Fatty acid composition of heart cells exposed to thermally oxidized fats

Corn oil and olive oil were thermally oxidized, and the free fatty acids from the fresh fats, and from the distillable non-urea-adductable (DNUA) fractions of the thermally oxidized fats were prepared. These were added as emulsions to the medium of primary cultures of heart endothelial and muscle cells from neonatal rats. After exposure for 24 hr, the fatty acid composition of the triacylglycerol (TC) and phospholipid (PL) fractions of the cells was determined. Reflecting the nature of the fat used, the corn oil treatment produced relatively higher concentrations of linoleic acid in the TG and PL fractions compared to the olive oil treatment, in which case the oleic acid level was influenced. Treatment of the cultured cells with components derived from oxidized corn oil or oxidized olive oil resulted in lower concentrations of linoleic and arachidonic acids in the PL moieties compared to the fresh fat controls. However, there were marked increases in arachidonic acid in the TG fractions of both the endothelial and muscle cells. These changes due to the DNUA from thermally oxidized fats indicate a distinct metabolic response to the derivatives formed during thermal oxidation of the fats.     

Nutritional and metabolic studies of distillable fractions from fresh and thermally oxidized corn oil and olive oil

A semisynthetic diet containing 15% by weight of dietary fat was fed to six groups of male Wistar rats for 28 days. Two groups received the distilable fraction of fresh corn oil (DCO) or fresh olive oil (DOO), two groups the distillable fraction of the thermally oxidized fats (OCO,OOO), and two groups received the respective fresh fats as controls (FCO, FOO). Substantial changes in the fatty acid composition occurred in the fats upon thermal oxidation. Only the rats that received OOO showed overt symptoms of heated fat toxicity. This was reflected in the histological scores of these animals, with the liver sustaining the most numerous and severe lesions. Tissue fatty acid changes of any significance were confined largely to the polar liver lipids of the rats that were fed OCO or OOO. The results of this study would suggest that the relatively greater toxicity of OOO, compared to OCO, may in part be due to the high oleic:linoleic acid ratio of the fresh olive oil and in part to a higher tocopherol content of the corn oil.     

Exacerbation of heart and liver lesions in rats by feeding of various mildly oxidized fats

Groups of 40 male Charles River rats were fed diets containing cottonseed oil, olive oil, corn oil, soybean oil, coconut oil, chicken fat, beef fat, butter oil, lard and saturated medium chain triglycerides. The fats were fed fresh and after 40 hr aeration at 60 C, which hardly changed peroxide values. In addition, fresh and aerated soybean oil and lard were fed to W/Fu rats. Body weights and life span were significantly influenced by the kind of fat fed, but not by aeration. Many hearts exhibited unspecific focal myocarditis and focal fibrosis. The latter was graded in a blind test, which revealed highly significant differences in the incidence of severe lesions; those fed corn oil had the most, followed by cottonseed oil, soybean oil, olive oil, beef fat, saturated medium chain triglycerides, butter, chicken fat and lard, in that order. Feeding of aerated fat resulted in an increased incidence with six of the eight fats. The W/Fu rats had lower incidences, but those fed soybean oil had more than those fed lard, and aeration led to a higher incidence. Some heart sections stained with Light Green SF Yellowish revealed areas of muscle fibrils that did not accept the stain, probably as a consequence of cellular damage. Higher incidences of this lesion were associated with the same fats as was severe fibrosis, and feeding of aerated fats led to higher incidences. Many livers revealed marked proliferation of bile ducts. The groups fed cottonseed, soybean, olive and corn oils had higher incidences of severe lesions, and feeding of the oxidized oils led to still higher incidences. None of the results appeared to be associated with the fatty acid composition of the fats, which suggested that these long term effects may have been due to minor constituents in the individual fats. One of seven papers presented in the symposium “Biological Significance of Autoxidized and Polymerized Oils,” JOCS-AOCS Joint Meeting, Los Angeles, April 1972.     

