Brassica campestris var. Span' II. Cardiopathogenicity of Fractions Isolated from Span Rapeseed Oil when Fed to Male Rats1 J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, Animal Research Institute, Research Branch, Agriculture Canada, Ottawa, Ontario, K1A 0C6, and A.H. CORNER, Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute, Ottawa, Ontario, K2H 8P9
ABSTRACT
in nontriglyceride constituents. These fractions were fed to weanling male rats for 16 weeks. Hearts of these rats were examined for myocardial lesions and fatty acid composition.
Rapeseed oils low in erucic acid caused myocardial lesions when fed to weanling male rats for 16 weeks. The cardiopathogenic properties appear to be associated with the triglycerides of the oil, and not to nontriglyceride components present in fully refined rapeseed oil. Cardiac lipid analysis confirmed that erucic acid accumulation was proportional to the concentration of this acid in the diet.
MATERIALS AND METHODS
INTRODUCTION
Recent reports in the literature indicate that feeding rapeseed oil (RSO) to weanling male rats causes lipidosis in the heart within the first w e e k and myocardial lesions after several months, even when RSO low in erucic acid is fed (1-4). It has been shown that the early lipidosis in the heart is directly related to the erucic acid concentration in the dietary oils (2, 5-8). However, at present the cause of the necrotic and fibrotic lesions which occur after feeding RSO for 16 weeks is not understood. Myocardial necrosis has been reported in rats under such stress as catecholamine injection, dietary vitamin K deficiency, severe muscular exertion, exposure to cold and digitalis intoxication (9), or seamin, an alcohol soluble substance found in sesame oil (10). Feeding cyclopropenoid fatty acids is known to produce liver and kidney necrosis (11). The possibility may exist, therefore, that a nontriglyceride component is present in the completely refined RSO which is responsible for the necrotic and fibrotic lesions. On the other hand, the lesion producing properties of RSO may be attributable to its triglycerides. To establish whether the long term lesions are caused by the trigiycerides or some nontriglyceride component present in the oil, Brassica campestris var. Span RSO was fractionated by molecular distillation and adsorption chromatography, as described previously (12), to yield highly purified triglycerides and fractions rich 1Contribution no. 575 Animal Research Institute.
Male Sprague-Dawley rats (Bio-Breeding, Ottawa, Ontario) 3 weeks of age and weighing 40-50 g, were randomly selected, identified by ear markings, assigned 2 per cage, and fed the experimental diets for 16 weeks. The sernisynthetic diet consisted of 20% casein, 20% sucrose, 30% cornstarch, 1% vitamin mixture, 4% U.S.P. XVII mineral mixture, 5% pure wood cellulose, and 20% test oil (w/w). The following otis were used: corn oil (St. Lawrence Starch Co. Ltd., Port Credit, Ontario); olive oil (Pastene Co. Ltd., Montreal, Quebec); safflower oil (Teklad Mills, Madison, Wisc.); B. napus var. Oro and B. campestris var. Tower (Cooperative Vegetable Oil Ltd., Altona, Manitoba); and B. campestris var. Span (Western Canada Processors, Lethbridge, Alberta). The fractions obtained from Span RSO by molecular distillation (MD) 5, 6, 7B, and 1" and b y adsorption chromatography F I , F2, and F3 have been described previously (12). Experiment I consisted of 2 groups of 20 rats fed Purina Laboratory Rat Chow or semisynthetic diet containing olive oil, and 6 groups of 50 rats fed sernisynthetic diet containing Oro RSO, Span RSO, or MD 5, 6, 7B and 1", from Span RSO (12). Because o f the limited supply of MD 1", 25 of the 50 rats were killed after 12 weeks and their hearts examined histologically. The remaining 25 rats were kept the entire 16 weeks on this diet. After 16 weeks, all rats were killed and their hearts examined histologically. One quarter of the heart was removed for lipid analyses from 10 rats on each diet; the remaining portion of these hearts was prepared for histological examination. Experiment II consisted of 4 groups of 10 rats fed chow or semisynthetic diet containing olive oil, Span RSO, or triglyceride fraction F2 obtained from Span RSO by adsorption chromatography (12). Rats were killed after 16 weeks on treatment and hearts removed for
511
512
J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, AND A.H. CORNER TABLE I Fatty Acid Composition of Chow and Dietary Oils Diets Fatty acids
Chowa
Olive
Corn
16.0 16:1 18:0 18:1 18:2 18:3 20:1 22:1 24:1
16.9 1.8 3.2 22.4 38.9 3.1 2.9 3.7
11.9 0.7 2.9 73.4 8.9 0.8 0.4 0.4
15.0 0.1 2.6 27.8 52.7 0.6 0.3
Safflower 9.9 0.1 3.4 13.9 71.9 0.2 0.2
Tower
Oro
Span
5.2 0.2 1.8 56.9 24.0 8.4 1.3 0.8 0.2
5.0 0.3 2.5 62.2 18.9 6.7 1.9 1.6 0.2
4.8 0.2 2.1 58.6 19.5 5.2 3.3 4.8 0.2
aChow contained fish meal as part of its formulation. The fatty acid composition is that of the total lipids extracted from the diet with chloroform: methanol (2:1). lipid analysis and histological examination. Experiment III consisted of 8 groups of 24 rats fed chow or sernisynthetic diet containing corn oil, safflower oil, Tower RSO, Span RSO, or fraction F1, F2, and F3 obtained from Span RSO by adsorption chromatography (12). All rats were killed after 16 weeks on these diets; 20 rats were examined histologically and 4 rats were used for lipid analyses. For histological examination hearts were fixed in 10% neutral buffered formalin. Three sections were prepared from each heart: a) one central section extending from apex to base and including interventricular septum, atrial and ventricular walls; b) a section parallel to the first and extending through the right ventricle; and c) a third section parallel to the other two and extending through the left ventricle. The 3 sections were equidistant from each other. From these, 6/a sections were made and stained with hematoxylin and eosin. All experimental rats were numerically coded and not identified until the histological examination was completed. The results were reported as the number of rats affected, as well as the number of lesions per affected heart. No attempt was made to grade individual lesion as to pathological severity. Instead, groups were made to include rats which had 1-2, 3-5, 6-10, and > 1 0 lesions per heart. Approximate Chisquare statistics, obtained following the approach of Fienberg (13) were used to examine the frequency tables (Tables II-IV) for evidence of differences in patterns of incidence and severity among the various diets. The techniques for homogenization of rat hearts, extraction of lipids, and preparation and analysis of fatty acid methyl esters have been described (2). Analysis of variance was performed on cardiac lipids and wt gains and significant difference at the 1% level (P < 0.01) LIPIDS, VOL. 10, NO. 9
were determined Range test (14).
