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Experimental atherosclerosis and hyperlipidemia in rats and rabbits: Influence of some alimentary fats
186
JOURNAL OF ATHEROSCLEROSIS RESEARCH
E X P E R I M E N T A L A T H E R O S C L E R O S I S AND H Y P E R L I P I D E M I A IN RATS AND RABBITS: I N F L U E N C E OF SOME A L I M E N T A R Y FATS O. L I B E R T AND C. ROGG-EFFRONT
Battelle Memorial Institute, Geneva (Switzerland) (Received December 22nd, 1961)
INTRODUCTION
An enormous amount of population statistics and of clinical and experimental data has led to the concept of the importance of diet, and especially of dietary fats, in the genesis of atherosclerosis 1-4, although it is now well recognised that lipids are not the cause, but probably a cause oi this illness 5. As cholesterolemia and atherosclerosis seemed to show a positive relationship in experimentation as well as in clinics, the action of those alimentary products which lower the level of cholesterol in blood was studied. KINSELL et al.6 showed that vegetable oils, which are rich in unsaturated f a t t y acids, could lower the level of cholesterolemia, whereas animal fats, which are rich in saturated f a t t y acids, augmented it. This fact was noted by m a n y authors, and BRONTE-STEWART et al. 7 showed that hydrogenated oils and animal fats augmented cholesterolemia in a male Bantu, whereas natural oils reduced it. I t was afterwards reported that corn oil could be particularly active in lowering the level of cholesterol in blood 7-12. However, RHOADS AND BARKER13 found corn oil to be only slightly active. The activity of corn oil was first related to its high content of linoleic acid and of other so-called "essential f a t t y acids". This theory was not clearly proved, and no definite relationship between the deficiency in essential f a t t y acids and vascular illnes was shown 14. Furthermore, soya oil, which is rich in unsaturated fatty acids, bears only very controversial activities against hypercholesterolemia and atherosclerosis in man 15. On account of these data it seemed of interest to make a comparative study of influences of different kinds of fat on experimental atherosclerosis and hypercholesterolemia. The main purpose of the present research was to compare, from this double point of view, the action of an oil which is rich in unsaturated f a t t y acids (corn oil), an equilibrated one (peanut oil), a hardened fat (margarine) and an animal fat (lard), on two animal species: (a) The rat, which is very resistant to atheromatosis, but in which it is possible to reproduce, with special diets or treatments, vascular abnormalities resembling those observed in manl6,17. Our data are concerned with 474 autopsied rats. (b) The rabbit, in which experimental atherosclerosis was first produced is, b y feeding cholesterol. This animal is very sensitive to dietary components: WIGAND19 showed J. Atheroscler. Res., 2 (1962) 186-198
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
187
in 1959 that feeding various fats in excess to rabbits without any cholesterol is sufficient to produce pronounced atherosclerotic lesions. Our data are concerned with 44 autopsied rabbits.
MATERIAL AND METHODS
Diets Rat. We chose to follow WILGRAM'S diet 16 as closely as possible; it was he who produced lipomatous coronary changes in 88 % of the rats he had treated for 6 to 12 months. The composition of our "inductive" diet was as follows:
basic diet egg yolk powder cholesterol sodium cholate propylthiouracil vitamin D (400,000 U/g)
85 % 12 % 2 % 0.2 % 0.075 % 0.35 %
Our basic diet was a mixture of cereals, alfalfa and fish flours (proteins 23 %; lipids 1.2 %; cellulose 5.5 %; carbohydrates 70 %). Rabbit. We followed approximately the work of JUSHCHENKO2°, the daily diet being as follows: each rabbit ate daily 500 g carrots and 50 g bran, in which 2 g cholesterol, dissolved b y gentle heating when there was an added oil, was mixed intimately. Fats added to tile diet consisted of lard, peanut oil, corn oil and margarine. Three different sources of peanut oil were used, namely: peanut oil (a) = natural peanut oil; peanut oil (b) = natural peanut oil submitted to a refinement process, consisting of demucilagination b y water and NaC1, neutralization of the free f a t t y acids with NaOH, water washing, decolorization with active earths at 90°C under vacuum, filtration and deodorization at 200°C under vacuum, then under water vapour, cooling to 40°C under vacuum to avoid oxidation; and peanut oil (c) = synthetic peanut triglycerides, imitating the fatty acid composition of natural peanut oil. The percentage fatty acid composition (main components) of the fats used in the experiment is shown below: Saturated acids
(C8+ Clo+ C1~) P e a n u t oil Corn oil Margarine Lard
0.5 1.0 32.1
Palmitie acid
lO.O 12.5 11.8 26.0
Stearic acid
2.9 2.0 3.2 19.4
Oleic acid
Linoleic acid
54 30 32.8 54.6
26 55 8.6 --
j . Atheroscler. Res., 2 (1962) 186-198
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O. LIBERT, C. ROGG-EFFRONT
Animal Groups Rat. 700 male adult rats were distributed at random in the following 14 groups: AC, absolute controls IC, "induction controls" L5 and L25 Pa 5 and Pa 25 Pb5 and Pb25 Pc5 and Pc25 CO5 and C025 M5 and M25
basic diet "inductive" "inductive" "inductive" "inductive" "inductive" "inductive" "inductive"
diet diet diet diet diet diet diet
+ + + + + +
lard, 5 % and 25 % resp. peanut oil (a), 5 % and 25 % resp. peanut oil (b), 5 % and 25 % resp. peanut oil (c), 5 % and 25 % resp. corn oil 5 % and 25 % resp. margarine 5 % and 25 % resp.
Rabbit. 48 adult rabbits (both sexes, distributed at random) were grouped as follows: AC CC Pa Pb Pc CO
absolute controls: basic diet (bran and carrots) "cholesterol" controls: basic diet + cholesterol (without fat) basic diet + cholesterol + peanut oil (a) basic diet + cholesterol + peanut oil (b) basic diet + cholesterol + peanut oil (c) basic diet + cholesterol + corn oil
Autopsies Rats were autopsied in 3 series; i.e. after 2, 4 and 6 months' treatment. Rabbits were all autopsied after the 2nd month. All the animals were autopsied after they had fasted for 24 hours. They were anesthetized with nembutal (50 mg/kg); blood was taken from the left carotid artery (5-10 ml for rats, 20 ml or more for rabbits); the aortas were removed immediately and were fixed in formalin 10 %. In every case the external and internal aspect of the animal, the extent of f a t t y depots and the appearance of steatosis in liver were observed; the animals were examined for various possible infections.
Chemical Determinations in Serum The following were measured in the sera of both rats and rabbits: total and esterified cholesterol21; esterified f a t t y acids22; phospholipids2~,24; lipoproteins, measured by paper electrophoresis 25. The ratio fl/a lipoproteins was calculated.
