Sam Hashimoto

A Controlled Clinical Trial of a Diet High in Unsaturated Fat in Preventing Complications of Atherosclerosis

This report describes a clinical trial, with domiciled male veterans, designed to determine whether a diet which lowers serum cholesterol concentration can prevent overt complications of coronary heart disease and other manifestations of atherosclerosis. Volunteers were allocated randomly to control and experimental groups. Participants numbered 422 in the control group and 424 in the experimental group. The two groups were indistinguishable at the outset of the study in almost all observations. These included age, racial characteristics, and religious affiliation; height, weight, and blood pressure; electrocardiographic findings; prevalence of pre-existing anginal syndrome, myocardial infarction, peripheral atherosclerosis, cerebral ischemia, or cerebral infarction; cardiac decompensation; utilization of relevant medications; serum cholesterol and serum total lipid levels; and prevalence of corneal arcus and xanthelasma. One significant difference between the groups in regard to a possibly influential characteristic was that they differed slightly in patterns of cigarette smoking habits. The control diet was similar to the regular institutional diet, which is a standard American diet. It provided, by analysis, 40.1% of calories as fat, having a mean iodine value of 53.5; cholesterol intake was 262 mg/1,000 calories (653 mg/day). The experimental diet provided 38.9% of calories as fat, with an iodine value of 102.4, and had a cholesterol content of 146 mg/1,000 calories (365 mg/day). Linoleic acid content of the two diets was 10% and 38% of total fatty acid, respectively. The experimental diet was prepared to simulate conventional food. Over-all adherence expressed as percentage of total possible meals taken, from introduction into the study to termination of the study, averaged 56% for the control subjects and 49% for the experimental group. The experimental diet induced a prompt drop in serum cholesterol level and sustained a difference between the experimental and control groups amounting to 12.7% of the starting level. It is estimated from published data that the change in saturation of dietary fat accounted for five-sixths of the diet-induced lowering of serum cholesterol in the experimental group, the remaining one-sixth having been due to decreased cholesterol intake. Mean linoleic acid concentration of adipose tissue was initially 10.9%. During the latter part of the trial, which lasted eight years for some subjects, linoleic acid concentration in adipose tissue approached an asymptotic level of 33.7% among good adherers. This variable was a good measure of adherence to the diet after five or more years (r=+0.71), but not earlier. Clinical follow-up was carried out on a double-blind basis. The number of men sustaining events in major categories, in the control and experimental groups, respectively, was: definite silent myocardial infarction, 4 and 9; definite overt myocardial infarction, 40 and 27; sudden death due to coronary heart disease, 27 and 18; definite cerebral infarction, 22 and 13. The difference in the primary end point of the study-sudden death or myocardial infarction -was not statistically significant. However, when these data were pooled with those for cerebral infarction and other secondary end points, the totals were 96 in the control group and 66 in the experimental group; P=0.01. Fatal atherosclerotic events numbered 70 in the control group and 48 in the experimental group; P<0.05. Life-table analysis in general confirmed these conclusions. For all primary and secondary end points combined, eight-year incidence rates were 47.7% and 31.3% for the control and experimental groups, respectively; P value for the difference between the two incidence curves was 0.02. Stratification of the data by age demonstrated that most of the prophylactic effect occurred in the younger half of the study population, less than 65.5 years old at the start of the study. Stratification by baseline serum cholesterol concentration revealed that most of the effect was encountered in men with starting levels above the median (233 mg/dl). Stratification on the basis of pre-existing atherosclerotic complications failed to yield consistent evidence that subjects without pre-existing complications responded differently to diet than did those with prior overt disease. Deaths due to nonatherosclerotic causes numbered 71 in the control group and 85 in the experimental group. Most of the difference occurred in the latter part of the study. Consideration of causes of death in this category suggested that this difference probably did not reflect a toxic effect of the experimental diet. Gross grading of the extent of atheromata in individuals who died and were autopsied failed to reveal significant differences between the two groups of subjects. The same was true of arterial total lipid and calcium concentrations. Relative abundance of major lipid fractions in coronary atheromata and circle of Willis appeared to be independent of diet. In general, most of the varieties of lesions just cited revealed increased concentrations of linoleic acid in triglyceride, cholesterol ester, and phosphatide among the experimental subjects. In individuals on the experimental diet who died after prolonged experience in the study and high adherence to the diet, arachidonic acid concentration in atheroma phosphatide was significantly depressed. A similar but less consistent decrease was observed in arachidonic acid in cholesterol ester and free fatty acid of atheromata. Observations of other investigators suggest that these changes were due to the effects of high &agr;-tocopherol intake on the experimental diet.