using Duncan's Multiple
RESULTS
The fatty acid composition of chow, corn oil, olive oil, safflower oil, Tower RSO, Oro RSO, and Span RSO is shown in Table I. The composition of the fractions isolated from Span RSO by molecular distillation and adsorption chromatography are fully described in an accompanying publication (12). Oro and Tower RSO were fully refined rapeseed oils low in 20:1 and 22:1 with a total sterol concentration of 0.29 and 0.23%, respectively. Growth rates of male rats fed MD 1" were significantly lower (P < 0.01) compared to rats fed the diet containing olive off (experiment I). The wt gains of rats fed Oro RSO, Span RSO, or MD 5, 6, and 7B (purified triglycerides) were slightly lower than, but not significantly different (P < , 0 . 0 1 ) from the control diets (chow and olive oil), or MD 1 ". Male rats fed the purified triglyceride fraction F2 obtained by adsorption chromatography (experiment II) performed as well as the control diets (chow or olive oil), whereas, feeding Span RSO gave a slightly depressed growth rate which was not significant at the 1% level. In experiment III, male rats fed safflower oil, Span RSO, and fractions F1, F2, and F3 obtained by adsorption chromatography gained significantly less (P < 0.01) compared to rats fed corn oil. Rats fed chow or Tower RSO gained less than those fed corn oil but more than those fed Span RSO or fractions F1, F2, or F3. However, none of these difference were significant. The inclusion of RSO in the diet resulted in myocardial accumulation of fatty acids characteristic of these oils, such as 20:1, 22:1, and 18:3. There was considerable evidence of a
CARDIOPATHOGENICITY OF RAPESEED OILS
513
12
1 0 84
22:1 t ~ Y = -1.20 + 5.95X
20:1 Y = 0.96 + 2.09X
/
o
f- 8ill r z m
/
O
6-
9 ./2/
iii .d O 4"
. OO
'5
'5 MOLE % IN CARDIAC LIPIDS
FIG. 1. Relationship of 20:1 and 22:1 in the diet compared to the relative concentration of these acids in total cardiac lipids after 16 weeks on diets containing rapeseed oil (RSO) or fractions isolated from RSO. Each point represents the mean of 4 or more rats. linear relationship (r -- 0.85) between the dietary level of 22:1 and that found in total cardiac lipids after 16 weeks (Figure 1). The linear relationship was less evident (r = 0.57) with 20:1 (Figure 1). A possible explanation for this could be that 20:1 is derived from the diet and by ~-oxidation of 22:1 (15). Linolenic acid was found in the cardiac lipids of rats fed diets contalning this fatty acid. A comparison of the cardiac lipids of rats fed diets containing olive oil or RSO showed a remarkable similarity in the fatty acid profile of principal fatty acids. Diets containing corn or safflower oil, when fed to rats, gave significantly higher levels (P < 0.01) of 16:0 and 18:2 in the heart compared to rats fed RSO, while the level of 18:1 was lower (P < 0.01) and the relative abundance of 18:0 and 20:4 was not significantly different. No major difference was observed in the principal fatty acids in the hearts of rats f e d Span RSO compared to MD 5, 6, 7B, and 1", other than 20:1 and 22:1 being lower (P < 0.01) when MD l " was fed. With few exceptions, the same was evident in experiment II and III; no major difference was observed between the cardiac lipid composition of rats fed Span RSO and rats fed fractions F1, F2, and F3 from adsorption chromatography. The exceptions were 20:1 and 22:1, which were signifi-
cantly higher, (P < 0.01) when F1 was fed, and 18:3 which was higher when F3 was fed. In accordance with our previous observations (2), the results in Table II show that rats fed control diets had heart lesions, but the incidence and the severity was generally less than that observed in rats fed RSO or its fractions. In this experiment, when rats fed Oro and Span RSO were compared to rats fed chow or olive oil, neither incidence nor severity were significantly different at the 5% probability level. However, it should be pointed out that the incidence of lesions approached significant difference (P --- 0.06). As shown in Table II, fractions MD 7B and MD l " were highly cardiopathogenic (P < 0.OO1) compared to MD 5, MD 6, Span RSO, and Oro RSO, both in incidence and severity. Finally, the 25 rats fed the MD l " diet for 12 weeks showed significantly fewer lesions (P < 0.O1) than rats fed the same diet for 16 weeks; no difference was observed in severity between these 2 groups. The results in Table III show that the incidence of myocardial lesions was not different in the rats fed the pure triglyceride F2 and the original Span RSO. However, the incidence for both was significantly higher (P ' ( 0 . 0 0 1 ) than that observed in rats fed the control diets. This observation was confirmed by the results shown in Table IV, which in addition showed no difLIPIDS, VOL. 10, NO. 9
514
J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, AND A.H. CORNER TABLE II Myocardial L e s i o n s in Male Rats: Experiment I
Diet
Chow Olive Oro RSO b Span RSO MD5 c MD6 MD7B MDI" MDI"(12 wks)
Incidence
1-2
6/20 4/20 19/50 23/50 31150 24/50 40/50 19/25 10/25
4 4 9 13 17 8 9 8 3
• Analysis d Control Treated Control vs. treated Control vs. Oro, Span, MD5 & MD6 Control vs. Oro and Span MD7B and MDI" vs. Oro, Span, MD5, and MD6 MDI"(12 wks vs. 16 wks)
Severity index a 3-5 6-10 2 0 7
> 10
0 0 3
0 0 0
4
6
0
9 10 11 5 4
4 3 9 0 1
1 3 11 6 2
Incidence (d.f.) e 0.53 (1) 27.25*** (5) 14.72"** (1) 7.91"* (1) 3.67 (1)
Severity (d.f.) e 2.38 (1) 41.25"** (15) 8.71" (S) 5.92 (3) 4.96 (2)
21.06"** 6.82**
20.26*** 0.62
(1) (1)
(3) (3)