Morphological Determination Rat. Histological sections were made b y means of a frozen microtome in three parts of the aorta (aortic arch = aorta 1, thoracic segment ----aorta 2, abdominal segment = aorta 3). These sections were stained with Sudan IV and with haematoxylin. About 30,000 slides were examined in all. The atheromatous lesions were defined as in RANDOIN AND BRUN2~ and were visualized on our slides as sudanophilic inclusions. J. Atheroscler. Res., 2 (1962) 186-198
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The statistical analyses were based on an "atheromatous incidence", defined as the percentage of rats bearing at least one atheroma in a given group. Quantitative analyses were also tentatively tried, and the "atheromatous coefficient" was calculated as the mean score of lesions observed in the three aortic segments in one animal, or in one group of animals (gradation: ± = 0.5; + --~ 1; + + = 2; + + + ----3). Rabbit. For the rabbits, aortas were observed in toto, after having been cut into two halves and fixed in formalin 10 %. The internal sides of the aortas were photographed and were then graded from ± (0.5) to + + + + (4). Statistical Analyses Statistical analyses were made of all data, using non-parametric methods, especially the WILCOXON-MANN-WHITNEYtest. RESULTS
Rat Growth and mortality. The animals which received the "inductive diet" alone continually lost weight; they also showed the highest mortality and it was not possible to draw the curves after the 4th month of treatment, the number of living animals being reduced to about 10 instead of 20 to 25 in the other groups*). This growth-inhibiting action may possibly be due to the presence of propylthiouracil in the diet as BEST et al. 27 observed a similar effect with thiouracil; it is well known that thyroid hypofunction generally inhibits growth. With diets containing any of the added fats, even in small doses, this weight loss was neutralized, until the 10th week of treatment; after this time we observed a constant weight loss in all the groups which received the "inductive diet" even with fat supplementation. There was no observable difference in mortality between the different fat-treated groups. Examination of rats at autopsy. This examination showed no difference in fatty depots, liver steatosis and various infections between the different groups of animals which received the "inductive diet". Lipids in serum. The large scatter of the results did not allow of any other interpretation than the statistical one. We compared statistically: (a) the six groups which received peanut oil with the six other groups of fat; (b) all the twelve fat-treated groups with the "induction" group. For the analysis of the statistical data, we defined: au = probability of error for the unilateral test (1-tailed test); ab = probability of error for the bilateral test (2-tailed test). The statistical results allow the following comments to be made: After two months of treatment, hyperlipidemia (esterified fatty acids, cholesterol and phospholipids) was higher if the animals had received peanut oil than if they had * Readers interested in further details of these and other data referred to should request copies of figures and tables, J. Atheroscler. Res., 2 (1962) 186, from the publishers. J. Atheroscler. Res., 2 (1962) 186-198
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O. LIBERT, C. ROGG-EFFRONT
received any of the three other fats. The significant data were much more numerous for the 25 % supplementation than for the 5 % supplementation ( a b < 5 %). All the six sorts of fat inhibited the hyperlipidenfia produced b y the "inductive" diet ( a b < 0 . 1 % in most cases, always < 5 %). After four months' treatment there was no longer a preferential hyperlipidemic activity of the three kinds of peanut oil, as compared with the other dietary fats; there were few statistically significant results for esterified fatty acids and cholesterol, and these few were contradictory; on the other hand we noticed a significant increase in the concentration of phospholipids with lard and margarine as compared with peanut oil ( a b < 0 . 1 % in every significant case). Lard, margarine and corn oil gave a more pronounced hyperlipidemia than did the inductive diet alone, for both supplementations (esterified fatty acids and cholesterol: 5 % > a b > 1 % ) . Considering the peanut oil group at high dosage, only Pa25 gave a significant augmentation as compared with the induction controls for esterified fatty acids (a~ = 0.002 %); there was no significant result for cholesterol; at low dosages, every augmentation was significant except with Pb5 (2 % > a 0 > 0 . 2 % for all the groups other than Pb5). Tbus at that moment of the experiment peanut oil showed in general less tendency to cause hyperlipidemia than the other groups of fat. After six months' treatment the only significant differences between groups could be found for peanut oil, which was generally less hyperlipidemic than the other fats. However, the C/P ratio was lower with lard than with peanut oil; this latter fact is due to the very high serum phospholipids shown b y the lard groups. The significant differences between peanut oil and the other fats were the following: Esterified fatty acids
Total cholesterol
Phospholipids
C/P ratio
Pa 25 < L 25 Pa 2 5 < C 0 25 Pa25< M25 Pa 2 5 < L 25 Pa 2 5 < C O 25 Pb25< L25 Pb 2 5 < C O 25 Pa 2 5 < L 25 Pb25< L25 Pb 2 5 < C 0 25 Pb25< M25 CO 5 < L 5 Pa 2 5 < C O 25 Pa5 > L5 Pb25> L25 Pc 2 5 > L 25
a~ - : 5 % ab = 5 % ab=2% a~ = 2 % ao = 2 % ab .... 2 % ab = 2 % ab = 2 % ab=0.1% a~ = 2 % a~=2% ab--0.1% a~ -- 3 % ab-5% ab--2% ab = 2 ~ .
As compared with the inductive diet alone, we observed very few significant data, the only definite result being a large augmentation of cholesterol esterification after J. Atheroscler. Res., 2 (1962) 186-198
191
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
six months' treatment by alimentary fats. Thus at the end of the experiment, fat supplementation did not show significant differences in serum lipids as compared with induction controls. Whenever there was a difference between the different groups of rats, this difference showed a lower hyperlipidemic influence of peanut oil than of the other fats. The evolution with time of the levels of esterified fatty acids and cholesterol for all the groups treated by the 25 % fat supplementation is shown in Fig. 1. mg
*to
700
---------. . . . . . . . . . . . . . . . . .
AC
ESTERIFIED
IC
FATTY
ACIDS
TOTAL
CFIOLESTEROL
L25 Pa 2 5 Pb 2 5 Pc 2 5
600
.f 500
400
300
200
7
100
I
i
i
i
i
t (months)
i
i
t (months)
Fig. 1. Serum lipids; evolution with time (high fat groups).
Lipoproteins. The ratio fl/a lipoproteins was calculated for the rats which were treated with 25 % additions of the various fats and were autopsied after six months. The ratio was increased by the inductive treatment. The presence of lard in the diet gave rise to the largest ratio. The difference Pb25 < L25 was nearly significant (an -- 5.2 %). There was no significant difference between groups CO 25 or M 25 and Pb 25. Morphological data. The incidence of aortic atheromata in rats after two and six months' treatment and the statistical comparison between these two for every group are gathered in Table I. The curve of the incidence of aortic atheromata as a function of time, for the 25% supplementations, is shown in Fig. 2. This figure, together with its statistical interpretation, allows the following observation to be made: (i) There is no significant difference in induction controls from two to four months' treatment (tendency to an augmentation: ab : l0 °/o), nor from four to six months
(ab>20 %). (ii) There seems to be a continuous increase in atheromatous severity in the lard and margarine groups (tendency to an augmentation from two to four months and J. Atheroscler. Res., 2 (1962) 186-198
192
O. LIBERT, C. ROGG-EFFRONT TABLE I INCIDENCE
OF AORTIC A T H E R O M A T A
Percentageofratsshowingatleastonesudanophiliclesion Groups
2 months
6 months
Observed difference
Probability of error (ab) in %
Controls IC L25 L5 Pa25 Pa5 Pb25 Pb5 Pc25 Pc5 C025 C05 M25 M5
0 38 44 20 20 10 60 60 22 10 20 0 22 30
0 69 100 100 80 95 85 92 77 92 88 73 84 71
0 not significant 6 months >2 months 6 months>2 months 6 months>2 months 6 months>2 months not significant not significant 6 months>2 months 6 months>2 months 6 months >2 months 6 months >2 months 6 months>2 months not significant
20 1 0.012 2.6 0.0028 >20 >20 2.4 0.038 0.2 0.07 1.2 20
from four to six m o n t h s ; significant increase from t w o to six m o n t h s ' t r e a t m e n t : (ab~ 1 % ) . (iii) I n t h e P a groups there seems to be an increase in a t h e r o m a t o u s s e v e r i t y from t wo to four m o n t h s ' t r e a t m e n t (nearly significant: ab ---- 7 %) a n d only a t e n d e n c y from four to six m o n t h s ; significant increase for the t r e a t m e n t as a whole (ab - - 2.6 %). 100
-----~
~a
..... --:--....-~
.... : ....
/
/~,~."~
/
/.-..-...-.: :y:--.-~
./"
60
"6® ~ g 2c I
!
[
I
t (months)
Fig. 2. Incidence of aortic atheromata as a function of time of dietary treatment (high fat groups).
