a t i o n could lead to m y o c a r d i a l fibrosis (13). The purpose of the present study was to d e t e r m i n e quantitative and qualitative changes in the fatty acid c o m p o s i t i o n of cardic TGs and F F A s during the first week of feeding and at 16 weeks using an i m p r o v e d extraction procedure minimizing autolysis of cardiac lipids (14). The diets included control oils, a low erucic acid RSO, oils to which about 5 % free erucic acid was added, and a high erucic acid rapeseed ( H E A R ) oil. The possibility that the changes in
1Contribution No. 739 Animal Research Institute.
MATERIALS AND METHODS Animals and Diets
Male Sprague-Dawley rats, 3 weeks of age, and weighing 40-50 g, were distributed rand o m l y to eight dietary treatments, 15 animals per t r e a t m e n t , and fed ad libitum the test diets for 3 days, 1 week, or 16 weeks. Water was available at all times. In addition, five animals were killed at the start of the e x p e r i m e n t to serve as 0 day control. The semisynthetic diets, prepared as described previously (15), contained 20% by weight of the following oils: soybean oil, olive oil, Brassica napus cv. T o w e r RSO, oil extracted from a seed m i x t u r e of B. campestris cv. Echo (85%) and Arlo (15%) designated H E A R (high erucic acid rapeseed). Free erucic acid was blended into soybean oil, olive oil, and T o w e r RSO to give the indicated level (Table I) of this acid in the dietary oil. Extraction and Analysis of Neutral Lipid Classes
Rats were killed by exsanguination under CO z anesthesia. The hearts were i m m e d i a t e l y r e m o v e d , weighed, and frozen b e t w e e n two blocks of dry ice. The hearts were quickly pulverized at dry ice temperature, and the lipids extracted with chilled C H C 1 3 / C H 3 O H (2: 1) as previously described (14). Total lipids were determined gravimetrically. A k n o w n aliquot o f total lipids was applied with a thin layer chromatography (TLC) s a m p l e streaker
438
CARDIAC NEUTRAL LIPID CHANGES
439
TABLE I Fatty Acid Composition of the Diets Diet a
16:0
16:1
18:0
Fatty acid b (wt %) 18:1 18:2 18:3
20:1
22:1
Soybean Soybean + EA (5.7%) Olive Olive + EA (4.5%) Tower RSO e Tower RSO + EA (0.8%) Tower RSO + EA (5.9%) HEAR d
12.4 13.5 11.6 11.5 6.1 6.5 7.1 4.0
0.1 0.1 1.2 1.2 0.1 0.1 0.1 0.3
3.7 4.3 2.5 2.7 2.0 2.4 2.5 1.7
25.4 25.4 75.5 71.2 56.5 55.7 54.2 36.2
0.8 0.4 0.7 1.5 1.6 1.9 12.3
5.7 0.1 4.5 0.3 0.8 5.9 22.3
50.6 44.1 7.3 6.8 26.0 25.7 22.2 15.1
7.9 6.0 0.7 0.6 7.1 6.5 5.7 5.9
aSemisynthetic diets containing the vegetable oils at 20% by weight of the diet. Free erucic acid (EA) was blended into the vegetable oils; final 22:1 level is indicated in parentheses. bThe major fatty acids are listed; minor amounts of 14:0, 15:0, 17:0, 20:0, and 20:2 are not shown. CTower RSO = Brassica napus cv. Tower rapeseed oil. dHEAR = high erucic acid rapeseed; oil extracted from a seed mixture containing 85% B. campestris cv. Echo and 15% B. campestris cv. Arlo. ( A p p l i e d Science L a b o r a t o r i e s Inc., State College, PA) o n Silica Gel G plates ( F i s h e r Scientific Co., O t t a w a , O n t a r i o ) , 5 0 0 m i c r o n s in thickness, a n d d e v e l o p e d using t h e solvent hexane-diethyl e t h e r - a c e t i c acid ( 8 5 : 1 5 : 1 ) . Bands were visualized u n d e r U V light a f t e r spraying the chromatogram with Rhodamine B in m e t h a n o l . T h e T G a n d F F A b a n d s were identified by co-chromatography with authentic s t a n d a r d s . T h e b a n d s c o r r e s p o n d i n g t o T G a n d F F A were r e m o v e d , m e t h y l h e p t a d e c a n o a t e was a d d e d as a n i n t e r n a l s t a n d a r d a n d t h e m i x t u r e was