Roslyn B. Alfin-Slater

Characteristics of the Cholesterol-Esterifying Activity in Normal and Atherosclerotic Rabbit Aortas

Esterification of cholesterol with [1-14C]palmityl-CoA by an atherosclerotic cell-free homogenate was approximately 16–50-fold greater than that by a normal cell-free homogenate for a given amount of protein in the homogenate. This difference was due to hyperactivity of the cholesterol-esterifying system in the atherosclerotic cell-free homogenate rather than to depletion of radioactive palmityl-CoA in the reaction mixture containing normal homogenate. Neither an activator of cholesterol esterification in the soluble fraction of the atherosclerotic aortic homogenate nor an inhibitor in the soluble fraction of the normal aortic homogenate could be demonstrated. The pH optimum within the pH range covered for esterification and the apparent Km values were approximately the same in normal and atherosclerotic microsomes, suggesting that the enzymes were probably the same. The results suggested a higher concentration or a higher activity of the enzyme in or on atherosclerotic microsomes. An alternative possibility is that high concentrations of free cholesterol in the atherosclerotic microsomes were responsible for the augmented cholesterol esterification. This possibility seems unlikely, because the observed 2.3-fold increase in the free cholesterol concentration should not produce a 25-fold increase in cholesterol esterification. The rate of cholesterol esterification by atherosclerotic microsomes varied with the substrate: oleyl-CoA > palmityl-CoA > linoleyl-CoA.

Esterification of cholesterol by homogenates of atherosclerotic and normal aortas

Abstract Cholesterol esterification with palmityl-CoA by cell-free homogenates from atherosclerotic rabbit aortas was 39 times greater than with cell-free homogenates from normal aortas. In a 2 1 2 hr time-trend study using atherosclerotic preparations, the specific activity curve of cholesteryl ester reached an early plateau at a level much lower than that of the fatty acid derived from the β-position of lecithin, with no tendency of the cholesteryl ester specific activity to rise further approaching that of lecithin. A similar type of experiment was done using normal aortic preparations. In contrast to the experiments with atherosclerotic tissue, the specific activity of cholesteryl ester was much higher than that of lecithin at each time interval after 4 minutes of incubation. The data indicate that lecithin is not an intermediate in the observed esterification of cholesterol in our preparations. Esterification of cholestrol occurs upon addition of labeled palmityl-CoA, and terminates after exhaustion of the substrate. Most of the cholesterol-esterifying activity is localized in the microsomes of normal and atherosclerotic aortas. Cholesterol esterification by atherosclerotic and normal microsomes requires ATP and CoA, when free fatty acid is provided as the acyl source. Very little esterification occurs without these factors. These observations together indicate that cholesterol esterification is accomplished by fatty acyl-CoA:cholesterol acyltransferase.

Effect of varying levels of dietary protein on tumor development and lipid metabolism in rats exposed to aflatoxin.

Reports in the literature concerning the relationship of protein nutrition to aflatoxicosis are contradictory. In an attempt to elucidate this relationship more clearly, we have examined the effects of low, normal, and high protein-containing diets on tumor incidence and development, as well as on several biochemical indices, in rats which have been exposed to low levels of aflatoxin in a “chronic” rather than “acute” situation. In our study, male weanling rats were place for 3 months on otherwise adequate diets containing either 8, 22, or 30% casein with and without aflatoxin B1 at 1.7 ppm. Half of the animals in each group received diets which were further supplemented with the amino acid, cystine, at 0.6% of the diet. (Sulfur-containing amino acids are the most limiting amino acids in casein, and the addition of cystine to the diet serves to improve the biological quality of the protein source.) After 3 months the animals were fed control diets without aflatoxin until they were killed at 1 year. Weight gain was markedly decreased and liver weights increased in response to aflatoxin in all groups except those on the low protein diets, where aflatoxin had no effect on these protein diets, where aflatoxin had no effect on these indices. No tumors were found in the livers of rats fed the low protein, aflatoxin-supplemented diet. In the other groups, the severity of the liver involvement increased progressively with increased protein levels in the diet. When cystine was included in the diet, tumors were observed also in the animals fed the low protein diet; furthermore, the livers of those animals on “normal” and high protein diets were much more severely involved than were the livers of animals on non-cystine supplemented diets. Plasma cholesterol levels were increased in response to aflatoxin when the diets containing 22 and 30% protein were fed and when cystine was included in the 8% protein diet. Liver cholesterol levels were increased in response to aflatoxin in all groups except in those receiving the low protein diets. Among these latter animals, aflatoxin administration had no effect on liver cholesterol values. Changes as a result of aflatoxin administration were also observed in the fatty acid composition of sterol esters, triglycerides, and phospholipids of liver and tumor tissue.

