Donald J. McNamara

Dietary cholesterol and atherosclerosis.

The perceived relationship between dietary cholesterol, plasma cholesterol and atherosclerosis is based on three lines of evidence: animal feeding studies, epidemiological surveys, and clinical trials. Over the past quarter century studies investigating the relationship between dietary cholesterol and atherosclerosis have raised questions regarding the contribution of dietary cholesterol to heart disease risk and the validity of dietary cholesterol restrictions based on these lines of evidence. Animal feeding studies have shown that for most species large doses of cholesterol are necessary to induce hypercholesterolemia and atherosclerosis, while for other species even small cholesterol intakes induce hypercholesterolemia. The species-to-species variability in the plasma cholesterol response to dietary cholesterol, and the distinctly different plasma lipoprotein profiles of most animal models make extrapolation of the data from animal feeding studies to human health extremely complicated and difficult to interpret. Epidemiological surveys often report positive relationships between cholesterol intakes and cardiovascular disease based on simple regression analyses; however, when multiple regression analyses account for the colinearity of dietary cholesterol and saturated fat calories, there is a null relationship between dietary cholesterol and coronary heart disease morbidity and mortality. An additional complication of epidemiological survey data is that dietary patterns high in animal products are often low in grains, fruits and vegetables which can contribute to increased risk of atherosclerosis. Clinical feeding studies show that a 100 mg/day change in dietary cholesterol will on average change the plasma total cholesterol level by 2.2-2.5 mg/dl, with a 1.9 mg/dl change in low density lipoprotein (LDL) cholesterol and a 0.4 mg/dl change in high density lipoprotein (HDL) cholesterol. Data indicate that dietary cholesterol has little effect on the plasma LDL:HDL ratio. Analysis of the available epidemiological and clinical data indicates that for the general population, dietary cholesterol makes no significant contribution to atherosclerosis and risk of cardiovascular disease.

The effect of abnormal plasma and cellular sterol content and composition on low density lipoprotein uptake and degradation by monocytes and lymphocytes in sitosterolemia with xanthomatosis

The hypothesis that abnormal low density lipoprotein (LDL) sterol content and composition in sitosterolemia with xanthomatosis affects LDL uptake and/or degradation was tested. Monocytes and lymphocytes from three patients and 12 age- and sex-matched controls were incubated at 37 degrees C in lipid-free medium with 125I-labeled LDL prepared from sitosterolemic patients (LDLs) and controls (LDLn) in the presence or absence of excess unlabeled lipoproteins. Normal monocytes and lymphocytes took up and degraded LDLs 13% to 30% less than LDLn (P less than .05). Sitosterolemic monocytes and lymphocytes degraded LDLn 13% and LDLs 67% more actively than control cells (P less than .05). Sitosterolemic monocytes contained three times more sterols and stanols than controls (P less than .01), of which 12% were plant sterols and 2% were 5 alpha-saturated stanols. In one patient, stimulating bile acid synthesis by ileal bypass surgery reduced plasma and monocyte sterol and stanol concentrations about 60%, and was associated with a 40% to 50% increase in LDLn and LDLs receptor-mediated degradation. The decreased uptake and degradation of LDLs relative to LDLn by normal cells suggest that abnormal plant sterols in LDLs may reduce its affinity for the native LDL receptor. Increased receptor-mediated uptake and degradation of LDLs by sitosterolemic cells in the presence of high cellular sterol content may result from failure of the sitosterolemic cells to down-regulate LDL receptor synthesis. Ileal bypass surgery increased cellular LDL receptor activity, reduced plasma and cellular sterol concentrations, and may diminish the risk of premature atherosclerosis in sitosterolemia.

Contributions of Absorbed Dietary Cholesterol and Cholesterol Synthesized in Small Intestine to Hypercholesterolemia in Diabetic Rats