A long-term nutritional study with fresh and mildly oxidized vegetable and animal fats

Fresh and oxidized cotton seed oil (CO) olive oil (OO), chicken fat (CF) and beef fat (BF) were fed to male weanling rats for 33 to 108 weeks. Groups fed oxidized fats except OO showed a higher death rate than those fed the corresponding fresh fats. Groups fed oxidized CO and BF had the highest death rate. Histological studies of animals dying from natural causes showed more pronounced cardiac lesions in the animals fed oxidized CO. Serum, liver and brain cholesterol levels were not influenced by oxidized fats. Fatty acid composition of depot fats and of heart and liver lipids did not show significant differences between groups fed fresh and the corresponding oxidized fats. Presented at the Spring Meeting of the AOCS 1964.     

Effect of dietary fish oil on the rate of very low density lipoprotein triacylglycerol formation and on the metabolism of chylomicrons

The mechanism by which ω3 fatty acids lower plasma triacylglycerol levels was investigated. Rats were fed fish oil, olive oil (10% fat by weight) or a nonpurified diet 4% fat by weight) for 15 days. Lipoprotein lipase was inhibited by intra-arterial administration of Triton WR 1339 to estimate hepatic triacylglycerol output. Rats fed the olive oil diet showed a higher rate of triacylglycerol formation than rats fed the ω3 fatty acid diet or the low-fat diet. All three groups showed identical rates of removal from plasma of intraarterially administered artificial chylomicrons that had simultaneously been labeled with cholesteryl [1-¹⁴C]oleate and [9,10(n)-³H]triolein. Liver radioactivity and total fat content were lowest in rats fed the fish oil diet, indicating that ω3 fatty acids were preferentially metabolized in liver. Chylomicrons obtained from donor rats fed either fish oil containg [¹⁴C]cholesterol or olive oil containing [³H]cholesterol were removed at similar rates when infused together intraarterially into recipient animals. A slower formation of plasma very low density lipoprotein triacylglycerols in rats fed fish oil is probably due to a faster rate of oxidation of the fatty acid chains in the liver resulting in decreased plasma triacylglycerol concentrations.     

Biological effects due to changes in fats during heating

In deep-fat frying the fat is used over and over again, and moisture and air are mixed into the hot oil. Substantial quantities of the heated fat are absorbed into the fried foods. Many reports from experimental observations with animals fed these fats have shown biological effects ranging from a slight depression in growth, all the way to very poor growth, diminished feed efficiency, increased liver size, fatty necrosis of the liver, and various other organ lesions. Obviously, certain fat constituents may be changed by frying conditions, and the adverse biological effects are relative. We are at the stage in studying these heated fats where selected techniques including biochemical parameters, histopathological evaluations, and tissue culture in monolayers can be good indicators of some of the specific effects on biological tissues. Isolated fractions from heated fat samples, which contained concentrations of cyclic monomer and dimer derivatives, were used in animal studies. Incorporation of the above materials into rat diets produced distended flatulent stomachs and intestines, gastric ulcers, and multiple focal hemorrhages. Histological evaluation of heart, liver, and kidney tissue sections indicated extensive cellular damage. Livers and kidneys exhibited the most severe lesions. Neonatal heart cells established as monolayers on glass cover slips were exposed to fractions from heated or fresh fats. Cellular damage including pyknosis, vacuolization of the cytoplasm, and mitotic aberrations were observed. Uptake of¹⁴C-labeled fatty acid by the triglyceride fraction of the cells was increased with heated fats.     

