J. K. G. Kramer

Comparative studies on composition of cardiac phospholipids in rats fed different vegetable oils

Male Sprague-Dawley rats were fed diets for 1 or 16 weeks, containing 20% by weight vegetable oils differing widely in their oleic, linoleic and linolenic acid content. No significant changes were observed in the level of the cardiac lipid classes. The fatty acid composition of the 2 major phospholipids, phosphatidylcholine and phosphatidylethanolamine, showed a remarkable similarity between diets in the concentration of total saturated, C22 polyunsaturated and arachidonic acids. Monounsaturated acids were incorporated depending on their dietary concentration, but the increases were moderate. Dietary linolenic acid rapidly substituted C22 polyunsaturated fatty acids of the linoleic acid family (n−6) with those from the linolenic acid family (n−3). The results suggest that dietary linolenic acid of less than 15% does not inhibit the conversion of linoleic to arachidonic acid but the subsequent conversion of arachidonic acid to the C22 polyunsaturates was greatly reduced. Significant amounts of dietary monounsaturated fatty acids were incorporated into cardiac cardiolipin accompanied by increases in polyunsaturated fatty acids, apparently to maintain an average of 2 double bonds/molecule. The cardiac sphingomyelins also accumulated monounsaturated fatty acids depending on the dietary concentration. It is quite evident from the results of this study that the incorporation of oleic acid and the substitution of linolenic for linoleic acid-derived C22 polyunsaturated fatty acids into cardiac phospholipids was related to the dietary concentration of these fatty acids and was not peculiar to any specific oil. Even though it is impossible to estimate the effect of such changes in cardiac phospholipids on membrane structure and function, results are discussed which suggest that the resultant membrane in the Sprague-Dawley male rat is more fragile, leading to greater cellular breakdown and focal necrosis.

Relationship between erucic acid and myocardial changes in male rats.

The back and belly fat of pigs fed a diet containing 20% by wt rapeseed oil (22% erucic acid) for 16 weeks was rendered into oil. This rendered pig fat, which contained 5.6% erucic acid, was fed to male rats in three separate experiments at 20% by wt of the diet for 16 weeks. In experiment I rendered pig fat was compared only toBrassica campestris var. Span rapeseed oil containing 4.8% erucic acid. In experiments II and III, rendered pig fat was compared to commerical lard containing 0.2% docosenoic acid, commercial lard to which 5.4% free erucic acid was added, and Span rapessed oil. There was no significant (P<0.01) differences observed in the level of erucic acid in the hearts of rats fed diets of rendered pig fat, Span rapeseed oil, or commercial lard plus erucic acid. However, the incidence (P<0.001) and severity (P<0.01) of cardiac lesions were significantly higher in Span rapeseed oil fed rats compared to rats fed control diets. The number of rats affected or the severity of lesions in the rendered pig fat fed group was not significantly different from controls. The results of this study indicate that the myocardial lesions associated with feeding 20% rapeseed oil diets are not related to the content of erucic acid per se. The possible reasons why rapeseed oil causes cardiac lesions in rats are discussed. It is suggested that a triglyceride imbalance in the oil might play an important role in causing these lesions in rats.

Brassica campestris var. Span: II. Cardiopathogenicity of fractions isolated from span rapeseed oil when fed to male rats

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.

Reduction of myocardial necrosis in male albino rats by manipulation of dietary fatty acid levels