t r a n s e s t e r i f i e d w i t h a n h y d r o u s H C 1 / C H 3 O H (5% m / m ) . The m e t h y l esters were purified b y TLC using 1 , 2 - d i c h l o r o e t h a n e as d e v e l o p i n g solvent. T h e f a t t y acids in t h e T G a n d F F A f r a c t i o n were resolved w i t h a H e w l e t t - P a c k a r d Model 5 8 3 0 A gas liquid c h r o m a t o g r a p h , e q u i p p e d w i t h a f l a m e i o n i z a t i o n d e t e c t o r a n d digital i n t e g r a t o r . Glass c o l u m n s (1.8 m x 2 r a m ) were packed w i t h 5% b u t a n e d i o l s u c c i n a t e o n 8 0 / 1 0 0 m e s h C h r o m o s o r b G, h i g h p e r f o r m a n c e ( C h r o m a t o g r a p h i c Specialties Ltd., Brockville, O n t a r i o ) . T h e c h r o m a t o g r a p h was o p e r a t e d isot h e r m a l l y at 190 C. Peaks were i d e n t i f i e d b y c o m p a r i n g t h e r e t e n t i o n time t o a u t h e n t i c methyl esters standards (Nu-Chek-Prep., Elysian, MN). Analysis of variance was p e r f o r m e d o n b o d y a n d h e a r t weights, cardiac lipid c o n t e n t , cardiac T G a n d F F A c o n c e n t r a t i o n , a n d m e t h y l ester data. T h e least significant d i f f e r e n c e at t h e I% level was d e t e r m i n e d f r o m t h e p o o l e d error estim a t e s of t h e analysis of variance (16). RESULTS
T h e f a t t y acid c o m p o s i t i o n s of t h e diets are
s h o w n in Table I. T h e erucic acid level of t h e n e w cultivar of r a p e s e e d ( T o w e r RSO) was o n l y 0.3%, while t h a t of t h e old c u l t i v a r ( H E A R ) was 22.3%. A d d i t i o n of free 22:1 t o each of t h e oils, s o y b e a n , olive, a n d T o w e r RSO, b r o u g h t t h e level of this acid to a b o u t 5%, t h e maxim u m level r e c o m m e n d e d in a n edible oil in Canada. Rats fed diets c o n t a i n i n g H E A R oil for 3 days a n d 1 week s h o w e d i n c r e a s e d h e a r t weights possibly d u e t o elevated fat levels (Table II). A f t e r 16 weeks o f feeding, t h e h e a r t weight a n d cardiac fat level a p p e a r e d n o r m a l , b u t t h e b o d y weights were significantly l o w e r (P
Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 ABSTRACT
Male Sprague-Dawley rats, 3 weeks of age, were fed semisynthetic diets containing test oils at 20% by weight for 3 days, 1 week, and 16 weeks. The test oils contained up to 22.3% erucic acid. Growth retardation was evident in rats fed rapeseed oil high in erucic acid, and soybean oil and Tower rapeseed oil diets containing about 5% erucic acid. Cardiac triglyceride accumulation was found in rats fed diets containing about 5% erucic acid but not in rats fed Tower rapeseed oil which contains 0.2% of this acid. The cardiac free fatty acid levels were low, 50-100 pg/g of wet heart tissue, and were not affected by feeding diets containing about 5% erucic acid. Feeding a diet containing a high erucic acid rapeseed oil did result in higher free fatty acid levels but only at 3 days and 1 week; the level at 16 weeks was similar to the other oils. The fatty acid analysis of cardiac triglycerides and free fatty acids showed high percentages of erucic acid at 3 days and 1 week; at 16 weeks these levels had declined significantly. The results indicate that the accumulated erucic and eicosenoic acids, at 3 days and 1 week, accounted for the increase in cardiac free fatty acids when rats were fed the high erucic acid rapeseed oil. There appears to be no evidence that the early cardiac triglyceride or free fatty acid accumulation is related to the formation of the long term myocardial lesions. INTRODUCTION
these lipid classes might lead to myocardial fibrosis will be discussed.