aSeverity index is the number of rats with lesion scores of 1-2, 3-5, 6-10 and >10 in 2 sections per heart. bRSO = Rapeseed oil. CMD = molecular distillate. dA comparison of rats affected to no. rats examined was used in the analysis of incidence. Only rats with heart lesions were compared in the analysis of severity. Significant difference: *(P < 0.05); **(P < 0.01); ***(P < 0.001). eThe degrees of freedom (d.f.) of these analyses were (r-l) (c-l), where r and c are the number of rows and columns, respectively. If an entire column was zero, the degrees of freedom were reduced accordingly. ference in t h e i n c i d e n c e o f m y o c a r d i a l lesions w i t h t h e f e e d i n g o f T o w e r RSO, S p a n R S O , F 1, F2, a n d F3. This t r e a t e d g r o u p s h o w e d a h i g h l y significant d i f f e r e n c e in i n c i d e n c e (P < 0 . 0 0 1 ) a n d severity (P < 0 . 0 1 ) o f h e a r t lesions w h e n c o m p a r e d t o t h e g r o u p o f rats fed c h o w , saff l o w e r oil, or c o r n oil. DISCUSSION
As i n d i c a t e d i n o u r p r e v i o u s p u b l i c a t i o n (2) a n d again e m p h a s i z e d h e r e , male r a t s fed c o n t r o l diets, i.e., c h o w , c o r n oil, olive oil, o r safflower oil, h a v e a d e f i n i t e i n c i d e n c e o f m y o c a r dial necrosis, w h i c h m a y in s o m e i n s t a n c e s app r o a c h t h a t o b s e r v e d in rats f e d O r o o r S p a n R S O ( T a b l e II). This w o u l d suggest t h a t t h e f a c t o r ( s ) r e s p o n s i b l e f o r m y o c a r d i a l lesions in male r a t s are n o t u n i q u e t o RSO. In general, h o w e v e r , w h e n R S O or f r a c t i o n s i s o l a t e d f r o m R S O were f e d t o male rats, t h e i n c i d e n c e o f m y o c a r d i a l necrosis was c o n s i d e r a b l y greater t h a n t h a t o b s e r v e d in rats f e d c o n t r o l diets (Tables III a n d IV). R o c q u e l i n a n d C l u z a n (1) were t h e first t o suggest t h a t a c a r d i o t o x i c f a c t o r m i g h t b e presLIPIDS, VOL. 10, NO. 9
e n t in t h e u n s a p o n i f i a b l e f r a c t i o n o f RSO. More r e c e n t l y , Beare-Rogers, et al., ( 1 6 ) c l a i m e d t o have c o n c e n t r a t e d this f a c t o r i n t h e d e o d o r i z e r c o n d e n s a t e f r o m B. campestris RSO. T h e resuits o f t h e p r e s e n t s t u d y deal w i t h t h e quest i o n o f w h i c h c o n s t i t u e n t o f R S O causes m y o cardial n e c r o s i s in male rats. F r o m t h e p r e s e n t w o r k , brassicasterol, characteristic o f t h e Cruciferae f a m i l y (17, 18), cert a i n l y c o u l d be e l i m i n a t e d as causing t h e s e p a t h o g e n i c results. This s t e r o l was n o t f o u n d i n triglyceride f r a c t i o n F 2 o b t a i n e d f r o m a d s o r p t i o n c h r o m a t o g r a p h y a n d was p r e s e n t in o n l y v e r y l o w levels i n MD 5, 6, a n d 7B, a n d y e t t h e s e f r a c t i o n s s h o w e d as great or greater incid e n c e of m y o c a r d i a l lesions t h a n t h e original oil. F u r t h e r m o r e , m a l e rats f e d f r a c t i o n s F1 a n d M D I " w h i c h were h i g h l y e n r i c h e d w i t h brassicasterol did n o t exclusively cause m y o c a r d i a l lesions c o m p a r e d t o f r a c t i o n s w h i c h were devoid o f this sterol. T h e g l u c o s i n o l a t e s ( 1 8 ) m a y also b e r u l e d o u t , b e c a u s e t h e s e c o m p o u n d s w o u l d be r e t a i n e d o n t h e a d s o r b e n t in a d s o r p t i o n c h r o m a t o g r a p h y a n d r e m a i n in t h e residue o f m o l e c u l a r distillation. Similarly, t e r p e n i c alc o h o l s ( 1 8 ) w o u l d be c o n c e n t r a t e d in t h e l i g h t
515
CARDIOPATHOGENICITY OF RAPESEED OILS TABLE III Myocardial Lesions in Male Rats: E x p e r i m e n t II Severity i nde xa Diet Chow Olive Span Rapeseed Oil F2b
Incidence
1-2
3-5
6-10
1/10 2/10 6/10
0 2 3
0 0 0
1 0 3
0 0 0
2
3
0
3
8/10
X2 Analysisc
Incidence (d.f.) d
Controls Treated Control vs. Treated
0.40 0.97 13.21"**
> 10
Severity (d.f.)
(1) (1) (1)
3.82 12.39"* 2.97
(2) (3) (3)
aSeverity index is the no. rats w i t h lesion scores of 1-2, 3-5, 6-10, or > 10 in 3 sections per heart. Note: 4 of the rats fed the diet contain i ng F2 had only 2 sections e xa mi ne d histologically. b F2 = pure triglyceride fraction obtained from Span Rapeseed oil by adsorption chromato grap hy. CSee F o o t n o t e d in Table II. dd.f. = degrees of freedom; see f o o t n o t e e in Table II. ** = (P < 0.01); *** = (P < 0.001). TABLE IV Myocardial Lesions in Male Rats: E x p e r i m e n t III Severity i nde x a Diet Chow Safflower Corn Tower RSO Span RSO F1 b F2 F3 X2 Analysisc Control Treated Control vs. trea ted
Incidence 11/20 7/20 9/20 15/20 17/20 19/20 16/20 16/20
1-2 5 7 4 8 2 3 10 7
3-5
6-10
4 0 3 2 1 6 1 2
1 0 1 2 3 3 2 4
Incidence (d.f.) d 1.63 3.80 24.99***
(2) (4) (1)
> 10 1 0 1 3 11 7 3 3
Severity (d.f.) 9.03 23.6 l * 11.55"*
(6) (12) (3)
aSeverity index is the no. rats with lesion scores of 1-2, 3-5, 6-10, or > 1 0 in 3 sections per heart. bF1, F2 an d F3 = fractions isolated from Span RSO by adsorption c hroma t ogra phy. CSee F o o t n o t e d in Table II. dd.f. = degrees of freedom; see f o o t n o t e e in Table II. * = (P < 0.05); ** = (P < 0.01); (*** = P < 0.00).
distillate MD 1" from molecular distillation and would be present in F3 obtained from adsorption chromatography. We conclude, therefore, because all the fractions tested caused myocardial lesions, and because triglycerides are the major constituents of all these fractions, that the triglycerides of RSO are the major components responsible for the myocardial lesions. In support of this, when RSO was tested for cardiotoxicity after various stages of commerical refinement, no decrease in cardiotoxicity was
observed (19), in spite of the fact, that during deodorization volatile compounds were removed.