A comprehensive statistical analysis had shown a significant correlation between the incidence of myocardial lesions in male albino rats and the concentration of certain dietary fatty acids. To test this result under controlled conditions, male rats were fed for 16 weeks diets containing 20% by weight soybean oil or a low erucic acid rapeseed (LEAR) oil. Both dietary oils contained substantial amounts of linolenic acid, and both groups developed a high incidence of myocardial necrosis. The addition of dietary saturated fatty acids to the oil in the form of cocoa butter significantly lowered the incidence of heart lesions in both groups. The addition of cocoa butter resulted in increased absorption of saturates and increased growth. Replacement of the cocoa butter by at least an equal amount of synthetic triolein resulted in no significant changes in the cardiopathogenic response compared to the original oils, thus ensuring that the reduction in heart lesions associated with the addition of cocoa butter was not due to dilution of cardiopathogenic compounds in the original vegetable oils. These results support the hypothesis that myocardial lesions in male rats are related to the balance of dietary fatty acids and not to cardiotoxic contaminants in the oils. Changes in the dietary fatty acids did not appear to influence the proportion of the cardiac phospholipids, but their fatty acid composition was markedly influenced. Dietary linolenic acid affected the C22 polyunsaturated fatty acids (PUFA) and dietary saturates increased the level of saturates in cardiac phospholipids. The level of arachidonic acid and total C22 PUFA did not appear to be affected by diet.

Quantitating heart lipids: Comparison of results obtained using the Iatroscan method with those from phosphorus and gas chromatographic techniques

The precision and accuracy of the Iatroscan method was evaluated by comparing the results obtained with established phosphorus and gas chromatographic techniques. A complete lipid class analysis of rat heart lipids was chosen in order to evaluate the performance of the Iatroscan method for biological samples which contained both neutral lipids and phospholipids. A partial scan and repeat development with chloroform/methanol/water (68.5∶29∶2.5) was introduced to achieve consistently good separations of the phospholipids on the Chromarods in the Iatroscan method. The results showed that the precision of the Iatroscan method for some lipid classes was comparable to that of phosphorus or gas chromatographic techniques, while for other lipid classes it was lower. Compared to the data obtained using the phosphorus method, the Iatroscan data were generally similar, while the gas chromatographic method generally gave lower values. These findings, together with the advantages of time required for analysis, size of sample, and universality of detection, suggest that the Iatroscan is a valuable complementary method for complex lipid analyses.

The effect of dietary fatty acid balance on myocardial lesions in male rats.

Three hundred (experiment I) and 350 (experiment II) weanling, 3-week-old male Sprague-Dawley rats weighing between 40–50 g were randomly assigned two per cage and 50 per dietary treatment to study the effect of dietary fatty acid balance on myocardial lesions. The following oils were tested: Experiment I.Brassica napus var. Tower rapeseed oil [Tower RSO, 1974 cultivar and 1975 cultivar, each containing 0.3% erucic (22∶1) acid];B. napus var. Zephyr RSO containing 0.9% 22∶1; corn oil; olive oil; and soybean oil. Experiment II.B. napus var. Tower RSO (1974 cultivar), olive oil, soybean oil, and the following oils to which was added the indicated level of free 22∶1; Tower +0.5% 22∶1; Tower +5.6% 22∶1; olive oil +4.4% 22∶1; soybean oil +5.7% 22∶1. In each case the oils were incorporated in a semisynthetic diet at a level of 20% by weight. Heart and heart lipid weights of rats fed the different oils did not differ statistically from each other. Fatty acid analyses of heart lipids revealed that the fatty acid composition of the cardiac lipids reflected that of the diet fed. In experiment I, there was a definite but significantly lower incidence (P<0.01) and severity (P<0.01) of heart lesions in rats fed control oils (corn, olive, soybean) than in rats fed rapeseed oils. Also, in experiment II, a definite but lower incidence and severity of heart lesions occurred in rats fed control oils (soybean, olive) compared to rats fed Tower RSO or this oil with added free 22∶1. Adding 22∶1 to an oil naturally high in 18∶3 (soybean) did not alter the incidence of heart lesions, whereas adding 22∶1 to an oil naturally high in 18∶1 (olive) increased significantly (P<0.01) both the incidence and severity of heart lesions. Thus, it appears that the background incidence of heart lesions that are found in the rat in any case, and which are increased by rapeseed oil feeding, is caused by the imbalanced fatty acid composition of the oil for the growing rat, i.e. high monoenes (18∶1, 20∶1, and 22∶1) and high 18∶3 and is not only due to the presence of excess 18∶3.