(iv)
In the Pb group there is only a t e n d e n c y to an a u g m e n t a t i o n during the first
two m o n t h s (ab ---- 8.6 %), a n d little or no difference afterwards ( a b > 2 0 %). This c ur v e is v e r y close to t h a t of the i n d u c t i o n controls. (v) In t h e Pc group a t h e r o m a t o s i s is not m a r k e d at the beginning, increases in s e v e r i t y from two to four m o n t h s (ab - - 1.5 %) an d shows no f u r t h e r significant difference l at er ( a ~ > 2 0 %). J. Atheroscler. Res., 2 (1962) 186-198
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
193
(vi) The CO group shows a curve very close to this latter: the a u g m e n t a t i o n from two to four m o n t h s is significant (ab = 0.6 %), a n d there is no or little difference d u r i n g
the last two m o n t h s of t r e a t m e n t (ab > 20 %). I t t h u s seems t h a t p e a n u t oil m a y give similar results to those given b y corn oil, as far as the tin-,e-course of e x p e r i m e n t a l atheromatosis is concerned. This similarity is the most p r o n o u n c e d in the case of the s y n t h e t i c p e a n u t triglycerides (Pc). There was no significant difference at a n y given time between the f a t - t r e a t e d groups a n d the i n d u c t i o n controls, nor was there a n y b e t w e e n the different f a t - t r e a t e d groups. The calculated m e a n scores for every group at various stages of the e x p e r i m e n t are shown in Table II. The use of the subjective gradings in order to arrive at a n atheromatosis coefficient is still in a trial period. TABLE II MEAN ATHEROMATOSlSCOEFFICIENT A theromatosis coefficient (aorta)
Group
2 months
4 months
6 months
0 0.39 0.44 0.20 0.40 0.15 0.70 0.55 0.22 0.29 0.33 0 0.10 0.33
0 1.23 1.55 1.80 2.15 1.55 2.30 1.40 -
0 1.27 2.10 3.14 1.43 1.96 1.40 1.50 1.53 2.25 1.88 1.31 1.70 1.43
Absolute controls Induction controls L25 L5 Pa25 Pa5 Pb25 Pb5 Pc25 Pc5 CO25 CO5 M25 M5
Rabbit S e r u m l @ i d s . The statistical analyses of our results, c o n d u c t e d in the same w a y as for the rat experiment, allow the following observations. The significant differences b e t w e e n a n i m a l s t r e a t e d with cholesterol plus fat a n d cholesterol alone were as follows: (i) I n the p e a n u t - o i l groups: P a > C C for ratio esterified: t o t a l cholesterol (EC/TC) (ab - - 0 . 3 2 %). (ii) I n the corn-oil group:
C O > C C for esterified f a t t y acids C O > C C for free cholesterol C O > C C for phospholipids
ab = 2.06 % ab = 1 % ab = 1.48 %
C 0 < C C for EC/TC ratio ab = 0.3 % The significant differences between p e a n u t oil a n d corn oil groups were the following: C 0 > P a for free cholesterol ab = 2.96 % J. Atheroscler. Res., 2 (1962) 186-198
194
O. LIBERT, C. ROGG-EFFRONT
CO>Pa COPb CO>Pb CO > Pb
for for for for for
phospholipids EC/TC ratio total cholesterol free cholesterol esterified cholesterol C O > P b for phospholipids C O < P c for EC/TC ratio C O < P c for C/P ratio
ab - - 4.5 %
ab = 0.16 % ab ~- 4.98 % ab = 4.98 % ab = 3.80 % ab ~- 2.82 % ab = 0.04 % ab - - 5.4 % (nearly significant)
It seems that, under our experimental conditions, peanut oil has no influence on the hyperlipidemia produced in rabbits by cholesterol feeding, while corn oil shows a definite enhancing effect. A t h e r o m a t o s i s . The morphological examination of rabbit aortas enabled us to calculate the atheromatous incidence and coefficient in the same way as in the rat experiment. The results are shown in Table III. TABLE III ATHEROMATOSIS IN CHOLESTEROL-TREATED RABBITS
Group Absolute controls Cholesterol c o n t r o l s Cholesterol + P a Cholesterol + P b Cholesterol + Pc Cholesterol + CO
Incidence (% of rabbits bearing atheromata) 0 62.5 50.0 62.5 71.4 100
Coe~cient (mean for the group)* 0 0.70 0.50 1.12 1.36 1.94
* R e a d e r s i n t e r e s t e d in d e t a i l s of t h e s e d a t a are i n v i t e d t o r e q u e s t t a b l e s from t h e p u b l i s h e r s of t h i s J o u r n a l .
The statistical analysis of the morphological data allows the following observations: (i) There is no significant difference between the peanut oil group and the cholesterol controls. (ii) There is a significant increase in severity of the atheromata when CO is added to cholesterol in the diet (cholesterol + CO>cholesterol controls, a u = 4.69 %). ( i i i ) There are significantly more atheromatous lesions when cholesterol is added in corn oil than when it is added in peanut oil (cholesterol + CO > cholesterol + Pa, a u = 1.11 °fo ). Thus our experimental data seem to show an enhancing effect of corn-oil on cholesterol atheromatosis in rabbit, an effect absent with peanut oil. DISCUSSION
Rat
It seemed to us (cf. also BRIOT e t a / . 2s) that the rat could be particularly valuable in a study of this nature, in spite of its resistivity to the atheromatous process, bearing J. Atheroscler. Res., 2 (1962} 186-198
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in mind its feeding habits which are close to man's, the necessity in both species of cellular factors for the development of the atheromata, and the very similar lesions found in rat and in man 16. CUTHBERTSONet al. 29 stated that sudanophilic lesions develop in the aortic arch of rats fed a high protein diet supplemented with 22 % hydrogenated peanut oil, but not with the same amount of natural peanut or corn oil. Our results show that sudanophilic lesions developed easily in the aortas of rats fed a normal protein diet, provided that cholesterol was added and that its degradation was inhibited by cholic acid and thyroid hypofunction, and provided that vascular sclerosis was produced by an excess of vitamin D in the diet. The aortic lesions may develop very rapidly even in the absence of lipid supplementation in the diet (inductive diet). There are also indications of differences in the evolution of atheromatosis with time caused by different fat supplements in the diet. Lard and margarine caused a continuous increase in the amount of lesions, whereas the plant oils seemed to show a "plateaued" curve or even a diminishing one after the fourth month of treatment. A preferential "anti-atheromatous" activity of corn oil as compared with peanut oil was never to be seen; the curves were very similar to one another and close to the curve given by "induction" controls (Fig. 2), especially if one compares the synthetic peanut-triglyceride curves with the corn-oil curves. Such a time difference between lard and margarine on the one hand, and the plant oils on the other, was not observable for the serum lipids, as can be seen comparing Figs. 1 and 2. However, if for the whole amount of our data one compares the incidence of atheromata in aorta (without considering the degree of the lesion) and the total serum cholesterol for every rat, there is an excellent statistical relationship between these parameters (less than one chance of error in 100,000!). The ratio fl/a lipoprotein in blood was higher when lard was present in the diet than when peanut oil was given to the rats; there was no comparable difference between corn oil or margarine supplementation and peanut oil supplementation. At that stage of the experiment (6 months) there was a slight indication towards a lower incidence of atheromatosis with peanut oil than with lard. Thus our results tend to confirm the idea of a relationship between the presence of atheromatous lesions and a high cholesterol content, or high fl/a ratio, in serum. They do not support the idea of a preferential "anti-atheromatous" effect of corn oil in rat.
