Antanas Butkus

Thromboxane production and platelet aggregation in diabetic subjects with clinical complications

Abstract The susceptibility of diabetic patients to pathophysiological complications and abnormalities in their platelet function prompted this investigation. A group of 180 insulin-dependent subjects were randomly selected: 38.9% with no other clinical complications; 16.7% with retinopathy; 15.0% - neuropathy; 3.9% - nephropathy; 4.4% - peripheral vascular disease; 21.1% - coronary arterial disease. Twenty seven subjects with no diabetic disease were used as controls. Thromboxane B 2 [stable metabolite of thromboxane A 2 (TXA 2 )], generated by exogenous arachidonic acid and quantitated by electron-capture gas chromatography (EC-GC) was significantly higher in platelets of diabetics with clinical complications than in controls. Platelets in platelet rich plasma (PRP) processed from citrated whole blood of diabetic subjects were more sensitive to ADP- or collagen-induced aggregation than those of non-diabetics. Platelets similarly obtained from diabetics, but separated on agarose gel column rather than obtained in PRP, aggregated with greater sensitivity to arachidonic acid than those of controls. Plasma total cholesterol and triglyceride concentrations were similar in diabetic and non-diabetic subjects. Platelet total phospholipid concentration, percent composition of molecular species and fatty acid distribution were similar in both groups of subjects indicating a similar substrate concentration for prostaglandin biosynthesis. It is postulated that in the platelets of diabetic subjects: a) the observed increase in TXA 2 synthesis is due to the increased activity of the thromboxane synthetase system at one or more sites; b) this increased enzyme activity is the mechanism of greater platelet sensitivity to aggregation.

Familial hypo-β-lipoproteinemia: A genetic disorder of lipid metabolism with nervous system involvement

Abstract A previously undescribed inborn error of lipid metabolism has been demonstrated in thirteen members of a kinship. The disorder was genetically transmitted in an autosomal dominant fashion. This abnormality, termed familial hypo-β-lipoproteinemia, consisted of a great reduction of serum β-lipoprotein content. Serum levels of cholesterol and phospholipid were low, whereas the concentrations of serum triglyceride were normal or low and that of α-lipoprotein normal. Vitamin E levels were decreased in five of the eleven persons so tested. Nine persons, all with serum cholesterol levels less than 100 mg. per 100 ml., showed acanthocytic red blood cells. These cells could be transformed to cells with normal forms by the in vitro addition of serum from hyperlipemic or hypercholesteremic persons. The clinical manifestations of this disorder are variable, and may be correlated with the severity of the biochemical abnormality. In its most severe form, as shown by the propositus, there is lack of responsiveness to local anesthetic agents, and signs and symptoms of a progressive demyelinating disorder affecting the central nervous system. Fasting peroral jejunal biopsy material from the propositus contained significant deposits of neutral fats beneath the lamina propria and in endothelial cells of capillaries containing lipid-laden mononuclear cells. The material presented suggests that hypo-β-lipoproteinemia may be an etiologic factor in some demyelinating disorders.

Prediction of coronary heart disease based on clinical suspicion, age, total cholesterol, and triglyceride.

Our results, based on the definition of coronary heart disease by cinearteriography, show that definite relationships exist among incidence of coronary heart disease (CHD), age, total cholesterol (TC), and total triglycerides, with less definite ones between free cholesterol, and phospholipids for a specific group of 450 male patients referred to the Cleveland Clinic because of suspected coronary heart disease. Equations and graphs of the association of incidence of CHD with these conditions are presented. The relationships were strong enough to be of aid in discriminating CHD from normal; the most useful being age, TC, and TG.Incidence determinations were presented in terms of total incidence and incidence associated with the variable itself. Coronary angiography demonstrated 350 CHD and 100 normals or a ratio of 3.5 to 1. This ratio is a reflection of the high accuracy with which physicians suspect CHD.Age and TC were good discriminators in these patients suspected of CHD. An improved relationship and better discrimination was obtained by relating incidence simultaneously to age, TC, and TG. The probability of having CHD is the total incidence corresponding to the patients age, TC, and TG. The probability level of 0.90 gave 95% assurance of a correct prediction for the physician-selected group of patients. Twenty-nine per cent of them had probabilities above 0.90.

Experimental arteriosclerosis in dogs. Evaluation of anatomopathological findings.