Nevertheless, feeding MD 1", a fraction which contains many of the compounds present in the deodorizer condensate which was fed by B e a r e - R o g e r s , e t al., ( 1 6 ) , d i d gi ve a h i g h e r i n c i dence of myocardial necrosis. Such results must be interpreted with caution because both the deodorizer condensate and MD 1" still cont a i n e d 8 0 - 9 0 % m o n o - , di-, a n d t r i g l y c e r i d e s . LIPIDS, VOL. 10, NO. 9
516
J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, AND A.H. CORNER
However, the possibility that a second cardiotoxic factor not related to triglycerides may be present in this fraction cannot be excluded at this time.
and G.J.F. Armstrong provided technical assistance B. Thompson performed statistical analysis.
A n early c o n c e p t p r o p o s e d b y A b d e l l a t i f a n d Vles ( 7 , 2 0 ) suggests t h a t 22:1 a n d 20:1 f a t t y acids are t h e p r i m a r y agents r e s p o n s i b l e f o r t h e p a t h o g e n i c p r o p e r t i e s of RSO, because m y o c a r d i a l lesions c o u l d be r e p r o d u c e d b y f e e d i n g a 1:1 m i x t u r e o f s u n f l o w e r oil a n d crude t r i e r u c i n (84%, 2 2 : 1 ) . This view is n o longer t e n a b l e b e c a u s e n e w RSO varieties very l o w in 20:1 a n d 22:1 still result in significant i n c i d e n c e o f m y o c a r d i a l lesions w h e n fed t o male rats. Nevertheless, as i n d i c a t e d b y t h e p r e s e n t w o r k , as well as b y t h e results of R o c q u e l i n , et al., (21), w h e n t h e level of l o n g c h a i n m o n o e n e s e x c e e d s an, as y e t , u n s p e c i f i e d level, it c a n b y itself cause a n i n c r e a s e d incid e n c e of m y o c a r d i a l necrosis. F o r t h a t reason, a dose r e s p o n s e of m y o c a r d i a l lesions t o 22:1 c o u l d be e x p e c t e d b y f e e d i n g a R S O high in 22: 1, b u t n o t o n e low i n 2 2 : 1 . T h e results of Beare-Rogers, et al., ( 1 6 ) a p p e a r t o c o n f i r m this hypothesis. We c o n c l u d e t h a t t h e p r i m a r y m y o c a r d i o t o x i c f a c t o r o f RSO is in t h e triglyceride f r a c t i o n , b u t it m a y n o t b e due solely t o l o n g c h a i n m o n o e n o i c acids, i.e., 2 0 : 1 , 2 2 : 1 , a n d 24: 1. As suggested in o u r earlier w o r k (2), R S O triglycerides h a v e a c o m p o s i t i o n w h i c h is diff e r e n t f r o m c o r n , safflower, olive, or s o y b e a n oil. T h e s e d i f f e r e n c e s i n c l u d e altered s a t u r a t e d t o u n s a t u r a t e d r a t i o s w i t h l o w levels o f 1.6:0, a high concentration of 18:3, a n d a l o w 1 8 : 2 / 1 8 : 3 ratio. It seems possible t h a t R S O triglycerides h a v e a c o m b i n a t i o n o f p r o p e r t i e s t h a t are r e s p o n s i b l e f o r c a r d i o t o x i c i t y in male rats. In a d d i t i o n t o t h e p r o p e r t i e s listed above, o t h e r c h a r a c t e r i s t i c s u n i q u e t o R S O m a y include t h e p r e s e n c e o f n-7 f a t t y acids ( 2 2 ) a n d a n a s y m m e t r i c d i s t r i b u t i o n o f 18:3 in t h e triglycerides (23).
1. Rocquelin, G., and R. Cluzan, Ann. Biol. Anirm Biochim. Biophys. 8:395 (1968). 2. Kramer, J.K.G., S. Mahadevan, J.R. Hunt, F.D. Sauer, A.H. Corner, and K.M. Chaflton, J. Nutr. 103:1696 (1973). 3. Rocquelin, G., J.-P. Sergiel, P.O. Astorg, and R. Cluzan, Ann. Biol. Anim. Bioehim. Biophys. 13:587 (1973). 4. Rocquelin, G., R. Cluzan, N. Vodovar, and R. Lev/llain, Cah. Nut. Di~t 8:103 (1973). 5. Beare~Rogers, J,~., E.A. Nera, and H.A. Heggtveit, Can. Inst. Food Technol. J. 4:120 (1971). 6. Beare-Rogers, J.L., E.A. Nera, and B.M. Craig, Lipids 7:46 (1972). q. AbdeUatif, A.M.M., and R.O. Vies, Nutr. Metabol. 15:219 (1973). 8. Mattson, F.H., and J.A. Streck, J. Nutr. 104:483 (1974). 9. Nirdlinger, E.L., and P.O. Bramante, J. MoL cell Cardiol. 6:49 (1974). 10. Budowski, P., and K.S. Markley, Chem. Rex,. 48:125 (1951). 11. Nixon, J.E., T.A. Eisele, J.H. Wales, and R.O. Sinnhuber, Lipids 9:314 (1974). 12. Kramer, J.K.G., H.W. Hulan, S. Mahadevan, and F.D. Sauer, Ibid. 10:000 (1975). 13. Fienberg, S.E., Ecology 51:419 (1970). 14. Steel, R.G.D., and J.H. Torrie, "Principles and Procedures of Statistics," McGraw-Hill Book Co., New York, N.Y., 1960, pp. 107-109. 15. Craig, B.M., and J.L. Beare, Can. J. Bioehenl. 45:1075 (1967). 16. Beare-Rogers, J.L., E.A. Nera, and H.A. Heggtveit, Nutr. Metabol. 17:213 (1974). 17. Jacini, G., E. Fedeli, and A. Lanzani, J. Ass. Offic. Anal. Chem. 50:84 (1967). 18. Appelqvist, L.-A., and R. Ohlson (Ed.) "Rap#seed," Elsevier Publishing Co., Amsterdam, The Netherlands, 1972, pp. 123-173. 19. Charlton, K.M., A.H. Corner, K. Davey, J.K.G. Kramer, S. Mahadevan, and F.D. Sauer, Can. J. Comp. Med. 39:261 (1975).
ACKNOWLEDGMENTS A. Meunier cared for the animals and R.C. Fouchard
LIPIDS, VOL. 10, NO. 9
REFERENCES
20. Abdellatif, A.M.M., and R.O. Vies, Nutr. Metabol.
12:285 (1970). 21. Rocquelin, G., B. Martin, and R. Cluzan, Proc. Inter. Conf. Sci. Tech. and Marketing of Rapeseed and Rapeseed Products, Sept. 20-23, St. Addle, Quebec, Canada, 1970, pp. 405-422. 22. Ackman, R.G., JAOCS 43:483 (1966). 23. Brockerhoff, H., and M. Yurkowski, J. Lipid Res. 7:62 (1966). [ R e c e i v e d May 9, 1 9 7 5 ]
ABSTRACT
in nontriglyceride constituents. These fractions were fed to weanling male rats for 16 weeks. Hearts of these rats were examined for myocardial lesions and fatty acid composition.