Rabbit The conclusions that can be drawn from our rabbit experiment are in agreement with those from the data regarding rats. Our data show a relationship between serum lipids and the degree of atheromatosis. Here corn oil gives the highest values for both chemical and morphological results. This fact must be considered in the light of the data for rabbits given in the literature, which are contradictory. KRITCHEVSKYet al.30, ~1 showed that the severity of aortic atheromatosis in rabbits fed cholesterol is inversely proportional to the iodine index of the fat used as cholesterol vehicle. On the contrary, serum cholesterol increased with the iodine index. In these authors' experiments corn oil diminished the J. Atheroscler. Res., 2 (1962) 186-198
196
O. LIBERT, C. ROGG-EFFRONT
atheromatosis produced by cholesterol, whereas it increased the amount of cholesterol in blood. COTTETet al. 32 and ENSELME eta/. 33,34 obtained results somewhat similar to KRITCHEVSKY'S as they noticed a lower degree of atheromatosis in rabbits which received natural corn oil than in those given hydrogenated corn oil (together with cholesterol in both cases). Cholesterolemia was similar in the two groups. These data thus confirm the fact that hydrogenation of an oil increases the experimental atheromatosis in rabbit, and they show a lack of correlation between vascular lesions and cholesterolemia. McMILLAN35 similarly found a lack of correlation between the two parameters. In STEINER'S experiment 36 the addition of plant oil rich in unsaturated f a t t y acids to a 0.5 g/day cholesterol diet slightly (and not significantly) increased the experimental atheromatosis, while it increased the serum cholesterol very markedly. Thus our results agree with those of some authors (STEINER) and somewhat disagree with others (KRITCHEVSKY, COTTET and ENSELME). The conclusion to be drawn is probably that the conditions of the experiment largely determine the results obtained. It is of interest that under our experimental conditions the results show peanut oils to be more anti-atheromatous than the much more unsaturated corn oil. Possibly the distribution of the f a t t y acids among the triglycerides is of importance. ACKNOWLEDGEMENTS
We gratefully express our thanks to Mrs. J. PONGRATZ-MEssI and Miss J. KROESE for technical assistance in this study; to Dr. H. GEISSB/dHLER for the lipoprotein determinations; and to Dr. J. HERSCH for carrying out the statistical analyses. SUMMARY
700 rats and 48 rabbits were fed atherogenic diets containing various fats in two dosages; a total amount of 474 rats was autopsied after 2, 4 and 6 months of treatment; 44 rabbits were autopsied after 2 months. A sample of blood was taken from each animal at autopsy for lipid determinations and the aorta was examined morphologically and histologically. The following points emerge from the data: In the rat, after a short period in which peanut oil increased the induced hyperlipidemia more than did the other fats, a reversal of this effect was observed; after 4 months, peanut oil was less hyperlipidemic than were the other fats, and especially than was corn oil. It was never possible, at any time of the experiment, to show a significant difference of the atherogenic activity of the diets containing different fats. Peanut oil gave rise to an evolution of aortic atheromatosis with time very sinfilar to the curves shown by the animals fed corn oil. All the plant oils used gave a m a x i m u m aortic atheromatosis within four months; lard and margarine showed a continuous increase in the severity of the lesions. A very close statistical relationship was shown between the presence of sudanophilic lesions in aorta and cholesterolemia. J. Atheroscler. Res., 2 (1962) 186-198
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197
In the rabbit, atheromatosis and hypercholesterolemia induced by feeding cholesterol were both significantly increased by the presence of corn oil in the diet and not by peanut oil. RI~SUM]~
700 rats et 48 lapins sont nourris avec un r6gime ath6rog~ne contenant diverses graisses ~ deux concentrations diff6rentes. Au total, 474 rats furent autopsi6s apr~s 2, 4 et 6 mois de traitement et 44 lapins apr~s 2 mois. Le sang et l'aorte de chaque animal sont pr61ev6s 5 l'autopsie et leur teneur en lipides mesur6e syst6matiquement. Les points suivants d6coulent des r6sultats obtenus: Chez le rat, l'huile d'arachide provoque, au d6but et pendant une courte p6riode, une hyperlipid6mie plus intense que les autres graisses, puis entralne ensuite un effet inverse; apr~s 4 mois cette huile est moins hyperlipid6miante que les autres et notamment que l'huile de mais. I1 ne fur jamais possible ~ aucun moment de l'exp6rience de mettre en 6videnee des differences significatives du pouvoir ath~rogSne des diff~rents r6gimes. L'6volution de l'ath6romatose aortique induite par l'huile d'arachide est trSs semblable ~t celle induite par l'huile de mais. Toutes les huiles v6g6tales utilis6es provoquent le maximum d'ath6romatose aortique en 4 mois; le lard et la margarine provoquent une augmentation continue de la gravit6 des 16sions. Une relation statistique tr~s 6troite est observ6e entre la pr6sence dans l'aorte de 16sions soudanophiles et la cholest6rol6mie. Chez le lapin, l'ath6romatose et l'hypercholest6rol6mie induites par un r6gime fiche en cholest6rol furent toutes deux significativement augment6es par la pr6sence d'huile de mais dans le r6gime mais pas par l'huile d'arachide. ZUSAMMENFASSUNG
700 Ratten und 48 Kaninchen wurden mit atherogenem Futter, das verschiedene Fette enth~ilt, geftittert. Insgesamt 474 Ratten kamen nach 2, 4 und 6-monatlicher Ffitterung zur Autopsie, 44 Kaninchen nach 2 Monaten. Blutproben wurden ftir die Lipidbestimmung von jedem Tier bei der Autopsie entnommen, und die Aorta wurde morphologisch und histologisch untersucht. Folgende Punkte ergaben sich aus den Befunden: Bei der Ratte verst/irkte zun~ichst Erdnuss61 kurzfristig die entstandene Hyperlipidaemie, sp~iter kehrte sich jedoch diese Wirkung urn; nach 4 Monaten wirkte Erdnuss61 weniger blntlipidsteigernd als die anderen Fette, besonders als Mais61. Zu keinem Zeitpunkt der Versuche war es m6glich, einen signifikanten Unterschied in der atherogenen Aktivit~it der verschiedenen Fettdi~iten nachzuweisen. Erdnuss61 liess mit der Zeit in ~ihnlicher Weise eine Aortenatheromatose entstehen, wie sich bei den Tieren zeigte, die Mais61 erhalten hatten. Alle verwendeten Pflanzen61e erzeugten den h6chsten Grad der Aortenatheromatose innerhalb vier Monaten. Nach Schweineschmalz und Margarine nahm die Schwere der Ver~inderungen kontinuierlich zu. J. Atheroscler. Res., 2 (1962) 186-198
198
o. LIBERT, C. ROGG-EFFRONT
Zwischen dem Auftreten sudanophiler cholesterinaemie Beim
VerAnderungen in der Aorta und der Hyper-
e r g a b s i c h e i n e eDge s t a t i s t i s c h e B e z i e h u n g .
Kaninchen
wurde
und Hypercholesterinaemie
die dutch
Cholesterinftitterung
durch Zugabe
erzeugte
Atheromatose
v o n Mais61 s i g n i f i k a n t g e s t e i g e r t , j e d o c h
nicht durch Erdnuss61.
REFERENCES 1 A. KEYS, F. FIDANZA, V. SCARDI AND G. BERGAMI, Lancet, ii (1952) 209. 2 A. KEYS, Am. J. Public Health, 44 (1954) 864. 3 A. KEYS, F. VIVANCO, J. L. RODRIGUEZ-MINON, M. H. KEYS AND H. CASTRO-MENDOZA, Metabolism, 3 (1954) 195. 4 B. BRONTE-STEWART, A. KEYS AND J. F. BROCK, Lancet, ii (1955) 1103. 5 I. H. PAGE, Postgrad. Med., (1960) 263. 6 L. W. KINSELL, J. PARTRIDGE, L. DOLING, S. MARGEN AND G. MICHAELS, J. Clin. Endocrinol., 12 (1952) 909. 7 B. BRONTE-STEWART, A. ANTONIS, L. BALES AND J. F. BROCK, Lancet, i (1956) 521. s E. H. AHRENS, JR., W. INSULL, JR., R. BLOMSTRAND, J. HIRSCH, T. T. TSALTAS AND M. L. PETERSON, Lancet, i (1957) 943. 9 H. MALMROS AND G. WIGAND, Lancet, ii (1959) 749. 10 A. KEYS, J. T. ANDERSON AND F. GRANDE, Lancet, ii (1957) 959. 11 j. M. BEVERIDGE, W. F. CONNEL, G. H. MAYER AND H. L. HAUST, Can. J. Biochem. and Physiol., 36 (1958) 895. 12 W. C. ALVAREZ, Geriatrics, 15 (1960) 72. 13 D. V. RHOADS AND N. W. BARKER, Proc. Staff Meetings Mayo Clinic, 34 (1959) 225. 14 A. T. JAMES AND J. E. LOVELOCK, Brit. Med. Bull., 14 (1958) 262. 15 R. WOLFF, R. HERBEUVAL, G. CUNY, J.-J. BRIGNON, J. GILGENKRANTZ AND P. ULRICH, Presse todd., 66 (1958) 1706. 16 G. F. WILGRAM, Am. J. Clin. Nutrition, 6 (1958) 274. 17 G. F. WILGRAM, Proc. Soc. Exptl. Biol. Med., 99 (1958) 496. 18 N. ANITSCHKOW, Beitr. pathol. Anat. u. aUgem. Pathol., 56 (1913) 379. 19 G. WIGAND, Acta Med. Scan&, 166 (1959) Suppl. 351. lo N. A. JUSHCHENKO, Byull. Eksptl. Biol. Meal., 47 (1959) 31. gl B. ZAK, R. C. DICKENMAN, E. G. WHITE, H. BURNETT, AND P. J. CHERNEY, Am. J. Clin. Pathol., 24 (1954) 1307. 22 I. STERN AND B. SHAPIRO, in N. L. ALLPORT AND J. W. KEYSER, Colorimetric Analysis. 1, 2rid ed., C h a p m a n and Hall, Ltd., 1957, p. 141. 28 j . B. SUMNER, Science, 1O0 (1944) 413. 24 M. L. EILERT, Metabolism, 2 (1953) 137. 25 B. SWAHN, Scan& J. Clin. Lab. Invest., 5 (1953) Suppl. 9, 5. 26 L. RANDOIN AND P. BRUN, Gaz. H6p., 645. 27 M. M. BEST AND C. H. DUNCAN, Am. Heart J., 58 (1959) 214. 28 C. BRIOT, J. CARLOTTI, PH. LAUDAT, M.-S. MOUKHTAR, j. BAILLET AND L. DE GENNES, Presse m~d., 67 (1959) 543. 29 W. F. J. CUTHBERTSON, P. V. ELCOATE, D. M. IRELAND, D. C. B. MILLS AND P. SHEARLY, BiDchem. J., 68 (1958) 25. a0 D. KRITCHEVSKY, A. W. MOYER, W. C. TESAR, J. B. LOGAN, R. A. BROWN, M. C. DAVIES AND H. R. Cox, Am. J. Physiol., 178 (1954) 30. 31 D. KRITCHEVSKY, A. W. MOYER, W. C. TESAR, R. F. J. MCCANDLESS, J. B. LOGAN, R. A. BROWN AND M. E. ENGLERT, Am. J. Physiol., 185 (1956) 279. 82 j . COTTET, J. ENSELME AND A. GUICHARD, Rev. Athdroscldr., 1 (1959) 256. 23 j . ENSELME, A. LEMAIRE, J. COTTET, A. GUICHARD AND J. TIGAUD, Presse todd., 67 (1959) 1227. 34 j . ENSELME, J. COTTET AND A. GUICHARD, Presse todd., 68 (1960) 81. 85 G. C. McMILLAN, B. I. WEIGENSBERG AND A. C. RITCmE, A . M . A . Arch. Pathol., 70 (1960) 220. 26 A. STEINER, A. VARSOS AND P. SAMUEL, Circulation Res., 7 (1959) 448.