Studies on the nutritional and physiological effects of thermally oxidized oils

Summary The present results indicated that the thermal oxidation products from the polyunsaturated fatty acids, primarily linoleic acid, are responsible for much of the loss of nutritional value in thermally oxidized edible oils. Oils which have a high linoleic acid content are more likely to undergo thermal oxidative damage than those with lower linoleic contents. Also the ratio of linoleic acid to total unsaturation has some effect on the nutritive stability of the oil when it has been thermally oxidized. An oil with a high iodine value but with a low linoleic acid value appears to be more stable to thermal oxidation than an oil with an iodine value one half as great but with most of the unsaturation in the oil caused by linoleic acid. The products formed during thermal oxidation which cause the loss of nutritional value are those which do not form urea-inclusion compounds. They are probably polymeric in nature, but thermally oxidized oils also contain carboxylic acids and carbonyl groups which might cause some of the nutritional loss observed when thermally oxidized oils are fed. The rate ofin vitro hydrolysis of the thermally oxidized corn oil by pancreatic lipase, also the rate of absorption from the intestine of the male rats, were found to be decreased. However the percentage of absorption in 24 hrs. was the same with both fresh and thermally oxidized oil. The liver-body weight ratio of rats fed a diet containing the thermally oxidized oil were found to be significantly larger than the liver-body weight ratio in animals fed diets containing fresh oil. However the livers of animals fed the thermally oxidized oil diets did not differ in lipide percentage or total solid content, and histopathological investigations did not show any abnormal conditions.     

In vivo determination of triglyceride secretion using radioactive glycerol in rats fed different dietary saturated fats

Our objective was to determine the relative rates ofin vivo triglyceride (TG) secretion and the composition of very low density lipoproteins (VLDL) in rats fed different dietary saturated fats. Male Sprague-Dawley rats (150–200 g) were fed diets containing 16% corn oil, or 14% butterfat, 14% beef tallow, 14% olive oil, or 14% coconut oil plus 2% corn oil for 5 wk. Changes in plasma TG specific radioactivity were determined in individual, unanesthetized fasted rats after injection of 100 μCi [2-³H]glycerol. Nonlinear regression analysis using a 2-compartment model was used to determine the fractional rate constant for TG turnover in plasma. The plasma TG pool was 33–40% larger with beef tallow than with corn, olive or coconut oil feeding (p<0.05), and 20% larger with beef tallow than with butterfat feeding. The rate of TG secretion into plasma (mg/min/100 g body weight) was 60% higher in animals fed beef tallow than corn or coconut oil (p<0.05) and 26–33% higher in animals fed beef tallow than olive oil or butterfat. Differences in VLDL composition (% wt) were also noted. Our data suggest that greater TG secretion is the primary factor contributing to the larger TG pool with ingestion of beef tallow relative to butterfat, corn or coconut oil. These results suggest that different dietary saturated fats have unique effects on TG metabolism in rats. Presented in part at the 1990 meeting of the Federation of American Societies for Experimental Biology in Washington, D.C. (see ref. 1).     

Effect of heated fat upon metabolism of 1-14C-acetate in the rat

Groups of rats were prefed for 18 weeks with fresh or heated corn oil (≈5% nonurea adduct) at 10, 20 and 30% protein levels and at 10% protein+2% cellulose; 1-¹⁴C-sodium acetate was injected intraperitonially into each animal and the radioactivity was measured in expelled CO₂ and also in the lipid fractions of liver. In the groups of rats fed 10% protein, one-half of the administered radioactivity was found 50 min after acetate injection into rats fed fresh oil, whereas 90 min were required to reach the same level in rats fed heated oil. The same trend was found in all the groups of rats receiving different protein levels. The conversion of acetate to CO₂ was significantly higher at a 10% dietary protein level than at the 20 or 30%, but there appeared to be no significant difference in the conversions at the 20 and 30% protein levels. However addition of 2% cellulose to the 10% dietary protein level significantly increased the conversion of the acetate to CO₂ in rats fed heated oil. The livers of animals receiving heated fat diets had a higher lipid content, mainly triglyceride. When the liver lipids from rats fed fresh corn oil were separated by argentation thin layer chromatography, the bulk of the radioactivity was found in the saturated fractions. Monoene, diene and triene fractions from the liver lipid of rats fed heated oil had almost twice the radioactivity of those from fresh oil, suggesting the preferential utilization of acetate in the synthesis of these unsaturated fatty acids. One of seven papers presented in the symposium “Biological Significance of Autoxidized and Polymerized Oils”, JOCS-AOCS Joint Meeting, Los Angeles, April 1972.     