Rapeseed oils low in erucic acid caused myocardial lesions when fed to weanling male rats for 16 weeks. The cardiopathogenic properties appear to be associated with the triglycerides of the oil, and not to nontriglyceride components present in fully refined rapeseed oil. Cardiac lipid analysis confirmed that erucic acid accumulation was proportional to the concentration of this acid in the diet.
MATERIALS AND METHODS
INTRODUCTION
Recent reports in the literature indicate that feeding rapeseed oil (RSO) to weanling male rats causes lipidosis in the heart within the first w e e k and myocardial lesions after several months, even when RSO low in erucic acid is fed (1-4). It has been shown that the early lipidosis in the heart is directly related to the erucic acid concentration in the dietary oils (2, 5-8). However, at present the cause of the necrotic and fibrotic lesions which occur after feeding RSO for 16 weeks is not understood. Myocardial necrosis has been reported in rats under such stress as catecholamine injection, dietary vitamin K deficiency, severe muscular exertion, exposure to cold and digitalis intoxication (9), or seamin, an alcohol soluble substance found in sesame oil (10). Feeding cyclopropenoid fatty acids is known to produce liver and kidney necrosis (11). The possibility may exist, therefore, that a nontriglyceride component is present in the completely refined RSO which is responsible for the necrotic and fibrotic lesions. On the other hand, the lesion producing properties of RSO may be attributable to its triglycerides. To establish whether the long term lesions are caused by the trigiycerides or some nontriglyceride component present in the oil, Brassica campestris var. Span RSO was fractionated by molecular distillation and adsorption chromatography, as described previously (12), to yield highly purified triglycerides and fractions rich 1Contribution no. 575 Animal Research Institute.
Male Sprague-Dawley rats (Bio-Breeding, Ottawa, Ontario) 3 weeks of age and weighing 40-50 g, were randomly selected, identified by ear markings, assigned 2 per cage, and fed the experimental diets for 16 weeks. The sernisynthetic diet consisted of 20% casein, 20% sucrose, 30% cornstarch, 1% vitamin mixture, 4% U.S.P. XVII mineral mixture, 5% pure wood cellulose, and 20% test oil (w/w). The following otis were used: corn oil (St. Lawrence Starch Co. Ltd., Port Credit, Ontario); olive oil (Pastene Co. Ltd., Montreal, Quebec); safflower oil (Teklad Mills, Madison, Wisc.); B. napus var. Oro and B. campestris var. Tower (Cooperative Vegetable Oil Ltd., Altona, Manitoba); and B. campestris var. Span (Western Canada Processors, Lethbridge, Alberta). The fractions obtained from Span RSO by molecular distillation (MD) 5, 6, 7B, and 1" and b y adsorption chromatography F I , F2, and F3 have been described previously (12). Experiment I consisted of 2 groups of 20 rats fed Purina Laboratory Rat Chow or semisynthetic diet containing olive oil, and 6 groups of 50 rats fed sernisynthetic diet containing Oro RSO, Span RSO, or MD 5, 6, 7B and 1", from Span RSO (12). Because o f the limited supply of MD 1", 25 of the 50 rats were killed after 12 weeks and their hearts examined histologically. The remaining 25 rats were kept the entire 16 weeks on this diet. After 16 weeks, all rats were killed and their hearts examined histologically. One quarter of the heart was removed for lipid analyses from 10 rats on each diet; the remaining portion of these hearts was prepared for histological examination. Experiment II consisted of 4 groups of 10 rats fed chow or semisynthetic diet containing olive oil, Span RSO, or triglyceride fraction F2 obtained from Span RSO by adsorption chromatography (12). Rats were killed after 16 weeks on treatment and hearts removed for
511
512
J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, AND A.H. CORNER TABLE I Fatty Acid Composition of Chow and Dietary Oils Diets Fatty acids
Chowa
Olive
Corn
16.0 16:1 18:0 18:1 18:2 18:3 20:1 22:1 24:1
16.9 1.8 3.2 22.4 38.9 3.1 2.9 3.7
11.9 0.7 2.9 73.4 8.9 0.8 0.4 0.4
15.0 0.1 2.6 27.8 52.7 0.6 0.3
Safflower 9.9 0.1 3.4 13.9 71.9 0.2 0.2
Tower
Oro
Span
5.2 0.2 1.8 56.9 24.0 8.4 1.3 0.8 0.2
5.0 0.3 2.5 62.2 18.9 6.7 1.9 1.6 0.2
4.8 0.2 2.1 58.6 19.5 5.2 3.3 4.8 0.2
aChow contained fish meal as part of its formulation. The fatty acid composition is that of the total lipids extracted from the diet with chloroform: methanol (2:1). lipid analysis and histological examination. Experiment III consisted of 8 groups of 24 rats fed chow or sernisynthetic diet containing corn oil, safflower oil, Tower RSO, Span RSO, or fraction F1, F2, and F3 obtained from Span RSO by adsorption chromatography (12). All rats were killed after 16 weeks on these diets; 20 rats were examined histologically and 4 rats were used for lipid analyses. For histological examination hearts were fixed in 10% neutral buffered formalin. Three sections were prepared from each heart: a) one central section extending from apex to base and including interventricular septum, atrial and ventricular walls; b) a section parallel to the first and extending through the right ventricle; and c) a third section parallel to the other two and extending through the left ventricle. The 3 sections were equidistant from each other. From these, 6/a sections were made and stained with hematoxylin and eosin. All experimental rats were numerically coded and not identified until the histological examination was completed. The results were reported as the number of rats affected, as well as the number of lesions per affected heart. No attempt was made to grade individual lesion as to pathological severity. Instead, groups were made to include rats which had 1-2, 3-5, 6-10, and > 1 0 lesions per heart. Approximate Chisquare statistics, obtained following the approach of Fienberg (13) were used to examine the frequency tables (Tables II-IV) for evidence of differences in patterns of incidence and severity among the various diets. The techniques for homogenization of rat hearts, extraction of lipids, and preparation and analysis of fatty acid methyl esters have been described (2). Analysis of variance was performed on cardiac lipids and wt gains and significant difference at the 1% level (P < 0.01) LIPIDS, VOL. 10, NO. 9
were determined Range test (14).