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.

Probucol reduces plasma and aortic wall oxysterol levels in cholesterol fed rabbits independently of its plasma cholesterol lowering effect

To understand further the antiatherogenic mechanism of probucol, the antioxidant effect of this agent was studied on specific cholesterol oxidation products in plasma and aortic wall in equally hypercholesterolemic New Zealand white rabbits. In order to maintain equal plasma total cholesterol levels, five control rabbits (C group) received a 1% followed by a 0.5% cholesterol enriched diet, while the probucol treated rabbits (C+P group) received a graded increase in the cholesterol supplemented diet from 1% to 3%; probucol supplementation was constant at 1%. After 9 weeks of feeding, the plasma oxysterols, cholest-5-ene-3 beta,7 alpha-diol, cholest-5-ene-3 beta,7 beta-diol, 5,6 beta-epoxy-5 alpha-cholestan-3 beta-ol, 5,6 alpha-epoxy-5 alpha-cholestan-3 alpha-ol and 5 alpha-cholestane-3 beta,5,6 beta-triol significantly increased over baseline levels in both experimental groups. However, the increase in all these products in plasma was 20-60% less in the C+P group than the C group (P < 0.05). Furthermore, the C+P aortic wall cholesterol oxide concentrations were 50-90% less than the C group (P < 0.05). The oxysterol pattern of the aortic wall was similar to plasma. Additionally, the aortic wall cholesterol content in the C+P group was 50% less than the C group (P < 0.05). The plasma cholesterol levels were not significantly different at any time point during the study and the cholesterol oxide content in the diets was the same. These results are consistent with the contention that the antioxidant properties of probucol serve as the basis for its antiatherogenic effects in vivo.

Movement of Labeled Cholesterol Between Plasma Lipoprotein and Normal Arterial Wall across the Intimal Surface

Normal rat aortas were incubated with their intimal surfaces in contact with a lipoprotein solution containing isotopically labeled free cholesterol. Uptake of cholesterol by the tissue increased with increasing concentration toward a plateau level. Maximum uptake of free cholesterol was estimated to be 13 μg/cm2 per 24 hours, and uptake at physiological cholesterol concentration was 7.9 μg/cm2 per 24 hours. Aortas containing labeled cholesterol were prepared by intragastric administration of cholesterol-7α-3H to normal rats. Experiments in vitro with these aortas demonstrated that efflux across the intimal surface was dependent upon presence of lipoprotein in the medium. This efflux was not retarded by prior boiling of the tissue. These results support suggestions that movement of labeled free cholesterol from plasma into the arterial wall may be largely the result of a physicochemical exchange between lipoprotein and endothelium.

Studies of the mechanism of augmented synthesis of cholesteryl ester in atherosclerotic rabbit aortic microsomes.

A study was undertaken to test the hypothesis that an abnormally high concentration of acyl-CoA:cholesterol acyltransferase in atherosclerotic microsomes is partly responsible for augmented esterification of cholesterol. We approached the problem indirectly by measuring the incorporation of radioactivity into cholesteryl ester from [1-14C]palmityl-CoA in normal microsomes after enrichment of their concentration of microsomal free cholesterol to levels characteristic of atherosclerotic microsomes. Elevation of free cholesterol content induced increased cholesterol esterification approximately linearly over the range studied. The cholesterol-esterifying activity of atherosclerotic microsomes was not greater than that of normal microsomes having the same concentration of cholesterol. The results suggest that, with acyl-CoA constant, augmented cholesterol esterification in atherosclerotic microsomes is an effect of high microsomal cholesterol concentrations and not due to an increase in the concentration of the enzyme.

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.