Abstract Young adult dogs fed a cholesterol supplemented semi-synthetic diet containing hydrogenated coconut oil without thiouracil for periods of 16 weeks to 16 months were studied in order to identify early vascular lesions as well as overt arteriosclerosis. In 16 weeks, ultrastructural medial cell changes were present in the abdominal aorta of all animals, while the same diet without cholesterol supplement failed to induce lesions. Arteriosclerotic lesions in the long-term study were characterized by extensive intra and extracellular lipid deposits in smooth muscle cells of the arterial media and to much less extent, of the intimal lining. These changes were usually more severe in small peripheral arteries than in the thoracic aorta or the coronary vessels. Although segmental stenotic lesions were common in severely involved vessels, thrombosis or infarction were not usual complications of advanced arterial disease.

Effects of diets rich in saturated fatty acids with or without added cholesterol on plasma lipids and lipoproteins.

Semi-synthetic diet I which contained 16% hydrogenated coconut oil and 5% cholesterol, and diet II, identical to I but without cholesterol supplement, were fed to dogs for four months to determine the effects of added cholesterol on lipemia produced by diets high in saturated fatty acids (FA) and lacking essential FA. In addition, diet I was fed to another group of dogs for 12 to 16 months. The initiation of lipemia was very similar in all experimental animals. Plasma from dogs on diets I and II showed significant increases in lipid concentration and changes in FA per cent composition within the first week, as compared to controls, while during the first month there was no difference in lipid concentration or FA distribution in all lipid fractions between I and II. At the 10th and 16th weeks plasma total and free cholesterol and phospholipid were significantly higher in the group on diet I, with the cholesterol supplement, than on diet II with no added cholesterol, but there was no difference in triglyceride concentration between these groups. Dogs on diet I for 12 to 16 months showed a further and substantial increase in plasma FA concentration; these changes were most marked in cholesteryl esters. Little or no lipoprotein with electrophoretic and ultracentrifugal properties of alpha-lipoprotein was present in the plasma. Immunotechniques showed that it was present. The composition of dietary FA had great influence in producing this hyperlipemia. Lipemia produced was not a simple reflection of the FA in these diets as evidenced by the increase in some FA, e.g., C16∶1, which was absent in the experimental diets and C18∶1, which contributed only 3.4% of the FA. Large increases in palmitoleate and oleate indicate synthesis or mobilization or both from other tissues. Diets composed predominantly of saturated medium chain length fatty acids, with or without added cholesterol were equally effective in the initiation of hyperlipemia. Data also suggest that added cholesterol is necessary for sustaining hyperlipemia.

Aortic lipids at different stages of canine experimental arteriosclerosis

Abstract Dogs were fed an arteriosclerosis-inducing diet which contained 16% hydrogenated coconut oil and 5% cholesterol, the same diet without cholesterol supplement, or meat-chow. The last diet group did not develop arteriosclerosis and was regarded as control. All animals showed severe arteriosclerotic lesions after 12–16 months on cholesterol supplement diet and after 4 months all showed ultrastructural changes in medial smooth muscle cells. Fatty acid patterns changed in all lipid fractions of intima-media of dogs on the supplemented diet, whereas in terms of fatty acid concentration, only triglyceride changed. All lipids showed their greatest increase in severely arteriosclerotic arteries of long-term dogs. Cholesteryl esters increased relatively more than other lipid fractions. Cholesteryl oleate increased most. Comparison of effects of short- and long-term feeding of cholesterol-supplemented diet indicates that some lipid changes in aortic intimamedia cannot be fully explained by simple filtration from plasma. Selective uptake, synthesis, degradation, or removal of lipids took place in situ . Changes in composition of lipids in the arterial wall, observed in this study, may alter arterial permeability, its enzymatic activity, or both. Greater deposition of plasma lipids probably then takes place at a later stage of arteriosclerotic involvement.

Quantitative and qualitative lipid correlation in experimental endogenous hyperlipemia

The reversible endogenous hyperlipemia in dogs, elicited by the detergent Triton which was given intravenously, was used to study the interrelations of serum lipids. In the cholesterol ester fraction an increase occurs in both monounsaturated and in saturated fatty acids, excepting myristic; while a decrease occurs in polyunsaturated fatty acids. The fatty acids of cholesterol esters of normal dogs contain 22% oleic acid, and only 24% when serum lipids are increased to almost double their normal value (TC=400–500 mg/100 ml). However there is a critical level above which a rapid rise in oleic acid occurs and, in severe hyperlipemia (TC=1500 ±430 mg/100 ml), this acid constitutes almost half of the esterified fatty acid component.Since there is no evidence that Triton directly regulates fatty acid synthesis, the lipid fraction-fatty acid interrelationship may be secondary to lipid mobilization from endogenous sources. This concept is supported by the fact that the increased serum fatty acids are only those which can be synthesized by animals. It is suggested, on the basis of a marked increased of endogenously produced fatty acids, that, at critical lipid levels, shortage of polyunsaturated fatty acids from exogenous sources occurs. This might be of sufficient degree to accelerate fatty acid synthesis to meet the need for fatty acids for energy requirements. There may also be need of fatty acid for esterification of chiefly the accumulated free cholesterol split from lipoprotein by Triton.Triton-induced changes in cholesterol ester fatty acids result in patterns which closely resemble those in the adipose tissue of dog and man and in the serum of human endogenous hyperlipemia.