J. Atheroscler. Res., 2 (1962) 186-198
JOURNAL OF ATHEROSCLEROSIS RESEARCH
E X P E R I M E N T A L A T H E R O S C L E R O S I S AND H Y P E R L I P I D E M I A IN RATS AND RABBITS: I N F L U E N C E OF SOME A L I M E N T A R Y FATS O. L I B E R T AND C. ROGG-EFFRONT
Battelle Memorial Institute, Geneva (Switzerland) (Received December 22nd, 1961)
INTRODUCTION
An enormous amount of population statistics and of clinical and experimental data has led to the concept of the importance of diet, and especially of dietary fats, in the genesis of atherosclerosis 1-4, although it is now well recognised that lipids are not the cause, but probably a cause oi this illness 5. As cholesterolemia and atherosclerosis seemed to show a positive relationship in experimentation as well as in clinics, the action of those alimentary products which lower the level of cholesterol in blood was studied. KINSELL et al.6 showed that vegetable oils, which are rich in unsaturated f a t t y acids, could lower the level of cholesterolemia, whereas animal fats, which are rich in saturated f a t t y acids, augmented it. This fact was noted by m a n y authors, and BRONTE-STEWART et al. 7 showed that hydrogenated oils and animal fats augmented cholesterolemia in a male Bantu, whereas natural oils reduced it. I t was afterwards reported that corn oil could be particularly active in lowering the level of cholesterol in blood 7-12. However, RHOADS AND BARKER13 found corn oil to be only slightly active. The activity of corn oil was first related to its high content of linoleic acid and of other so-called "essential f a t t y acids". This theory was not clearly proved, and no definite relationship between the deficiency in essential f a t t y acids and vascular illnes was shown 14. Furthermore, soya oil, which is rich in unsaturated fatty acids, bears only very controversial activities against hypercholesterolemia and atherosclerosis in man 15. On account of these data it seemed of interest to make a comparative study of influences of different kinds of fat on experimental atherosclerosis and hypercholesterolemia. The main purpose of the present research was to compare, from this double point of view, the action of an oil which is rich in unsaturated f a t t y acids (corn oil), an equilibrated one (peanut oil), a hardened fat (margarine) and an animal fat (lard), on two animal species: (a) The rat, which is very resistant to atheromatosis, but in which it is possible to reproduce, with special diets or treatments, vascular abnormalities resembling those observed in manl6,17. Our data are concerned with 474 autopsied rats. (b) The rabbit, in which experimental atherosclerosis was first produced is, b y feeding cholesterol. This animal is very sensitive to dietary components: WIGAND19 showed J. Atheroscler. Res., 2 (1962) 186-198
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
187
in 1959 that feeding various fats in excess to rabbits without any cholesterol is sufficient to produce pronounced atherosclerotic lesions. Our data are concerned with 44 autopsied rabbits.
MATERIAL AND METHODS
Diets Rat. We chose to follow WILGRAM'S diet 16 as closely as possible; it was he who produced lipomatous coronary changes in 88 % of the rats he had treated for 6 to 12 months. The composition of our "inductive" diet was as follows:
basic diet egg yolk powder cholesterol sodium cholate propylthiouracil vitamin D (400,000 U/g)
85 % 12 % 2 % 0.2 % 0.075 % 0.35 %
Our basic diet was a mixture of cereals, alfalfa and fish flours (proteins 23 %; lipids 1.2 %; cellulose 5.5 %; carbohydrates 70 %). Rabbit. We followed approximately the work of JUSHCHENKO2°, the daily diet being as follows: each rabbit ate daily 500 g carrots and 50 g bran, in which 2 g cholesterol, dissolved b y gentle heating when there was an added oil, was mixed intimately. Fats added to tile diet consisted of lard, peanut oil, corn oil and margarine. Three different sources of peanut oil were used, namely: peanut oil (a) = natural peanut oil; peanut oil (b) = natural peanut oil submitted to a refinement process, consisting of demucilagination b y water and NaC1, neutralization of the free f a t t y acids with NaOH, water washing, decolorization with active earths at 90°C under vacuum, filtration and deodorization at 200°C under vacuum, then under water vapour, cooling to 40°C under vacuum to avoid oxidation; and peanut oil (c) = synthetic peanut triglycerides, imitating the fatty acid composition of natural peanut oil. The percentage fatty acid composition (main components) of the fats used in the experiment is shown below: Saturated acids
(C8+ Clo+ C1~) P e a n u t oil Corn oil Margarine Lard
0.5 1.0 32.1
Palmitie acid
lO.O 12.5 11.8 26.0
Stearic acid
2.9 2.0 3.2 19.4
Oleic acid
Linoleic acid
54 30 32.8 54.6
26 55 8.6 --
j . Atheroscler. Res., 2 (1962) 186-198
188
O. LIBERT, C. ROGG-EFFRONT
Animal Groups Rat. 700 male adult rats were distributed at random in the following 14 groups: AC, absolute controls IC, "induction controls" L5 and L25 Pa 5 and Pa 25 Pb5 and Pb25 Pc5 and Pc25 CO5 and C025 M5 and M25
basic diet "inductive" "inductive" "inductive" "inductive" "inductive" "inductive" "inductive"
diet diet diet diet diet diet diet
+ + + + + +
lard, 5 % and 25 % resp. peanut oil (a), 5 % and 25 % resp. peanut oil (b), 5 % and 25 % resp. peanut oil (c), 5 % and 25 % resp. corn oil 5 % and 25 % resp. margarine 5 % and 25 % resp.