Influence of cabbage juices on oxidative changes of rapeseed oil and lard

The influence of white cabbage on oxidative and thermooxidative changes of plant fats (rapeseed oil) and animal fats (lard) was determined by an array of standard tests. The fats were heated at 100 °C for 6 h in a model system mimicking normal cooking conditions in the presence or absence of fresh cabbage juice (FCJ) or sauerkraut juice (SJ). After thermal processing of fats in the presence of cabbage juices, regardless of the kind, the formation of oxidized triacylglycerols (TAG) was decreased by about 40% compared to fats heated alone. Juices also inhibited the formation of TAG dimers and polymers. Such parameters as peroxide value, extinction coefficient at 232 nm (E^1%_1cm), and losses of polyene fatty acids revealed the protective effect of cabbage juices as well; however, in this case FCJ displayed a much stronger antioxidant effect than SJ. The protection was more visible for lard which contains much lower amounts of natural antioxidants.     

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[³H]glycerol/diacyl[³H]glycerol ratio was observed. The distribution of [1-¹⁴C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-¹⁴C]EPA was oxidized to a much greater extent than [1-¹⁴C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.     

Biosynthesis of protein relative to the accumulation of fat in the liver of EFA deficient rats

Effect of an essential fatty acid (EFA) deficiency in the rat on the incorporation of leucine-¹⁴C and glucosamine-¹⁴C into serum and liver protein are reported. Weanling male rats of the Sprague-Dawley strain were raised on a fat-free diet for 10–12 weeks and then switched to diets supplemented with 10% corn oil or 10% hydrogenated coconut oil. Leucine-¹⁴C or glucosamine-¹⁴C was injected into the tail veins of the animals of each group. At selected intervals up to 120 min after the injections, the animals were sacrificed and the radioactivity of the liver and serum proteins was measured. The levels of triglyceride (TG) in the serum and the liver were also determined. Less radio-activity was incorporated into the serum β-lipoprotein (β-LP) fraction of the hydrogenated coconut oil than the corn oil fed animals injected with leucine-¹⁴C, but no differences were observed in the incorporation of radioactivity into the liver protein and both albumin and globulin fractions of the serum of these groups of animals. In the similar experiments with glucosamine-¹⁴C less radioactivity was incorporated into the β-LP fraction of the serum and into the smooth endoplasmic reticulum of the liver in the hydrogenated coconut oil (EFA deficient) than the corn oil fed animals. Time course studies also indicated that less radioactivity was incorporated into the β-LP fraction than into the albumin and globulin fractions of the serum of the hydrogenated coconut oil group. These findings suggest that an EFA deficiency results in an impairment of the synthesis or release of lipoprotein.     

Factors influencing the formation of complexes between oxidized lipids and proteins

Complexes were formed between egg albumin and oxidized lipids, such as thermally oxidized corn oil and autoxidized linoleic acid, under a standardized set of conditions. No complex could be obtained under these conditions when lysine, glycine, bactopeptone, gelatin or sodium caseinate were substituted for egg albumin. Lactalbumin was equally as reactive, casein much less and fresh egg white superior to egg albumin in their complexing ability with oxidized corn oil. As the time of reaction was increased, the amount of the complex formed also increased. Optimum complex formation took place at a concentration of 1% protein, a pH of 7 and 60C. The ease of formation of these complexesin vitro indicated that similar complexing could readily take placein vivo under suitable pathological conditions. This work was supported by research grant no. C-1932 from the National Institutes of Health, U. S. Public Health Service, Department of Health, Education, and Welfare.     