Effects of Dietary Saturated Fat on Erucic Acid Induced Myocardial Lipidosis in Rats

Male Sprague-Dawley rats were fed for one week diets containing 20% by weight fat/oil mixtures with different levels of erucic acid (22∶1n−9) (∼2.5 or 9%) and total saturated fatty acids (∼8 or 35%). Corn oil and high erucic acid rapeseed (HEAR) oil were fed as controls. The same hearts were evaluated histologically using oil red O staining and chemically for cardiac triacylglycerol (TAG) and 22∶1n−9 content in cardiac TAG to compare the three methods for assessing lipid accumulation in rat hearts. Rats fed corn oil showed trace myocardial lipidosis by staining, and a cardiac TAG content of 3.6 mg/g wet weight in the absence of dietary 22∶1n−9. An increase in dietary 22∶1n−9 resulted in significantly increased myocardial lipidosis as assessed histologically and by an accumulation of 22∶1n−9 in heart lipids; there was no increase in cardiac TAG except when HEAR oil was fed. An increase in saturated fatty acids showed no changes in myocardial lipid content assessed histologically, the content of cardiac TAG or the 22∶1n−9 content of TAG at either 2.5 or 9% dietary 22∶1n−9. The histological staining method was more significantly correlated to 22∶1n−9 in cardiac TAG (r=0.49;P<0.001) than to total cardiac TAG (r=0.40;P<0.05). The 22∶1n−9 content was highest in cardiac TAG and free fatty acids. Among the cardiac phospholipids, the highest incorporation was observed into phosphatidylserine, followed by sphingomyelin. With the addition of saturated fat, the fatty acid composition showed decreased accumulation of 22∶1n−9 and increased levels of arachidonic and docosahexaenoic acids in most cardiac phospholipids, despite decreased dietary concentrations of their precursor fatty acids, linoleic and linolenic acids.

Lipid composition ofNeurospora crassa

The lipids ofNeurospora crassa, isolated in pure form from freeze-dried mycelium, were found to contain squalene, sterol esters, triglycerides, free fatty acids, geranylgeraniol, free sterols, carotenoids, cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The above compounds were isolated in pure form by column and thin layer chromatography and were characterized by infrared spectroscopy and chromatographic mobilities. Fatty acid moieties were characterized by gas liquid chromatographic retention times of their methyl esters relative to those of authentic standards. The fatty acid composition of the triglycerides was found to be similar to that of phosphatidic acid, cardiolipin, and lecithin.

Changes in liver lipid composition of male rats fed rapeseed oil diets

Studies are reported on the effect of feeding diets containing rapessed oils differing in their erucic acid content to male weanling rats for 16 weeks. Rapeseed oil high in erucic acid depressed growth. Total lipids, lipid phosphorous and cholesterol, in the livers were not significantly different between the experimental groups. The fatty acid composition of the total liver lipids, the neutral lipids, phosphatidylethanolamine and phosphatidylcholine are documented. Erucic and eicosenoic acids were found in all lipid classes at the same relative concentration; the amount being incorporated was proportional to that found in the dietary oil. The positional analysis of phosphatidylethanolamine and phosphatidylcholine are presented. Erucic acid was incorporated preferentially at position two of these phospholipids, whereas, twice the level of eicosenoic acid was found at position one, compared to that which occurred at the two position.

Cardiopathogenicity of soybean oil and tower rapessed oil triglycerides when fed to male rats

The triglycerides of soybean oil were purified by molecular distillation and those of Tower rapeseed oil by molecular distillation and adsorption chromatography. The original oils and the purified triglycerides were incorporated in semisynthetic diets at 20% by weight and fed for 16 weeks to weanling male Sprague-Dawley rats to compare the nutritional and pathological effects of the oils and their triglyceride fractions on rats. The study was carried out at two independent laboratories. No significant differences were observed between the results of the two establishments. The incidence of myocardial lesions was significantly higher in rats fed Tower rapeseed oil than in those fed soybean oil. Purification of the triglycerides by molecular distillation and adsorption chromatography appeared to have no major effect on the incidence of myocardial lesions. This supports our previous findings that the cardiopathogenicity of the test oils to rats resides in the triglycerides of these oils.