Rabbit. 48 adult rabbits (both sexes, distributed at random) were grouped as follows: AC CC Pa Pb Pc CO
absolute controls: basic diet (bran and carrots) "cholesterol" controls: basic diet + cholesterol (without fat) basic diet + cholesterol + peanut oil (a) basic diet + cholesterol + peanut oil (b) basic diet + cholesterol + peanut oil (c) basic diet + cholesterol + corn oil
Autopsies Rats were autopsied in 3 series; i.e. after 2, 4 and 6 months' treatment. Rabbits were all autopsied after the 2nd month. All the animals were autopsied after they had fasted for 24 hours. They were anesthetized with nembutal (50 mg/kg); blood was taken from the left carotid artery (5-10 ml for rats, 20 ml or more for rabbits); the aortas were removed immediately and were fixed in formalin 10 %. In every case the external and internal aspect of the animal, the extent of f a t t y depots and the appearance of steatosis in liver were observed; the animals were examined for various possible infections.
Chemical Determinations in Serum The following were measured in the sera of both rats and rabbits: total and esterified cholesterol21; esterified f a t t y acids22; phospholipids2~,24; lipoproteins, measured by paper electrophoresis 25. The ratio fl/a lipoproteins was calculated.
Morphological Determination Rat. Histological sections were made b y means of a frozen microtome in three parts of the aorta (aortic arch = aorta 1, thoracic segment ----aorta 2, abdominal segment = aorta 3). These sections were stained with Sudan IV and with haematoxylin. About 30,000 slides were examined in all. The atheromatous lesions were defined as in RANDOIN AND BRUN2~ and were visualized on our slides as sudanophilic inclusions. J. Atheroscler. Res., 2 (1962) 186-198
V A R I O U S FATS A N D E X P E R I M E N T A L A T H E R O S C L E R O S I S
189
The statistical analyses were based on an "atheromatous incidence", defined as the percentage of rats bearing at least one atheroma in a given group. Quantitative analyses were also tentatively tried, and the "atheromatous coefficient" was calculated as the mean score of lesions observed in the three aortic segments in one animal, or in one group of animals (gradation: ± = 0.5; + --~ 1; + + = 2; + + + ----3). Rabbit. For the rabbits, aortas were observed in toto, after having been cut into two halves and fixed in formalin 10 %. The internal sides of the aortas were photographed and were then graded from ± (0.5) to + + + + (4). Statistical Analyses Statistical analyses were made of all data, using non-parametric methods, especially the WILCOXON-MANN-WHITNEYtest. RESULTS
Rat Growth and mortality. The animals which received the "inductive diet" alone continually lost weight; they also showed the highest mortality and it was not possible to draw the curves after the 4th month of treatment, the number of living animals being reduced to about 10 instead of 20 to 25 in the other groups*). This growth-inhibiting action may possibly be due to the presence of propylthiouracil in the diet as BEST et al. 27 observed a similar effect with thiouracil; it is well known that thyroid hypofunction generally inhibits growth. With diets containing any of the added fats, even in small doses, this weight loss was neutralized, until the 10th week of treatment; after this time we observed a constant weight loss in all the groups which received the "inductive diet" even with fat supplementation. There was no observable difference in mortality between the different fat-treated groups. Examination of rats at autopsy. This examination showed no difference in fatty depots, liver steatosis and various infections between the different groups of animals which received the "inductive diet". Lipids in serum. The large scatter of the results did not allow of any other interpretation than the statistical one. We compared statistically: (a) the six groups which received peanut oil with the six other groups of fat; (b) all the twelve fat-treated groups with the "induction" group. For the analysis of the statistical data, we defined: au = probability of error for the unilateral test (1-tailed test); ab = probability of error for the bilateral test (2-tailed test). The statistical results allow the following comments to be made: After two months of treatment, hyperlipidemia (esterified fatty acids, cholesterol and phospholipids) was higher if the animals had received peanut oil than if they had * Readers interested in further details of these and other data referred to should request copies of figures and tables, J. Atheroscler. Res., 2 (1962) 186, from the publishers. J. Atheroscler. Res., 2 (1962) 186-198
190
O. LIBERT, C. ROGG-EFFRONT
received any of the three other fats. The significant data were much more numerous for the 25 % supplementation than for the 5 % supplementation ( a b < 5 %). All the six sorts of fat inhibited the hyperlipidenfia produced b y the "inductive" diet ( a b < 0 . 1 % in most cases, always < 5 %). After four months' treatment there was no longer a preferential hyperlipidemic activity of the three kinds of peanut oil, as compared with the other dietary fats; there were few statistically significant results for esterified fatty acids and cholesterol, and these few were contradictory; on the other hand we noticed a significant increase in the concentration of phospholipids with lard and margarine as compared with peanut oil ( a b < 0 . 1 % in every significant case). Lard, margarine and corn oil gave a more pronounced hyperlipidemia than did the inductive diet alone, for both supplementations (esterified fatty acids and cholesterol: 5 % > a b > 1 % ) . Considering the peanut oil group at high dosage, only Pa25 gave a significant augmentation as compared with the induction controls for esterified fatty acids (a~ = 0.002 %); there was no significant result for cholesterol; at low dosages, every augmentation was significant except with Pb5 (2 % > a 0 > 0 . 2 % for all the groups other than Pb5). Tbus at that moment of the experiment peanut oil showed in general less tendency to cause hyperlipidemia than the other groups of fat. After six months' treatment the only significant differences between groups could be found for peanut oil, which was generally less hyperlipidemic than the other fats. However, the C/P ratio was lower with lard than with peanut oil; this latter fact is due to the very high serum phospholipids shown b y the lard groups. The significant differences between peanut oil and the other fats were the following: Esterified fatty acids
Total cholesterol
Phospholipids
C/P ratio
Pa 25 < L 25 Pa 2 5 < C 0 25 Pa25< M25 Pa 2 5 < L 25 Pa 2 5 < C O 25 Pb25< L25 Pb 2 5 < C O 25 Pa 2 5 < L 25 Pb25< L25 Pb 2 5 < C 0 25 Pb25< M25 CO 5 < L 5 Pa 2 5 < C O 25 Pa5 > L5 Pb25> L25 Pc 2 5 > L 25
a~ - : 5 % ab = 5 % ab=2% a~ = 2 % ao = 2 % ab .... 2 % ab = 2 % ab = 2 % ab=0.1% a~ = 2 % a~=2% ab--0.1% a~ -- 3 % ab-5% ab--2% ab = 2 ~ .
As compared with the inductive diet alone, we observed very few significant data, the only definite result being a large augmentation of cholesterol esterification after J. Atheroscler. Res., 2 (1962) 186-198
191
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
six months' treatment by alimentary fats. Thus at the end of the experiment, fat supplementation did not show significant differences in serum lipids as compared with induction controls. Whenever there was a difference between the different groups of rats, this difference showed a lower hyperlipidemic influence of peanut oil than of the other fats. The evolution with time of the levels of esterified fatty acids and cholesterol for all the groups treated by the 25 % fat supplementation is shown in Fig. 1. mg
*to
700
---------. . . . . . . . . . . . . . . . . .
AC
ESTERIFIED
IC
FATTY
ACIDS
TOTAL
CFIOLESTEROL
L25 Pa 2 5 Pb 2 5 Pc 2 5
600
.f 500
400
300
200
7
100
I
i
i
i
i
t (months)
i
i
t (months)
Fig. 1. Serum lipids; evolution with time (high fat groups).