Digestibility of fatty acid monomers,dimers and polymers in the rat

This study was designed to determine digestibilities of fatty acid monomers, dimers and polymers as components of diets containing thermally oxidized oils. Male Wistar rats were fed semipurified diets supplemented with unheated, heated and a 1:1 mixture of unheated/heated olive oils at 6, 12 and 20% w/w of diet. In a 14-d experimental period, fecal lipids were extracted and analyzed by a combination of adsorption and high-performance size-exclusion chromatographies. Thus, it was possible to separate and quantitate five groups of fatty acids—nonpolar monomers, oxidized monomers, nonpolar dimers, oxidized dimers and polymers. Nonpolar fatty acid monomers showed high digestibilities, although significantly influenced by the alteration level of the dietary oil. The apparent absorption of oxidized fatty acid monomers averaged 76.6%. Among polymeric fatty acids, the lowest digestibilities were found for nonpolar dimers (10.9% on average), whereas oxidized dimers and polymers possessed higher apparent absorbability than expected, ranging from 22.7% to 49.6%. Chemical modifications prior to absorption, leading to less complex products, may have contributed to enhanced digestibility of polymers.     

Supercritical fluid fractionation of thermally oxidized canola oil

Supercritical fluid extraction procedures were developed to fractionate thermally oxidized canola oil. Canola oil was heated in a sealed glass ampoule for 72 h at 200°C in a forced-convection oven. After heating, the percentages of triacylglycerol (TAG) trimer, dimer, monomer and lowermolecular weight (LMW) compounds in the heated oil sample (as determined by high-performance size-exclusion chromatography) were 3.0, 8.4, 81.3 and 7.3%, respectively. The LMW compounds included diacylglycerols, monoacylglycerols and free fatty acids. Fractions of TAG monomer of approximately 98% purity were isolated after extraction at 408 atm for 45 min. After a second extraction at 442 atm for 45 min, a TAG dimer fraction of 95% purity was isolated. Recoveries of TAG monomer and dimer were 96 and 87%, respectively.     

Column and high-performance size exclusion chromatography applications to the in vivo digestibility study of a thermoxidized and polymerized olive oil

This study aimed (i) to design an in vivo model to study fat digestibility, and (ii) to apply this design to test the in vivo digestibility of a highly thermoxidized olive oil. True digestibility of unheated olive oil was tested 2, 4, 6, and 7 h after administering 1 g of olive oil/100 g body weight to young adult Wistar rats by means of esophageal probes. Remaining gastrointestinal lumen fat showed an inversely linear relationship (t=−0.9932; P<0.001) with the length of the experiment. A 4-h test was considered adequate because after this period, half of the oil administer still temains in the lumen, making it possible to accurately measure the different nondigested, nonabsorbed themoxidized compounds. In a second experiment, fresh olive oil (3.6 mg polar content/100 mg oil) was heated at 180°C for 50 h in the presence of air; the polar content in this oil rose to 46.0 mg/100 mg oil. After 4 h, the true digestibility coefficient of 50-h heated olive oil did not significantly change, although it tended to decrease (24%) with respect to the unheated oil. Silica gel column chromatography and high-performance size exclusion chromatography were used to quantify nonthermoxidized and thermoxidized products present in the oils and in the gastrointestinal lumen after these test periods. True digestibility of the different thermoxidized compounds from the heated oil was 30–40%, whereas that of thermoxidized compounds from the fresh oil was much higher (∼80%). Nonoxidized triacylglycerol hydrolysis was negatively affected by the presence of large amounts of thermoxidized compounds. The present proposed model seems to be a useful tool for the study of thermoxidized oils. Data also show that thermoxidized compounds from abused olive oil are poorly but actively hydrolyzed and absorbed in vivo.     