To determine how insulin deficiency leads to hypercholesterolemia, we examined cholesterol absorption, synthesis, and utilization in streptozocin-induced diabetic rats fed a grain-based diet ad libitum. Absorbed dietary cholesterol was determined from measurement of dietary cholesterol intake and previous data for cholesterol fractional absorption. Daily rates of cholesterol synthesis in the small intestine, liver, and periphery were calculated from recovery of labeled sterols after injection of 3H2O at six times during 24 h. Utilization of cholesterol for growth, fecal excretion, and bile acid synthesis were also determined. Absorbed dietary cholesterol in diabetic rats was double that in control rats. The contribution of absorbed cholesterol to total cholesterol production (sum of absorbed dietary cholesterol) and endogenous cholesterol synthesis) in control rats was 24% compared to 48% in diabetic rats. The increase in diabetic rats was due to overeating and, to a lesser extent, to increased fractional absorption. Overeating also induced intestinal hypertrophy and a twofold increase in cholesterol synthesis by the small intestine to 24% of whole-body production in diabetic rats. Consequently, the small intestine accounted for 72% of daily cholesterol input in diabetic rats compared to 37% in control rats, with diet accounting for two-thirds of the cholesterol input via the small intestine in both groups. In response to this increased input from the intestine, cholesterol synthesis in the periphery was 39% of whole-body production in control rats compared to 22% in diabetic rats, and synthesis in the liver was 26 and 6% of total cholesterol production in control and diabetic rats, respectively. The net effect of these changes was a decrease in whole-body synthesis equal to the increase in absorbed dietary cholesterol and no change in whole-body cholesterol production in diabetic rats. The need for cholesterol as substrate for bile acid synthesis was met largely by absorption from the diet in diabetic rats and by synthesis in the liver in control rats. The need for cholesterol in the periphery was entirely met by synthesis in situ in both groups. We conclude that 1) an increased fraction of the net cholesterol production is derived from intestinal sources and transported through the blood in diabetic rats, and 2) cholesterol absorbed from the diet contributes more to the hypercholesterolemia of diabetic rats than the increased cholesterol synthesis in the small intestine.

Sterol synthesis in vitro in freshly isolated blood mononuclear leukocytes from familial hypercholesterolemic patients treated with probucol

Sterol synthesis rates were measured in freshly isolated blood mononuclear leukocytes obtained from familial hypercholesterolemic patients undergoing treatment with either probucol alone or probucol plus cholestyramine. Subjects with heterozygous familial hypercholesterolemia on probucol had a significant 31% reduction in mononuclear cell sterol synthesis rates as compared to control patients; sterol synthesis in cells from homozygous familial hypercholesterolemic patients on probucol did not differ from that in control subjects. Addition of cholestyramine to probucol therapy in heterozygous familial hypercholesterolemic patients caused an increase in sterol synthesis rates equal to but not greater than control values, thus negating the decreased mononuclear leukocyte sterol synthesis associated with probucol administration alone. Probucol treatment effectively decreased plasma cholesterol levels in both homozygous and heterozygous familial hypercholesterolemic subjects; however, the data suggest that the drug may exert different effects on sterol synthesis in peripheral tissues depending upon the presence or absence of cellular receptors for low-density lipoproteins.

Hyperphagia Alters Cholesterol Dynamics in Diabetic Rats

Rats with streptozotocin-induced diabetes stop growing and start eating more chow. These two events elicit an interacting series of changes in cholesterol dynamics. Hyperphagia increases dietary cholesterol intake and cholesterol synthesis by the small intestine. These increases are balanced by a decrease in cholesterol synthesis in the rest of the body so that total cholesterol input is normal. With growth failure, utilization of cholesterol for formation of new tissue ceases. This decrease is balanced by an increase in bile acid synthesis by the liver. The bile acid pool in the contents of the small intestine is enlarged by hyperphagia. Despite these changes, fecal sterol excretion and total utilization of cholesterol are normal. During the course of changes in growth and food intake and the attendant changes in cholesterol flux, the total tissue cholesterol pool does not change. Therefore influx equals efflux and the systems regulating cholesterol and bile acid synthesis are responding appropriately and are themselves unperturbed by insulin deficiency. However, plasma cholesterol level increases threefold. This elevation is due to increased influx of cholesterol from the small intestine and decreased synthesis in the rest of the body, so that a larger portion of total body cholesterol influx passes through the blood.

Increased hepatic cholesterol production due to liver hypertrophy in rat experimental nephrosis

Control and nephrotic rats were compared as to the liver contents of cholesterol, phospholipid and the activity of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Whole liver homogenates as well as endoplasmic reticulum membrane samples showed increased free cholesterol and phospholipid mass in the nephrotics. Correction of the values by the protein content indicated membrane expansion, i.e. liver hypertrophy. However, total hepatic cholesterol synthesis as measured by the reductase activity was increased in the nephrotic rat. These results are in accordance with previous studies showing enhanced cholesterol production in experimental nephrosis. In short, enhanced cholesterol mass in the liver coexists with increased hepatic synthesis in the experimental model used.

Effects of dietary pectin and cellulose on hepatic and intestinal mixed-function oxidations and hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the rat.