Effects of caerulein and bombesin on insulin and glucagon secretion from the isolated, perfused rat pancreas

Caerulein nd bombesin, peptides first isolated from amphibian skin, act upon the mammalian gastrointestinal tract. To determine if these peptides influence mammalian endocrine pancreatic function, we tested their effects on the isolated, perfused rat pancreas. In these experiments, we examined effects of constant infusions of either caerulein (10(-11) M through 10(-8) M) or bombesin (10(11) M through 3.0 . 10(-7) M) which were superimposed upon glucose. Secretory studies consisted of a 20-min basal period (60 mg/dl glucose), then a 15-min infusion of glucose (150 mg/dl). Effects of the peptides (10(-9) M) upon the response to post-glucose arginine (168 mg/dl), confusion with glucose (60 mg/dl) for 15 min. were also studied. Neither peptide had any effect on basal insulin secretion. However, both peptides had distinct effects on insulin responses to the stimuli. Both caerulein and bombesin produced enhancement of glucose-induced insulin secretion, increasing total insulin secretion in a dose-dependent manner. Only caerulein enhanced arginine-induced insulin secretion. These peptides had no effect upon arginine-induced glucagon secretion.

Vitamin E and Lipid Metabolism

Publisher Summary This chapter discusses the relationship of vitamin E to lipid and other metabolisms. The identification of vitamin E as a fat-soluble vitamin, its occurrence in vegetable oils, its storage in association with body lipids, and its possible function as a biological antioxidant suggest that a close relationship exists between vitamin E and various phases of lipid metabolism. The observation that atheromatous lesions contain relatively high concentrations of organic peroxides, and cholesterol, and the fact that vitamin E protects essential fatty acids from being oxidized to organic peroxides implicate vitamin E as possible inhibitor in atherogenesis. A significant amount of evidence indicates that the primary, if not the sole, function of vitamin E in metabolism is of an in vivo lipid antioxidant. Probably the most direct evidence to substantiate this theory is that lipoperoxides have been found in the tissues of vitamin E-deficient animals. It is assumed that vitamin E acts as an in vivo lipid antioxidant, protecting unsaturated fatty acids in tissue lipids against peroxidation.

Dietary fat composition and tocopherol requirement: III. Quantitative studies on the relationship between dietary linoleate and vitamin E

The present study has involved biologically titrating linoleate vs. vitamin E using the male rat as the indicator. In the first of the titration studies, the dietary tocopherol level was held constant, while in the second study the linoleate intake was held constant. The investigation was conducted with male rats since these have a much higher linoleate requirement than females. By first depleting such animals of their stores of essential fatty acids by feeding a fat-free diet from weaning, a sensitive test organism was provided. These animals have an immediate need for linoleate during the repletion periods. If an imbalance between linoleate and vitamin E content existed in any of the dietary regimens, such an imbalance would have been more likely noted in test animals actively metabolizing the ingested linoleate. Based upon various nutritional and biochemical indices, the amount of tocopherol ordinarily included in the basic diets fed to our rats, 0.01% as dl-alphatocopheryl acetate, was adequate even when the diet provided up to 5% linoleate; an amount corresponding to ca. 12% of the total calories and providing a ratio of linoleate to the tocopherol of ca. 500:1. In the reverse biological titration with all test diets now providing the constant level of 5% linoleate, ratios of linoleate to vitamin E were satisfactory even in a ratio of as much as 2500:1 (or 0.4 mg gram of vitamin E per polyunsaturated fatty acid). The control animals continued on the fat-free diet indicated that there is a need for added tocopherols even in the absence of linoleate according to a number of biochemical indices. Based upon a number of accepted bioanalytical approaches, the minimum requirement for linoleate by the fat-depleted male rat was found to be between 100–200 mg/day or ca. 1–2% of the caloric intake. Although the fatty acid composition of tissue lipid fractions is markedly affected by the amount of linoleate in the diet, dietary tocopherol supplements have little effect on these values.