Rats fed diets containing rapeseed oil (RSO) high in erucic acid (22: 1), developed early m y o cardial lipidosis (1-6), characterized by pron o u n c e d intracellular lipid deposition mainly in the form of triglycerides (TG) (2,6-11) and free fatty acids ( F F A ) (2,7,9). F a t t y acid analysis showed the relative c o n c e n t r a t i o n of 22:1 to be very high in cardiac TGs (2,6-11) with lower levels r e p o r t e d in cardiac F F A s (2,7,9). On the other hand, rats fed diets containing RSO l o w in 22:1 showed no myocardial lipidosis as determined gravimetrically (4), and no TG a c c u m u l a t i o n (6,10), although, histologically a scanty fat deposition was d e m o n s t r a t e d by oil redO staining (3-5). The cause of the early m y o cardial lipidosis was a t t r i b u t e d to 22:1 (1-8,12) and to a lesser extent to eicosenoic acid (12). It was suggested that the TG a n d / o r the F F A a c c u m u l a t i o n could lead to m y o c a r d i a l fibrosis (13). The purpose of the present study was to d e t e r m i n e quantitative and qualitative changes in the fatty acid c o m p o s i t i o n of cardic TGs and F F A s during the first week of feeding and at 16 weeks using an i m p r o v e d extraction procedure minimizing autolysis of cardiac lipids (14). The diets included control oils, a low erucic acid RSO, oils to which about 5 % free erucic acid was added, and a high erucic acid rapeseed ( H E A R ) oil. The possibility that the changes in
1Contribution No. 739 Animal Research Institute.
MATERIALS AND METHODS Animals and Diets
Male Sprague-Dawley rats, 3 weeks of age, and weighing 40-50 g, were distributed rand o m l y to eight dietary treatments, 15 animals per t r e a t m e n t , and fed ad libitum the test diets for 3 days, 1 week, or 16 weeks. Water was available at all times. In addition, five animals were killed at the start of the e x p e r i m e n t to serve as 0 day control. The semisynthetic diets, prepared as described previously (15), contained 20% by weight of the following oils: soybean oil, olive oil, Brassica napus cv. T o w e r RSO, oil extracted from a seed m i x t u r e of B. campestris cv. Echo (85%) and Arlo (15%) designated H E A R (high erucic acid rapeseed). Free erucic acid was blended into soybean oil, olive oil, and T o w e r RSO to give the indicated level (Table I) of this acid in the dietary oil. Extraction and Analysis of Neutral Lipid Classes
Rats were killed by exsanguination under CO z anesthesia. The hearts were i m m e d i a t e l y r e m o v e d , weighed, and frozen b e t w e e n two blocks of dry ice. The hearts were quickly pulverized at dry ice temperature, and the lipids extracted with chilled C H C 1 3 / C H 3 O H (2: 1) as previously described (14). Total lipids were determined gravimetrically. A k n o w n aliquot o f total lipids was applied with a thin layer chromatography (TLC) s a m p l e streaker
438
CARDIAC NEUTRAL LIPID CHANGES
439
TABLE I Fatty Acid Composition of the Diets Diet a
16:0
16:1
18:0
Fatty acid b (wt %) 18:1 18:2 18:3
20:1
22:1
Soybean Soybean + EA (5.7%) Olive Olive + EA (4.5%) Tower RSO e Tower RSO + EA (0.8%) Tower RSO + EA (5.9%) HEAR d
12.4 13.5 11.6 11.5 6.1 6.5 7.1 4.0
0.1 0.1 1.2 1.2 0.1 0.1 0.1 0.3
3.7 4.3 2.5 2.7 2.0 2.4 2.5 1.7
25.4 25.4 75.5 71.2 56.5 55.7 54.2 36.2
0.8 0.4 0.7 1.5 1.6 1.9 12.3
5.7 0.1 4.5 0.3 0.8 5.9 22.3
50.6 44.1 7.3 6.8 26.0 25.7 22.2 15.1