Abstract The aim of this study was to determine if feeding dietary fiber (cellulose or pectin) to male rats could influence hepatic and intestinal mixed-function oxidation. We simultaneously compared hepatic drug-oxidizing activity with the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for cholesterol biosynthesis. Three groups of six animals were fed a purified diet containing by weight either 10.4% cellulose or 10.4% pectin, or a standard cereal-based diet containing 4.5% crude fiber; the caloric contributions by carbohydrate, protein and fat in the three diets were similar. In the cellulose-fed rats, the hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were significantly lower when compared with those of rats fed pectin or the cereal-based diet. The hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were similar in the pectin-fed and cereal diet-fed rats. Hepatic HMG-CoA reductase activity, hepatic microsomal cytochrome b5 content, and intestinal benzo[a]pyrene hydroxylase activity were comparably lower in rats fed the purified diet with either dietary fiber when compared to those fed the cereal diet. It is concluded that dietary pectin and cellulose exert distinctly different influences on the hepatic microsomal mixed-function oxidase system for drug metabolism, but not on liver cholesterol synthesis or intestinal benzo[a]pyrene hydroxylation, suggesting that different physiological mechanisms control these enzyme systems.

Dietary fat, plasma lipoproteins, and immune function in middle‐aged American men

Dietary fat has been incriminated as a positive risk factor for the development of neoplasia in human populations. We used adipose tissue fatty acid analysis as an index of dietary fat intake to study the association between dietary fat and immune function in a group of 94 free-living American males (avg age 47 years). Immunocompetence was tested by a battery of T- and B-lymphocyte stimulation tests and also by natural killer (NK) cell activity. Correlations were sought between fatty acid composition, plasma lipids, and immune responsivity. The degree of unsaturation of the diet over a polysaturated-to-saturated fat ratio range of 0.54-1.01 had no predictable effect on the immune function. Stepwise regression analysis showed that the concentrations of plasma triglycerides and cholesterol and its subfractions did not explain any of the variance in the immune tests. Palmitic acid (16:0) was associated with 7% of the variance of the response to C. albicans and E. coli, perhaps through influencing B-cell activity. Stearic acid (18:0) was correlated negatively to concanavalin A responsivity (18% of the variance) and positively to NK activity (20% of the variance). If impaired in vitro immune function is a marker of increased risk for carcinogenesis, then our data do not support a role for dietary fat influencing in any systematic manner lymphocyte function in vitro, as reflected by proliferative response or NK activity. Further, plasma lipoproteins, in particular cholesterol levels, did not appear to affect any immune function test. It remains to be studied whether dietary fat, lipoproteins, or fat-soluble substances may influence membrane structure and function and prostaglandin formation as alternative pathways in the promotion of neoplasia.

Cholesterol homeostasis in lean and obese male zucker rats

Studies were performed in male Zucker rats to determine the metabolic effect of genetic obesity on whole body cholesterol homeostasis. Lean and obese mature Zucker rats were studied during intake of either a chow diet or a semisynthetic diet containing 10% corn oil; in addition growing animals were studied during constant body weight gain on a chow diet. Under all conditions the obese Zucker rats had significantly higher levels of total plasma cholesterol and triglyceride; however, measurements of the specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and of the rate of whole body cholesterol synthesis by sterol balance techniques demonstrated that the lean and obese animals did not differ in their endogenous rates of cholesterol synthesis. When sterol balance data were calculated per kilogram body weight, lean male Zucker rats synthesized a greater amount of cholesterol per day than obese animals. These studies demonstrate that the obese male Zucker rat, in many ways a model of human obesity, does not overproduce cholesterol and thus fails to exhibit one of major characteristics of the obese human.

Cholesterol homeostasis in rats fed a purified diet

The rate of whole bodyb cholesterol synthesis was measured in male Sprague-Dawley rats fed either a standard chow, cereal-based diet or a semi-synthetic purified diet consisting of casein, sucrose and lard. The purified diet significantly decreased daily fecal excretion of neutral and acidic sterols, the specific acitvity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the bile acid pool size, and total daily cholesterol synthesis in the rat, while increasing plasma cholesterol concentrations and the total body content of cholesterol. The increased body content of cholesterol occurred primarily in muscle and connective tissue and not in the liver. The data demonstrate the importance of quantitating the net tissue accumulatin of cholesterol for accurate measurement of daily sterol synthesis in growing animals when sterol balance measurements are used. Tissue accumulation accounted for 7% of total daily cholesterol synthesis in rats fed the cereal diet, and 20% of daily synthesis in animals fed the purified diet.