Analysis of A, B, E, and F prostaglandins as pentafluorobenzyl esters by electron-capture gas-liquid chromatography

A new and sensitive method is described for the simultaneous analysis of a mixture containing PGE1, PGE2, PGF1alpha, and PGF2alpha by electron-capture gas-liquid chromatography. During derivatization of the mixture, PGE1 and PGE2 were converted to PGB1 and PGB2, respectively, yielding a mixture of PGB1, PGB2, PGF1alpha, and PGF2alpha trimethylsilyl ether pentafluorobenzyl esters. Gas chromatographic resolution of all four derivatives is sufficient for quantitation of each prostaglandin. The A prostaglandins were analyzed by similar conversion to the respective B prostaglandin derivatives. Minimum detection limits for the B and F prostaglandin derivatives were 10 pg and 1 pg, respectively. Samples of rabbit kidney medulla were incubated and analyzed for A, B, E, and F prostaglandins. The results indicate that the method is capable of high recovery and reproducibility.

Plasma and aortic lipids in experimental canine atherosclerosis

Abstract Twenty-four dogs were split into three equal groups for an experimental period of 1 year. Group A was fed a semisynthetic diet containing 5% cholesterol and 16% hydrogenated coconut oil (HCO). Another eight dogs were fed diet B which differed from diet A only in that one-quarter of the HCO was replaced by safflower oil. Group C ate a controlled diet of meat and kibble. Diet A contained no essential fatty acids while diets B and C contained sufficient amounts of linoleic acid to satisfy established nutritional requirements. Severe aortic, coronary, and cerebral atherosclerosis was present at autopsy in group A dogs but no lesions were observed in any of the dogs fed diets B and C. All dogs of group A had profound elevations in plasma lipid concentrations when compared with dogs of control group C. Dogs of group B had more moderate increases. The largest elevation occurred in the cholesteryl esters of group A. The predominant ester in the plasma of group A dogs was cholesteryl oleate. In contrast, the major plasma cholesteryl ester in dogs of groups B and C was linoleate. Intima-media samples from atherosclerotic aortic segments showed large increases in lipid concentrations. The bulk of the increases was due to the accumulation of cholesteryl esters and the major cholesteryl ester within the lesion was oleate. The predominant cholesteryl ester in the normal intima-media of groups B and C was cholesteryl linoleate. Fatty acid analyses suggested that much of the lesion cholesteryl ester could have been derived directly from plasma, but the preferential accumulation of cholesteryl oleate and eicosatrienoate suggested that there was also considerable local cholesteryl ester synthesis. Phospholipid eicosatrienoate:arachidonate ratios in excess of 0.4, indicative of essential fatty acid deficiency, were found consistently in the plasma and aortic tissue of group A dogs but not in dogs of group B or C. Essential fatty acid deficiency appeared to be most severe within atherosclerotic intimal plaques.

Hepatic cholesterol metabolism in vitro in the obese spontaneously hypertensive, hyperlipemic and atherosclerotic rat

Abstract Hepatic cholesterol synthesis and esterification as well as cholesteryl ester hydrolysis and cholesterol-7α-hydroxylation in vitro were studied in a strain of spontaneously hypertensive rats (SHR) in which phenotypic obesity is inherited as a recessive trait. The obese rats also develop hyperlipemia and arterial lesions while on Purina Chow diet, but their heterozygous littermates do not. Measurements of cholesterol synthesis from [ 14 C]mevalonate by microsomal fraction of liver homogenates from obese SHR gave evidence of elevated hepatic cholesterogenesis as compared to nonobese SHR. Other experiments in vitro also showed that the obese SHR liver had a decreased cholesterol esterifying capacity and a reduced capacity of cholesterol-7α-hydroxylation as a function of free cholesterol concentration, as well as a depressed cholesteryl ester hydrolytic capacity as a function of cholesteryl ester concentration when compared with nonobese SHR. These changes in enzymatic activities may, in part, contribute to the elevated total cholesterol concentrations in plasma and liver of these obese SHR.