Influence of medium-chain triglycerides on lipid metabolism in the rat

Lipid metabolism was studied in rats fed diets containing corn oil, coconut oil, or medium-chain triglyceride (MCT), a glyceride mixture containing fatty acids of 8 and 10 carbons in length. The ingestion of MCT-supplemented, cholesterolfree diets depressed plasma and liver total lipids and cholesterol as compared with corn oil-supplemented diets. In rats fed cholesterol-containing diets, plasma cholesterol levels were not influenced by dietary MCT, but liver cholesterol levels were significantly lower than in animals fed corn oil. In vitro cholesterol synthesis from acetate-1-¹⁴C was lower in liver slices of rats that consumed MCT than in similar preparations from corn oil-fed rats. Studies of fatty acid carboxyl labeling from acetate-1-¹⁴C and the conversion of palmitate-1-¹⁴C to C₁₈ acids by liver slices showed that chain-lengthening activity is greater in the liver tissue of rats fed MCT than in the liver of animals fed corn oil. The hepatic fatty acid desaturation mechanisms, evaluated by measuring the conversion of stearate-2-¹⁴C to oleate, was also enhanced by feeding MCT. Adipose tissue of rats fed MCT converts acetate-1-¹⁴C to fatty acids at a much faster rate than does tissue from animals fed corn oil. Evidence is presented to show that the enhanced incorporation of acetate into fatty acids by the adipose tissue of rats fed MCT represents de novo synthesis of fatty acids and not chain-lengthening activity. Data are also presented on the fatty acid composition of plasma, liver, and adipose tissue lipids of rats fed the different fats under study.     

The nutritive value of the Brazil nut oil

The chemical composition of the decorticated Brazil nut in the natural and fat-free state was determined, and the results confirm that this seed contains significant amounts of oil and protein. In three growth experiments carried out in rats, the Brazil nut oil had a nutritive value comparable to that of butterfat and the common table oils, olive oil, cottonseed oil, and corn oil. The use of levels of the Brazil nut oil up to 20% of the diet did not reduce growth or food consumption of rats. Heat treatment of the oil up to 120 min. at 140°C. did not reduce its nutritive value. The coefficient of digestibility of the Brazil nut oil was similar to that of the other oils, fresh and heated. The digestibility of the Brazil nut oil averaged 98% and was not affected by the heat treatment. The data indicate that Brazil nut oil is comparable to other animal and vegetable fats and oils in common use for human consumption. International Cooperation Administration fellow and Research Assistant. INCAP Publication I-150.     

Studium der UV-Spektren oxydierter Fette III

Studies on the UV-Spectra of Autoxidized Fats III The author has determined the various properties of oxidized oils and fats with relation to the first maximum of extinction E₁ according the direct UV-method (E_1cm^1%). Following fats and oils were investigated: sunflower oil, rapeseed oil, peanut oil, soybean oil, corn oil, lard, butter, beef tallow and hardened edible fat.     

Complex formation between oxidized lipids and egg albumin

The nature of the reactions leading to the formation of complexes between oxidized lipids and proteins is little understood. Complexes were formed between thermally oxidized and thermally polymerized corn oils and egg albumin. The results indicated that the thermally oxidized oil was much more reactive than the thermally polymerized oil. The nature and extent of oxidative groups in the lipid were varied and reacted withegg albumin. The keto and epoxy groups seemed to have a pronounced influence on complex formation while the hydroxy and hydroperoxide groups were observed to be less reactive. Acetylation of egg albumin did not significantly decrease its complexing ability with thermally oxidized corn oil. Further confirmation that the reactive groups in the protein were not covalently tied up to the lipid was obtained by estimating these groups in the original protein and in the complexed protein. IR analyses of oxidized lipid-alumina, oxidized lipid-egg albumin and fatty acid-urea complexes indicated that the characteristic absorption was due to bonded O-H stretching vibration in the first two complexes and the bonded N-H stretching vibration in the two latter complexes.