Influence of a Diet High in Unsaturated Fat upon Composition of Arterial Tissue and Atheromata in Man

Detailed chemical analyses have been carried out on aorta, and on coronary and aortic atheromata, of men who died during a study of prolonged use of diets rich in unsaturated fat and of control subjects. Concentrations of total aortic lipid and of total aortic calcium were not significantly different for the two groups of subjects. Concentrations of cholesterol and cholesterol esters, triglyceride, and phosphatide showed no difference between the two groups in analyses of aorta, uncomplicated aortic atheromata, complicated aortic atheromata, and coronary atheromata. Coronary atheroma lipid contained substantially more triglyceride than did aortic atheroma lipid, whether derived from complicated or uncomplicated plaques.Atheroma triglyceride in experimental subjects contained more linoleic acid than in control subjects, in all types of plaques. Lesser but significant increases in linoleic acid were seen in cholesterol ester and phosphatide of coronary atheromata and complicated aortic atheromata. In the case of uncomplicated aortic atheroma, changes in linoleic acid of cholesterol ester and phosphatide were smaller and not significant. In both groups of subjects, coronary atheroma contained more cholesteryl oleate and less linoleate than did aortic atheroma.Linoleic acid contents of atheroma lipid fractions were positively correlated with the linoleic acid figures for corresponding fractions of the most recent antemortem serum samples. Ratio of linoleic acid in an atheroma lipid fraction to the linoleic acid content of the corresponding serum fraction was, in most cases, constant after the time of the first sample, obtained after 495 days on experimental diet. It is postulated that this ratio constitutes an estimate of the fraction of atheroma fatty acid derived from plasma. In cholesterol ester and triglyceride, the ratio was about 0.65 in experimental subjects, higher in subjects on the control diet. The ratio for phosphatide was about 0.5 in both groups of subjects.

Origin of cholesteryl oleate and other esterified lipids of rabbit atheroma

Rabbit were fed a diet containing [9,10-3H]oleic acid starting at weaning, After 14 weeks, 2% cholesterol was added to the diet to induce atheroma formation, and the experiment was continued for 12 additional weeks. During these 12 weeks serial serum samples were obtained, and at the end of the period the animals were autopsied and aortic atheromata were dissected out. From all serum and atheroma samples, cholesteryl ester, triglyceride, lecithin and free fatty acid were isolated. Oleic acid was obtained from each component and assayed for 3H. The ratio of the atheroma oleic acid specific radioactivity in a given component to the mean serum oleic acid specific radioactivity in that component, designated A/S, was calculated. 1.0-A/S was taken as an estimate of the minimum fractional contribution of local synthesis to the oleate in that component. For cholesteryl ester, the mean value for 1.0-A/S was 0.19 with standard error of the mean ± 0.05; if one outlying value is omitted, the mean is 0.13 with standard error ± 0.02. It is concluded that a measurable and significant fraction of atheroma cholesteryl oleate is derived by local synthesis, and reasons are given for considering the observed value a probable underestimate.

Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum.

Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.

Activity of acyl-CoA: Cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in subfractions of hepatic microsomes enriched with cholesterol

The influence of membrane cholesterol on the activities of acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase was examined in three microsomal subfractions (RNA-rich, RNA-poor, and smooth) that had been enriched with cholesterol by incubation with mixed lipoproteins from hypercholesterolemic rabbit serum. Acyl-CoA: cholesterol acyltransferase activity was significantly stimulated in the three subfractions, particularly in the RNA-rich microsomal component. 3-Hydroxy-3-methylglutaryl-CoA reductase, on the other hand, was suppressed (30%) in only one (RNA-poor) of the three microsomal subfractions, despite a 1.4-fold increase in the concentration of membrane cholesterol. An attempt was made to distinguish between an effect based exclusively on an increase in available cholesterol substrate and an activation of acyl-CoA: cholesterol acyltransferase in RNA-rich microsomes enriched with cholesterol. An experimental design was devised so that substrate cholesterol was provided in the form of heated smooth microsomes and acyl-CoA: cholesterol acyltransferase was provided as a separate preparation in the form of RNA-rich microsomes. Appropriate controls were carried out to test for transfer of cholesteryl ester between the two sets of particles. The results suggested that cholesterol enhanced acyl-CoA: cholesterol acyltransferase activity by serving both as a substrate and as a non-substrate modulator.