Dietary factors and aflatoxin toxicity: I. comparison of the effect of two diets supplemented with aflatoxin B1 upon two different strains of rats

Previous studies in this laboratory with rats fed low levels of aflatoxin as a component of peanut discards, suggested either a strain tolerance for aflatoxin or a possible protective factor present in the diets used. In this study, two strains of rats, the Charles-River strain and the former USC strain, were used to test the effect of 1.7 ppm purified aflatoxin B1 included for 3 months in two different diets; one previously used in this laboratory and one used by other investigators in aflatoxin studies. After an experimental period of either 12 or 18 months, growth, mortality, gross pathology, and organ wt were measured, and histopathological examination and biochemical analyses were performed. Plasma and liver cholesterol levels, total liver lipids, and fatty acids in the various lipid fractions of plasma, liver, and liver tumor lipids were measured. Both strains of rats proved to be susceptible to aflatoxin toxicity at this level as manifested by the appearance of hepatomas; however, liver involvement was more extensive in the Charles-River rats. The diet used by other investigators produced symptoms similar to those observed as a result of essential fatty acid deficiency and also affected the response to aflatoxin through an aggravation of symptoms, i.e. an inhibition of growth and increased size and severity of the liver tumors.

Dietary fat composition and tocopherol requirement. I. Lack of correlation between nutritional indices and results of in vitro peroxide hemolysis tests.

In general, the native tocopherols in polyunsaturated vegetable oils such as cottonseed oil, corn oil and their lightly hydrogenated products include sufficient vitamin E for growth, reproduction, lactation and normal lipid metabolism in the rat. The administration of vitamin E to animals fed diets deficient in essential fatty acids (e.g., a hydrogenated coconut oil or a fat-free diet) does not stimulate growth or reproductive performance per se, although testes development in the male rats is improved and some improvement in lipid metabolism is also noted. Hemolysis of the erythrocytes in vitro by hydrogen peroxide is increased in animals on diets rich (30%) in polyunsaturated vegetable oils or on diets providing no essential fatty acids at all. However, the conditions of the in vitro hemolysis test are not related to those in vivo and the in vitro test is not a measure of erythrocyte fragility. In addition, the in vitro hemolysis test does not necessarily reflect plasma tocopherol levels nor an abnormal nutritional state as a result of subsistence on high linoleate, low tocopherol intake, but rather measures the susceptibility to oxidation of a labile biological substrate and indicates the effective balance between potentially oxidizable lipids (polyunsaturates) in the stroma of the red blood cell and the antioxidant present (tocopherol or vitamin E). The labile lipid substrate may be either of exogenous origin (diet) or may be formed endogenously through tissue synthesis (as a result of an essential fatty acid deficiency). It is concluded that the in vitro hemolysis test may not be a valid indicator of vitamin E nutriture unless it is used in conjunction with other nutritional tests.

A comparison of the effects of the polyunsaturated fatty acids of cuttlefish liver oil and cottonseed oil on cholesterol metabolism.

Rats red ad lib. for 12 weeks either a fat-free diet, a diet containing 15% cottonseed oil, or a diet containing 15% cuttlefish liver oil, with or without exogenous (1%) cholesterol, were studied to evaluate and compare the effect of polyunsaturated fatty acids of cuttlefish liver oil and cottonseed oil on cholesterol metabolism. The results indicate that the longer chain polyunsaturated fatty acids contained in the fish oil cannot substitute for the essential fatty acid, linoleic, either as far as effect on various aspects of cholesterol metabolism are concerned or in the ability to form arachidonic acid. The observed interference of cuttlefish liver oil with the absorption of exogenous cholesterol may be caused by the presence in this oil of the highly unsaturated long chain fatty acids.

The Effect of Plant Sterols on Cholesterol Levels in the Rat

Simultaneous feeding of plant sterols and cholesterol evoked no change in plasma, levels of cholesterol or in the carcass. However, total cholesterol and lipid contents of the liver were both less than when cholesterol was fed alone.