using Duncan's Multiple
RESULTS
The fatty acid composition of chow, corn oil, olive oil, safflower oil, Tower RSO, Oro RSO, and Span RSO is shown in Table I. The composition of the fractions isolated from Span RSO by molecular distillation and adsorption chromatography are fully described in an accompanying publication (12). Oro and Tower RSO were fully refined rapeseed oils low in 20:1 and 22:1 with a total sterol concentration of 0.29 and 0.23%, respectively. Growth rates of male rats fed MD 1" were significantly lower (P < 0.01) compared to rats fed the diet containing olive off (experiment I). The wt gains of rats fed Oro RSO, Span RSO, or MD 5, 6, and 7B (purified triglycerides) were slightly lower than, but not significantly different (P < , 0 . 0 1 ) from the control diets (chow and olive oil), or MD 1 ". Male rats fed the purified triglyceride fraction F2 obtained by adsorption chromatography (experiment II) performed as well as the control diets (chow or olive oil), whereas, feeding Span RSO gave a slightly depressed growth rate which was not significant at the 1% level. In experiment III, male rats fed safflower oil, Span RSO, and fractions F1, F2, and F3 obtained by adsorption chromatography gained significantly less (P < 0.01) compared to rats fed corn oil. Rats fed chow or Tower RSO gained less than those fed corn oil but more than those fed Span RSO or fractions F1, F2, or F3. However, none of these difference were significant. The inclusion of RSO in the diet resulted in myocardial accumulation of fatty acids characteristic of these oils, such as 20:1, 22:1, and 18:3. There was considerable evidence of a
CARDIOPATHOGENICITY OF RAPESEED OILS
513
12
1 0 84
22:1 t ~ Y = -1.20 + 5.95X
20:1 Y = 0.96 + 2.09X
/
o
f- 8ill r z m
/
O
6-
9 ./2/
iii .d O 4"
. OO
'5
'5 MOLE % IN CARDIAC LIPIDS
FIG. 1. Relationship of 20:1 and 22:1 in the diet compared to the relative concentration of these acids in total cardiac lipids after 16 weeks on diets containing rapeseed oil (RSO) or fractions isolated from RSO. Each point represents the mean of 4 or more rats. linear relationship (r -- 0.85) between the dietary level of 22:1 and that found in total cardiac lipids after 16 weeks (Figure 1). The linear relationship was less evident (r = 0.57) with 20:1 (Figure 1). A possible explanation for this could be that 20:1 is derived from the diet and by ~-oxidation of 22:1 (15). Linolenic acid was found in the cardiac lipids of rats fed diets contalning this fatty acid. A comparison of the cardiac lipids of rats fed diets containing olive oil or RSO showed a remarkable similarity in the fatty acid profile of principal fatty acids. Diets containing corn or safflower oil, when fed to rats, gave significantly higher levels (P < 0.01) of 16:0 and 18:2 in the heart compared to rats fed RSO, while the level of 18:1 was lower (P < 0.01) and the relative abundance of 18:0 and 20:4 was not significantly different. No major difference was observed in the principal fatty acids in the hearts of rats f e d Span RSO compared to MD 5, 6, 7B, and 1", other than 20:1 and 22:1 being lower (P < 0.01) when MD l " was fed. With few exceptions, the same was evident in experiment II and III; no major difference was observed between the cardiac lipid composition of rats fed Span RSO and rats fed fractions F1, F2, and F3 from adsorption chromatography. The exceptions were 20:1 and 22:1, which were signifi-
cantly higher, (P < 0.01) when F1 was fed, and 18:3 which was higher when F3 was fed. In accordance with our previous observations (2), the results in Table II show that rats fed control diets had heart lesions, but the incidence and the severity was generally less than that observed in rats fed RSO or its fractions. In this experiment, when rats fed Oro and Span RSO were compared to rats fed chow or olive oil, neither incidence nor severity were significantly different at the 5% probability level. However, it should be pointed out that the incidence of lesions approached significant difference (P --- 0.06). As shown in Table II, fractions MD 7B and MD l " were highly cardiopathogenic (P < 0.OO1) compared to MD 5, MD 6, Span RSO, and Oro RSO, both in incidence and severity. Finally, the 25 rats fed the MD l " diet for 12 weeks showed significantly fewer lesions (P < 0.O1) than rats fed the same diet for 16 weeks; no difference was observed in severity between these 2 groups. The results in Table III show that the incidence of myocardial lesions was not different in the rats fed the pure triglyceride F2 and the original Span RSO. However, the incidence for both was significantly higher (P ' ( 0 . 0 0 1 ) than that observed in rats fed the control diets. This observation was confirmed by the results shown in Table IV, which in addition showed no difLIPIDS, VOL. 10, NO. 9
514
J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, AND A.H. CORNER TABLE II Myocardial L e s i o n s in Male Rats: Experiment I
Diet
Chow Olive Oro RSO b Span RSO MD5 c MD6 MD7B MDI" MDI"(12 wks)
Incidence
1-2
6/20 4/20 19/50 23/50 31150 24/50 40/50 19/25 10/25
4 4 9 13 17 8 9 8 3
• Analysis d Control Treated Control vs. treated Control vs. Oro, Span, MD5 & MD6 Control vs. Oro and Span MD7B and MDI" vs. Oro, Span, MD5, and MD6 MDI"(12 wks vs. 16 wks)
Severity index a 3-5 6-10 2 0 7
> 10
0 0 3
0 0 0
4
6
0
9 10 11 5 4
4 3 9 0 1
1 3 11 6 2
Incidence (d.f.) e 0.53 (1) 27.25*** (5) 14.72"** (1) 7.91"* (1) 3.67 (1)
Severity (d.f.) e 2.38 (1) 41.25"** (15) 8.71" (S) 5.92 (3) 4.96 (2)
21.06"** 6.82**
20.26*** 0.62
(1) (1)
(3) (3)
aSeverity index is the number of rats with lesion scores of 1-2, 3-5, 6-10 and >10 in 2 sections per heart. bRSO = Rapeseed oil. CMD = molecular distillate. dA comparison of rats affected to no. rats examined was used in the analysis of incidence. Only rats with heart lesions were compared in the analysis of severity. Significant difference: *(P < 0.05); **(P < 0.01); ***(P < 0.001). eThe degrees of freedom (d.f.) of these analyses were (r-l) (c-l), where r and c are the number of rows and columns, respectively. If an entire column was zero, the degrees of freedom were reduced accordingly. ference in t h e i n c i d e n c e o f m y o c a r d i a l lesions w i t h t h e f e e d i n g o f T o w e r RSO, S p a n R S O , F 1, F2, a n d F3. This t r e a t e d g r o u p s h o w e d a h i g h l y significant d i f f e r e n c e in i n c i d e n c e (P < 0 . 0 0 1 ) a n d severity (P < 0 . 0 1 ) o f h e a r t lesions w h e n c o m p a r e d t o t h e g r o u p o f rats fed c h o w , saff l o w e r oil, or c o r n oil. DISCUSSION