Lipoproteins. The ratio fl/a lipoproteins was calculated for the rats which were treated with 25 % additions of the various fats and were autopsied after six months. The ratio was increased by the inductive treatment. The presence of lard in the diet gave rise to the largest ratio. The difference Pb25 < L25 was nearly significant (an -- 5.2 %). There was no significant difference between groups CO 25 or M 25 and Pb 25. Morphological data. The incidence of aortic atheromata in rats after two and six months' treatment and the statistical comparison between these two for every group are gathered in Table I. The curve of the incidence of aortic atheromata as a function of time, for the 25% supplementations, is shown in Fig. 2. This figure, together with its statistical interpretation, allows the following observation to be made: (i) There is no significant difference in induction controls from two to four months' treatment (tendency to an augmentation: ab : l0 °/o), nor from four to six months
(ab>20 %). (ii) There seems to be a continuous increase in atheromatous severity in the lard and margarine groups (tendency to an augmentation from two to four months and J. Atheroscler. Res., 2 (1962) 186-198
192
O. LIBERT, C. ROGG-EFFRONT TABLE I INCIDENCE
OF AORTIC A T H E R O M A T A
Percentageofratsshowingatleastonesudanophiliclesion Groups
2 months
6 months
Observed difference
Probability of error (ab) in %
Controls IC L25 L5 Pa25 Pa5 Pb25 Pb5 Pc25 Pc5 C025 C05 M25 M5
0 38 44 20 20 10 60 60 22 10 20 0 22 30
0 69 100 100 80 95 85 92 77 92 88 73 84 71
0 not significant 6 months >2 months 6 months>2 months 6 months>2 months 6 months>2 months not significant not significant 6 months>2 months 6 months>2 months 6 months >2 months 6 months >2 months 6 months>2 months not significant
20 1 0.012 2.6 0.0028 >20 >20 2.4 0.038 0.2 0.07 1.2 20
from four to six m o n t h s ; significant increase from t w o to six m o n t h s ' t r e a t m e n t : (ab~ 1 % ) . (iii) I n t h e P a groups there seems to be an increase in a t h e r o m a t o u s s e v e r i t y from t wo to four m o n t h s ' t r e a t m e n t (nearly significant: ab ---- 7 %) a n d only a t e n d e n c y from four to six m o n t h s ; significant increase for the t r e a t m e n t as a whole (ab - - 2.6 %). 100
-----~
~a
..... --:--....-~
.... : ....
/
/~,~."~
/
/.-..-...-.: :y:--.-~
./"
60
"6® ~ g 2c I
!
[
I
t (months)
Fig. 2. Incidence of aortic atheromata as a function of time of dietary treatment (high fat groups).
(iv)
In the Pb group there is only a t e n d e n c y to an a u g m e n t a t i o n during the first
two m o n t h s (ab ---- 8.6 %), a n d little or no difference afterwards ( a b > 2 0 %). This c ur v e is v e r y close to t h a t of the i n d u c t i o n controls. (v) In t h e Pc group a t h e r o m a t o s i s is not m a r k e d at the beginning, increases in s e v e r i t y from two to four m o n t h s (ab - - 1.5 %) an d shows no f u r t h e r significant difference l at er ( a ~ > 2 0 %). J. Atheroscler. Res., 2 (1962) 186-198
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
193
(vi) The CO group shows a curve very close to this latter: the a u g m e n t a t i o n from two to four m o n t h s is significant (ab = 0.6 %), a n d there is no or little difference d u r i n g
the last two m o n t h s of t r e a t m e n t (ab > 20 %). I t t h u s seems t h a t p e a n u t oil m a y give similar results to those given b y corn oil, as far as the tin-,e-course of e x p e r i m e n t a l atheromatosis is concerned. This similarity is the most p r o n o u n c e d in the case of the s y n t h e t i c p e a n u t triglycerides (Pc). There was no significant difference at a n y given time between the f a t - t r e a t e d groups a n d the i n d u c t i o n controls, nor was there a n y b e t w e e n the different f a t - t r e a t e d groups. The calculated m e a n scores for every group at various stages of the e x p e r i m e n t are shown in Table II. The use of the subjective gradings in order to arrive at a n atheromatosis coefficient is still in a trial period. TABLE II MEAN ATHEROMATOSlSCOEFFICIENT A theromatosis coefficient (aorta)
Group
2 months
4 months
6 months
0 0.39 0.44 0.20 0.40 0.15 0.70 0.55 0.22 0.29 0.33 0 0.10 0.33
0 1.23 1.55 1.80 2.15 1.55 2.30 1.40 -
0 1.27 2.10 3.14 1.43 1.96 1.40 1.50 1.53 2.25 1.88 1.31 1.70 1.43
Absolute controls Induction controls L25 L5 Pa25 Pa5 Pb25 Pb5 Pc25 Pc5 CO25 CO5 M25 M5
Rabbit S e r u m l @ i d s . The statistical analyses of our results, c o n d u c t e d in the same w a y as for the rat experiment, allow the following observations. The significant differences b e t w e e n a n i m a l s t r e a t e d with cholesterol plus fat a n d cholesterol alone were as follows: (i) I n the p e a n u t - o i l groups: P a > C C for ratio esterified: t o t a l cholesterol (EC/TC) (ab - - 0 . 3 2 %). (ii) I n the corn-oil group:
C O > C C for esterified f a t t y acids C O > C C for free cholesterol C O > C C for phospholipids
ab = 2.06 % ab = 1 % ab = 1.48 %
C 0 < C C for EC/TC ratio ab = 0.3 % The significant differences between p e a n u t oil a n d corn oil groups were the following: C 0 > P a for free cholesterol ab = 2.96 % J. Atheroscler. Res., 2 (1962) 186-198
194
O. LIBERT, C. ROGG-EFFRONT
CO>Pa CO
for for for for for
phospholipids EC/TC ratio total cholesterol free cholesterol esterified cholesterol C O > P b for phospholipids C O < P c for EC/TC ratio C O < P c for C/P ratio
ab - - 4.5 %
ab = 0.16 % ab ~- 4.98 % ab = 4.98 % ab = 3.80 % ab ~- 2.82 % ab = 0.04 % ab - - 5.4 % (nearly significant)
It seems that, under our experimental conditions, peanut oil has no influence on the hyperlipidemia produced in rabbits by cholesterol feeding, while corn oil shows a definite enhancing effect. A t h e r o m a t o s i s . The morphological examination of rabbit aortas enabled us to calculate the atheromatous incidence and coefficient in the same way as in the rat experiment. The results are shown in Table III. TABLE III ATHEROMATOSIS IN CHOLESTEROL-TREATED RABBITS
Group Absolute controls Cholesterol c o n t r o l s Cholesterol + P a Cholesterol + P b Cholesterol + Pc Cholesterol + CO
Incidence (% of rabbits bearing atheromata) 0 62.5 50.0 62.5 71.4 100
Coe~cient (mean for the group)* 0 0.70 0.50 1.12 1.36 1.94
* R e a d e r s i n t e r e s t e d in d e t a i l s of t h e s e d a t a are i n v i t e d t o r e q u e s t t a b l e s from t h e p u b l i s h e r s of t h i s J o u r n a l .
The statistical analysis of the morphological data allows the following observations: (i) There is no significant difference between the peanut oil group and the cholesterol controls. (ii) There is a significant increase in severity of the atheromata when CO is added to cholesterol in the diet (cholesterol + CO>cholesterol controls, a u = 4.69 %). ( i i i ) There are significantly more atheromatous lesions when cholesterol is added in corn oil than when it is added in peanut oil (cholesterol + CO > cholesterol + Pa, a u = 1.11 °fo ). Thus our experimental data seem to show an enhancing effect of corn-oil on cholesterol atheromatosis in rabbit, an effect absent with peanut oil. DISCUSSION
Rat
It seemed to us (cf. also BRIOT e t a / . 2s) that the rat could be particularly valuable in a study of this nature, in spite of its resistivity to the atheromatous process, bearing J. Atheroscler. Res., 2 (1962} 186-198
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
195
in mind its feeding habits which are close to man's, the necessity in both species of cellular factors for the development of the atheromata, and the very similar lesions found in rat and in man 16. CUTHBERTSONet al. 29 stated that sudanophilic lesions develop in the aortic arch of rats fed a high protein diet supplemented with 22 % hydrogenated peanut oil, but not with the same amount of natural peanut or corn oil. Our results show that sudanophilic lesions developed easily in the aortas of rats fed a normal protein diet, provided that cholesterol was added and that its degradation was inhibited by cholic acid and thyroid hypofunction, and provided that vascular sclerosis was produced by an excess of vitamin D in the diet. The aortic lesions may develop very rapidly even in the absence of lipid supplementation in the diet (inductive diet). There are also indications of differences in the evolution of atheromatosis with time caused by different fat supplements in the diet. Lard and margarine caused a continuous increase in the amount of lesions, whereas the plant oils seemed to show a "plateaued" curve or even a diminishing one after the fourth month of treatment. A preferential "anti-atheromatous" activity of corn oil as compared with peanut oil was never to be seen; the curves were very similar to one another and close to the curve given by "induction" controls (Fig. 2), especially if one compares the synthetic peanut-triglyceride curves with the corn-oil curves. Such a time difference between lard and margarine on the one hand, and the plant oils on the other, was not observable for the serum lipids, as can be seen comparing Figs. 1 and 2. However, if for the whole amount of our data one compares the incidence of atheromata in aorta (without considering the degree of the lesion) and the total serum cholesterol for every rat, there is an excellent statistical relationship between these parameters (less than one chance of error in 100,000!). The ratio fl/a lipoprotein in blood was higher when lard was present in the diet than when peanut oil was given to the rats; there was no comparable difference between corn oil or margarine supplementation and peanut oil supplementation. At that stage of the experiment (6 months) there was a slight indication towards a lower incidence of atheromatosis with peanut oil than with lard. Thus our results tend to confirm the idea of a relationship between the presence of atheromatous lesions and a high cholesterol content, or high fl/a ratio, in serum. They do not support the idea of a preferential "anti-atheromatous" effect of corn oil in rat.