7.9 6.0 0.7 0.6 7.1 6.5 5.7 5.9
aSemisynthetic diets containing the vegetable oils at 20% by weight of the diet. Free erucic acid (EA) was blended into the vegetable oils; final 22:1 level is indicated in parentheses. bThe major fatty acids are listed; minor amounts of 14:0, 15:0, 17:0, 20:0, and 20:2 are not shown. CTower RSO = Brassica napus cv. Tower rapeseed oil. dHEAR = high erucic acid rapeseed; oil extracted from a seed mixture containing 85% B. campestris cv. Echo and 15% B. campestris cv. Arlo. ( A p p l i e d Science L a b o r a t o r i e s Inc., State College, PA) o n Silica Gel G plates ( F i s h e r Scientific Co., O t t a w a , O n t a r i o ) , 5 0 0 m i c r o n s in thickness, a n d d e v e l o p e d using t h e solvent hexane-diethyl e t h e r - a c e t i c acid ( 8 5 : 1 5 : 1 ) . Bands were visualized u n d e r U V light a f t e r spraying the chromatogram with Rhodamine B in m e t h a n o l . T h e T G a n d F F A b a n d s were identified by co-chromatography with authentic s t a n d a r d s . T h e b a n d s c o r r e s p o n d i n g t o T G a n d F F A were r e m o v e d , m e t h y l h e p t a d e c a n o a t e was a d d e d as a n i n t e r n a l s t a n d a r d a n d t h e m i x t u r e was t r a n s e s t e r i f i e d w i t h a n h y d r o u s H C 1 / C H 3 O H (5% m / m ) . The m e t h y l esters were purified b y TLC using 1 , 2 - d i c h l o r o e t h a n e as d e v e l o p i n g solvent. T h e f a t t y acids in t h e T G a n d F F A f r a c t i o n were resolved w i t h a H e w l e t t - P a c k a r d Model 5 8 3 0 A gas liquid c h r o m a t o g r a p h , e q u i p p e d w i t h a f l a m e i o n i z a t i o n d e t e c t o r a n d digital i n t e g r a t o r . Glass c o l u m n s (1.8 m x 2 r a m ) were packed w i t h 5% b u t a n e d i o l s u c c i n a t e o n 8 0 / 1 0 0 m e s h C h r o m o s o r b G, h i g h p e r f o r m a n c e ( C h r o m a t o g r a p h i c Specialties Ltd., Brockville, O n t a r i o ) . T h e c h r o m a t o g r a p h was o p e r a t e d isot h e r m a l l y at 190 C. Peaks were i d e n t i f i e d b y c o m p a r i n g t h e r e t e n t i o n time t o a u t h e n t i c methyl esters standards (Nu-Chek-Prep., Elysian, MN). Analysis of variance was p e r f o r m e d o n b o d y a n d h e a r t weights, cardiac lipid c o n t e n t , cardiac T G a n d F F A c o n c e n t r a t i o n , a n d m e t h y l ester data. T h e least significant d i f f e r e n c e at t h e I% level was d e t e r m i n e d f r o m t h e p o o l e d error estim a t e s of t h e analysis of variance (16). RESULTS
T h e f a t t y acid c o m p o s i t i o n s of t h e diets are
s h o w n in Table I. T h e erucic acid level of t h e n e w cultivar of r a p e s e e d ( T o w e r RSO) was o n l y 0.3%, while t h a t of t h e old c u l t i v a r ( H E A R ) was 22.3%. A d d i t i o n of free 22:1 t o each of t h e oils, s o y b e a n , olive, a n d T o w e r RSO, b r o u g h t t h e level of this acid to a b o u t 5%, t h e maxim u m level r e c o m m e n d e d in a n edible oil in Canada. Rats fed diets c o n t a i n i n g H E A R oil for 3 days a n d 1 week s h o w e d i n c r e a s e d h e a r t weights possibly d u e t o elevated fat levels (Table II). A f t e r 16 weeks o f feeding, t h e h e a r t weight a n d cardiac fat level a p p e a r e d n o r m a l , b u t t h e b o d y weights were significantly l o w e r (P