As i n d i c a t e d i n o u r p r e v i o u s p u b l i c a t i o n (2) a n d again e m p h a s i z e d h e r e , male r a t s fed c o n t r o l diets, i.e., c h o w , c o r n oil, olive oil, o r safflower oil, h a v e a d e f i n i t e i n c i d e n c e o f m y o c a r dial necrosis, w h i c h m a y in s o m e i n s t a n c e s app r o a c h t h a t o b s e r v e d in rats f e d O r o o r S p a n R S O ( T a b l e II). This w o u l d suggest t h a t t h e f a c t o r ( s ) r e s p o n s i b l e f o r m y o c a r d i a l lesions in male r a t s are n o t u n i q u e t o RSO. In general, h o w e v e r , w h e n R S O or f r a c t i o n s i s o l a t e d f r o m R S O were f e d t o male rats, t h e i n c i d e n c e o f m y o c a r d i a l necrosis was c o n s i d e r a b l y greater t h a n t h a t o b s e r v e d in rats f e d c o n t r o l diets (Tables III a n d IV). R o c q u e l i n a n d C l u z a n (1) were t h e first t o suggest t h a t a c a r d i o t o x i c f a c t o r m i g h t b e presLIPIDS, VOL. 10, NO. 9
e n t in t h e u n s a p o n i f i a b l e f r a c t i o n o f RSO. More r e c e n t l y , Beare-Rogers, et al., ( 1 6 ) c l a i m e d t o have c o n c e n t r a t e d this f a c t o r i n t h e d e o d o r i z e r c o n d e n s a t e f r o m B. campestris RSO. T h e resuits o f t h e p r e s e n t s t u d y deal w i t h t h e quest i o n o f w h i c h c o n s t i t u e n t o f R S O causes m y o cardial n e c r o s i s in male rats. F r o m t h e p r e s e n t w o r k , brassicasterol, characteristic o f t h e Cruciferae f a m i l y (17, 18), cert a i n l y c o u l d be e l i m i n a t e d as causing t h e s e p a t h o g e n i c results. This s t e r o l was n o t f o u n d i n triglyceride f r a c t i o n F 2 o b t a i n e d f r o m a d s o r p t i o n c h r o m a t o g r a p h y a n d was p r e s e n t in o n l y v e r y l o w levels i n MD 5, 6, a n d 7B, a n d y e t t h e s e f r a c t i o n s s h o w e d as great or greater incid e n c e of m y o c a r d i a l lesions t h a n t h e original oil. F u r t h e r m o r e , m a l e rats f e d f r a c t i o n s F1 a n d M D I " w h i c h were h i g h l y e n r i c h e d w i t h brassicasterol did n o t exclusively cause m y o c a r d i a l lesions c o m p a r e d t o f r a c t i o n s w h i c h were devoid o f this sterol. T h e g l u c o s i n o l a t e s ( 1 8 ) m a y also b e r u l e d o u t , b e c a u s e t h e s e c o m p o u n d s w o u l d be r e t a i n e d o n t h e a d s o r b e n t in a d s o r p t i o n c h r o m a t o g r a p h y a n d r e m a i n in t h e residue o f m o l e c u l a r distillation. Similarly, t e r p e n i c alc o h o l s ( 1 8 ) w o u l d be c o n c e n t r a t e d in t h e l i g h t
515
CARDIOPATHOGENICITY OF RAPESEED OILS TABLE III Myocardial Lesions in Male Rats: E x p e r i m e n t II Severity i nde xa Diet Chow Olive Span Rapeseed Oil F2b
Incidence
1-2
3-5
6-10
1/10 2/10 6/10
0 2 3
0 0 0
1 0 3
0 0 0
2
3
0
3
8/10
X2 Analysisc
Incidence (d.f.) d
Controls Treated Control vs. Treated
0.40 0.97 13.21"**
> 10
Severity (d.f.)
(1) (1) (1)
3.82 12.39"* 2.97
(2) (3) (3)
aSeverity index is the no. rats w i t h lesion scores of 1-2, 3-5, 6-10, or > 10 in 3 sections per heart. Note: 4 of the rats fed the diet contain i ng F2 had only 2 sections e xa mi ne d histologically. b F2 = pure triglyceride fraction obtained from Span Rapeseed oil by adsorption chromato grap hy. CSee F o o t n o t e d in Table II. dd.f. = degrees of freedom; see f o o t n o t e e in Table II. ** = (P < 0.01); *** = (P < 0.001). TABLE IV Myocardial Lesions in Male Rats: E x p e r i m e n t III Severity i nde x a Diet Chow Safflower Corn Tower RSO Span RSO F1 b F2 F3 X2 Analysisc Control Treated Control vs. trea ted
Incidence 11/20 7/20 9/20 15/20 17/20 19/20 16/20 16/20
1-2 5 7 4 8 2 3 10 7
3-5
6-10
4 0 3 2 1 6 1 2
1 0 1 2 3 3 2 4
Incidence (d.f.) d 1.63 3.80 24.99***
(2) (4) (1)
> 10 1 0 1 3 11 7 3 3
Severity (d.f.) 9.03 23.6 l * 11.55"*
(6) (12) (3)
aSeverity index is the no. rats with lesion scores of 1-2, 3-5, 6-10, or > 1 0 in 3 sections per heart. bF1, F2 an d F3 = fractions isolated from Span RSO by adsorption c hroma t ogra phy. CSee F o o t n o t e d in Table II. dd.f. = degrees of freedom; see f o o t n o t e e in Table II. * = (P < 0.05); ** = (P < 0.01); (*** = P < 0.00).
distillate MD 1" from molecular distillation and would be present in F3 obtained from adsorption chromatography. We conclude, therefore, because all the fractions tested caused myocardial lesions, and because triglycerides are the major constituents of all these fractions, that the triglycerides of RSO are the major components responsible for the myocardial lesions. In support of this, when RSO was tested for cardiotoxicity after various stages of commerical refinement, no decrease in cardiotoxicity was
observed (19), in spite of the fact, that during deodorization volatile compounds were removed.
Nevertheless, feeding MD 1", a fraction which contains many of the compounds present in the deodorizer condensate which was fed by B e a r e - R o g e r s , e t al., ( 1 6 ) , d i d gi ve a h i g h e r i n c i dence of myocardial necrosis. Such results must be interpreted with caution because both the deodorizer condensate and MD 1" still cont a i n e d 8 0 - 9 0 % m o n o - , di-, a n d t r i g l y c e r i d e s . LIPIDS, VOL. 10, NO. 9
516
J.K.G. KRAMER, H.W. HULAN, S. MAHADEVAN, F.D. SAUER, AND A.H. CORNER
However, the possibility that a second cardiotoxic factor not related to triglycerides may be present in this fraction cannot be excluded at this time.
and G.J.F. Armstrong provided technical assistance B. Thompson performed statistical analysis.