Rabbit The conclusions that can be drawn from our rabbit experiment are in agreement with those from the data regarding rats. Our data show a relationship between serum lipids and the degree of atheromatosis. Here corn oil gives the highest values for both chemical and morphological results. This fact must be considered in the light of the data for rabbits given in the literature, which are contradictory. KRITCHEVSKYet al.30, ~1 showed that the severity of aortic atheromatosis in rabbits fed cholesterol is inversely proportional to the iodine index of the fat used as cholesterol vehicle. On the contrary, serum cholesterol increased with the iodine index. In these authors' experiments corn oil diminished the J. Atheroscler. Res., 2 (1962) 186-198
196
O. LIBERT, C. ROGG-EFFRONT
atheromatosis produced by cholesterol, whereas it increased the amount of cholesterol in blood. COTTETet al. 32 and ENSELME eta/. 33,34 obtained results somewhat similar to KRITCHEVSKY'S as they noticed a lower degree of atheromatosis in rabbits which received natural corn oil than in those given hydrogenated corn oil (together with cholesterol in both cases). Cholesterolemia was similar in the two groups. These data thus confirm the fact that hydrogenation of an oil increases the experimental atheromatosis in rabbit, and they show a lack of correlation between vascular lesions and cholesterolemia. McMILLAN35 similarly found a lack of correlation between the two parameters. In STEINER'S experiment 36 the addition of plant oil rich in unsaturated f a t t y acids to a 0.5 g/day cholesterol diet slightly (and not significantly) increased the experimental atheromatosis, while it increased the serum cholesterol very markedly. Thus our results agree with those of some authors (STEINER) and somewhat disagree with others (KRITCHEVSKY, COTTET and ENSELME). The conclusion to be drawn is probably that the conditions of the experiment largely determine the results obtained. It is of interest that under our experimental conditions the results show peanut oils to be more anti-atheromatous than the much more unsaturated corn oil. Possibly the distribution of the f a t t y acids among the triglycerides is of importance. ACKNOWLEDGEMENTS
We gratefully express our thanks to Mrs. J. PONGRATZ-MEssI and Miss J. KROESE for technical assistance in this study; to Dr. H. GEISSB/dHLER for the lipoprotein determinations; and to Dr. J. HERSCH for carrying out the statistical analyses. SUMMARY
700 rats and 48 rabbits were fed atherogenic diets containing various fats in two dosages; a total amount of 474 rats was autopsied after 2, 4 and 6 months of treatment; 44 rabbits were autopsied after 2 months. A sample of blood was taken from each animal at autopsy for lipid determinations and the aorta was examined morphologically and histologically. The following points emerge from the data: In the rat, after a short period in which peanut oil increased the induced hyperlipidemia more than did the other fats, a reversal of this effect was observed; after 4 months, peanut oil was less hyperlipidemic than were the other fats, and especially than was corn oil. It was never possible, at any time of the experiment, to show a significant difference of the atherogenic activity of the diets containing different fats. Peanut oil gave rise to an evolution of aortic atheromatosis with time very sinfilar to the curves shown by the animals fed corn oil. All the plant oils used gave a m a x i m u m aortic atheromatosis within four months; lard and margarine showed a continuous increase in the severity of the lesions. A very close statistical relationship was shown between the presence of sudanophilic lesions in aorta and cholesterolemia. J. Atheroscler. Res., 2 (1962) 186-198
VARIOUS FATS AND EXPERIMENTAL ATHEROSCLEROSIS
197
In the rabbit, atheromatosis and hypercholesterolemia induced by feeding cholesterol were both significantly increased by the presence of corn oil in the diet and not by peanut oil. RI~SUM]~
700 rats et 48 lapins sont nourris avec un r6gime ath6rog~ne contenant diverses graisses ~ deux concentrations diff6rentes. Au total, 474 rats furent autopsi6s apr~s 2, 4 et 6 mois de traitement et 44 lapins apr~s 2 mois. Le sang et l'aorte de chaque animal sont pr61ev6s 5 l'autopsie et leur teneur en lipides mesur6e syst6matiquement. Les points suivants d6coulent des r6sultats obtenus: Chez le rat, l'huile d'arachide provoque, au d6but et pendant une courte p6riode, une hyperlipid6mie plus intense que les autres graisses, puis entralne ensuite un effet inverse; apr~s 4 mois cette huile est moins hyperlipid6miante que les autres et notamment que l'huile de mais. I1 ne fur jamais possible ~ aucun moment de l'exp6rience de mettre en 6videnee des differences significatives du pouvoir ath~rogSne des diff~rents r6gimes. L'6volution de l'ath6romatose aortique induite par l'huile d'arachide est trSs semblable ~t celle induite par l'huile de mais. Toutes les huiles v6g6tales utilis6es provoquent le maximum d'ath6romatose aortique en 4 mois; le lard et la margarine provoquent une augmentation continue de la gravit6 des 16sions. Une relation statistique tr~s 6troite est observ6e entre la pr6sence dans l'aorte de 16sions soudanophiles et la cholest6rol6mie. Chez le lapin, l'ath6romatose et l'hypercholest6rol6mie induites par un r6gime fiche en cholest6rol furent toutes deux significativement augment6es par la pr6sence d'huile de mais dans le r6gime mais pas par l'huile d'arachide. ZUSAMMENFASSUNG
700 Ratten und 48 Kaninchen wurden mit atherogenem Futter, das verschiedene Fette enth~ilt, geftittert. Insgesamt 474 Ratten kamen nach 2, 4 und 6-monatlicher Ffitterung zur Autopsie, 44 Kaninchen nach 2 Monaten. Blutproben wurden ftir die Lipidbestimmung von jedem Tier bei der Autopsie entnommen, und die Aorta wurde morphologisch und histologisch untersucht. Folgende Punkte ergaben sich aus den Befunden: Bei der Ratte verst/irkte zun~ichst Erdnuss61 kurzfristig die entstandene Hyperlipidaemie, sp~iter kehrte sich jedoch diese Wirkung urn; nach 4 Monaten wirkte Erdnuss61 weniger blntlipidsteigernd als die anderen Fette, besonders als Mais61. Zu keinem Zeitpunkt der Versuche war es m6glich, einen signifikanten Unterschied in der atherogenen Aktivit~it der verschiedenen Fettdi~iten nachzuweisen. Erdnuss61 liess mit der Zeit in ~ihnlicher Weise eine Aortenatheromatose entstehen, wie sich bei den Tieren zeigte, die Mais61 erhalten hatten. Alle verwendeten Pflanzen61e erzeugten den h6chsten Grad der Aortenatheromatose innerhalb vier Monaten. Nach Schweineschmalz und Margarine nahm die Schwere der Ver~inderungen kontinuierlich zu. J. Atheroscler. Res., 2 (1962) 186-198
198
o. LIBERT, C. ROGG-EFFRONT
Zwischen dem Auftreten sudanophiler cholesterinaemie Beim
VerAnderungen in der Aorta und der Hyper-
e r g a b s i c h e i n e eDge s t a t i s t i s c h e B e z i e h u n g .
Kaninchen
wurde
und Hypercholesterinaemie
die dutch
Cholesterinftitterung
durch Zugabe
erzeugte
Atheromatose
v o n Mais61 s i g n i f i k a n t g e s t e i g e r t , j e d o c h
nicht durch Erdnuss61.
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J. Atheroscler. Res., 2 (1962) 186-198