A n early c o n c e p t p r o p o s e d b y A b d e l l a t i f a n d Vles ( 7 , 2 0 ) suggests t h a t 22:1 a n d 20:1 f a t t y acids are t h e p r i m a r y agents r e s p o n s i b l e f o r t h e p a t h o g e n i c p r o p e r t i e s of RSO, because m y o c a r d i a l lesions c o u l d be r e p r o d u c e d b y f e e d i n g a 1:1 m i x t u r e o f s u n f l o w e r oil a n d crude t r i e r u c i n (84%, 2 2 : 1 ) . This view is n o longer t e n a b l e b e c a u s e n e w RSO varieties very l o w in 20:1 a n d 22:1 still result in significant i n c i d e n c e o f m y o c a r d i a l lesions w h e n fed t o male rats. Nevertheless, as i n d i c a t e d b y t h e p r e s e n t w o r k , as well as b y t h e results of R o c q u e l i n , et al., (21), w h e n t h e level of l o n g c h a i n m o n o e n e s e x c e e d s an, as y e t , u n s p e c i f i e d level, it c a n b y itself cause a n i n c r e a s e d incid e n c e of m y o c a r d i a l necrosis. F o r t h a t reason, a dose r e s p o n s e of m y o c a r d i a l lesions t o 22:1 c o u l d be e x p e c t e d b y f e e d i n g a R S O high in 22: 1, b u t n o t o n e low i n 2 2 : 1 . T h e results of Beare-Rogers, et al., ( 1 6 ) a p p e a r t o c o n f i r m this hypothesis. We c o n c l u d e t h a t t h e p r i m a r y m y o c a r d i o t o x i c f a c t o r o f RSO is in t h e triglyceride f r a c t i o n , b u t it m a y n o t b e due solely t o l o n g c h a i n m o n o e n o i c acids, i.e., 2 0 : 1 , 2 2 : 1 , a n d 24: 1. As suggested in o u r earlier w o r k (2), R S O triglycerides h a v e a c o m p o s i t i o n w h i c h is diff e r e n t f r o m c o r n , safflower, olive, or s o y b e a n oil. T h e s e d i f f e r e n c e s i n c l u d e altered s a t u r a t e d t o u n s a t u r a t e d r a t i o s w i t h l o w levels o f 1.6:0, a high concentration of 18:3, a n d a l o w 1 8 : 2 / 1 8 : 3 ratio. It seems possible t h a t R S O triglycerides h a v e a c o m b i n a t i o n o f p r o p e r t i e s t h a t are r e s p o n s i b l e f o r c a r d i o t o x i c i t y in male rats. In a d d i t i o n t o t h e p r o p e r t i e s listed above, o t h e r c h a r a c t e r i s t i c s u n i q u e t o R S O m a y include t h e p r e s e n c e o f n-7 f a t t y acids ( 2 2 ) a n d a n a s y m m e t r i c d i s t r i b u t i o n o f 18:3 in t h e triglycerides (23).
1. Rocquelin, G., and R. Cluzan, Ann. Biol. Anirm Biochim. Biophys. 8:395 (1968). 2. Kramer, J.K.G., S. Mahadevan, J.R. Hunt, F.D. Sauer, A.H. Corner, and K.M. Chaflton, J. Nutr. 103:1696 (1973). 3. Rocquelin, G., J.-P. Sergiel, P.O. Astorg, and R. Cluzan, Ann. Biol. Anim. Bioehim. Biophys. 13:587 (1973). 4. Rocquelin, G., R. Cluzan, N. Vodovar, and R. Lev/llain, Cah. Nut. Di~t 8:103 (1973). 5. Beare~Rogers, J,~., E.A. Nera, and H.A. Heggtveit, Can. Inst. Food Technol. J. 4:120 (1971). 6. Beare-Rogers, J.L., E.A. Nera, and B.M. Craig, Lipids 7:46 (1972). q. AbdeUatif, A.M.M., and R.O. Vies, Nutr. Metabol. 15:219 (1973). 8. Mattson, F.H., and J.A. Streck, J. Nutr. 104:483 (1974). 9. Nirdlinger, E.L., and P.O. Bramante, J. MoL cell Cardiol. 6:49 (1974). 10. Budowski, P., and K.S. Markley, Chem. Rex,. 48:125 (1951). 11. Nixon, J.E., T.A. Eisele, J.H. Wales, and R.O. Sinnhuber, Lipids 9:314 (1974). 12. Kramer, J.K.G., H.W. Hulan, S. Mahadevan, and F.D. Sauer, Ibid. 10:000 (1975). 13. Fienberg, S.E., Ecology 51:419 (1970). 14. Steel, R.G.D., and J.H. Torrie, "Principles and Procedures of Statistics," McGraw-Hill Book Co., New York, N.Y., 1960, pp. 107-109. 15. Craig, B.M., and J.L. Beare, Can. J. Bioehenl. 45:1075 (1967). 16. Beare-Rogers, J.L., E.A. Nera, and H.A. Heggtveit, Nutr. Metabol. 17:213 (1974). 17. Jacini, G., E. Fedeli, and A. Lanzani, J. Ass. Offic. Anal. Chem. 50:84 (1967). 18. Appelqvist, L.-A., and R. Ohlson (Ed.) "Rap#seed," Elsevier Publishing Co., Amsterdam, The Netherlands, 1972, pp. 123-173. 19. Charlton, K.M., A.H. Corner, K. Davey, J.K.G. Kramer, S. Mahadevan, and F.D. Sauer, Can. J. Comp. Med. 39:261 (1975).
ACKNOWLEDGMENTS A. Meunier cared for the animals and R.C. Fouchard
LIPIDS, VOL. 10, NO. 9
REFERENCES
20. Abdellatif, A.M.M., and R.O. Vies, Nutr. Metabol.
12:285 (1970). 21. Rocquelin, G., B. Martin, and R. Cluzan, Proc. Inter. Conf. Sci. Tech. and Marketing of Rapeseed and Rapeseed Products, Sept. 20-23, St. Addle, Quebec, Canada, 1970, pp. 405-422. 22. Ackman, R.G., JAOCS 43:483 (1966). 23. Brockerhoff, H., and M. Yurkowski, J. Lipid Res. 7:62 (1966). [ R e c e i v e d May 9, 1 9 7 5 ]
