D.N. Kim

Effects of a soy protein product on serum and tissue cholesterol concentratins in swine fed high-fat, high-cholesterol diets

Abstract Hypocholesterolemic effect of a soy protein product was studied in swine fed a high-fat, high-cholesterol diet. In the first experiment, a group of swine were fed 42% butter (by calories) and 1055 mg cholesterol daily, with casein as the source for protein, for 6 weeks and this diet resulted in moderately high serum cholesterol concentrations (219 ± 33 mg/dl). Another group fed the same diet except with soy protein product as the protein source instead of casein showed virtual normocholesterolemia at the end (107 ± 3 mg/dl). Cholesterol balance was studied under non-steady state conditions using methods designed for this purpose. Reflecting the serum cholesterol concentration, the total body cholesterol concentration (excluding CNS) was also significantly lower in soy protein group. However, parameters of cholesterol balance, such as fecal neutral and acidic steroid excretions, dietary cholesterol absorption, and whole body cholesterol synthesis were studied and no differences were demonstrated between the casein- and soy protein-fed swine. The experiment was repeated and in Experiment II virtually the same results were obtained. When swine were given the same high-fat, high-cholesterol diets with 1 2 casein + 1 2 soy protein or casein + soy protein, hypocholesterolemic effects were also observed. Therefore, such action is probably caused principally by soy protein per se rather than simply by replacement of casein by soy protein. Addition of dl -methionine to soy protein containing diet did not alter the hypocholesterolemic effect of soy protein indicating that the effect was not the result of methionine deficiency. In conclusion, we can state that the hypocholesterolemic action of soy protein was clearly demonstrated in swine fed a high-fat, high-cholesterol diet, but that the mechanism of action is yet to be established.

Modification of lipoprotein patterns and retardation of atherogenesis by a fish oil supplement to a hyperlipidemic diet for swine

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased steroid excretion in swine fed high-fat, high-cholesterol diet with soy protein

Abstract Hypocholesterolemic mechanism of soy protein when added to high-fat, high-cholesterol (HC) diet as compared to casein was studied in young male Yorkshire swine (10 kg) in two experiments. Three soy protein products were used: Soy Protein A, B, and C. Soy Protein A is Pro-Lean™ (Miles Laboratories) and contains 62.2% protein. Soy Protein B is more purified than Soy Protein A and contains 92% protein. Soy Protein C is the same as Soy Protein A, except that it contains less salt than Soy Protein A. The first experiment with 43 swine was designed to observe: (1) the effects of two soy protein products (Soy Protein A and B) vs casein on serum cholesterol concentrations and hepatic microsomal HMG-CoA reductase activities when added to a mash diet, and (2) the effect of more purified soy protein (Soy Protein B) when added to an HC diet. The second experiment with 10 swine was designed to compare serum cholesteol concentrations and fecal steroid excretions on an individual basis in two groups of swine fed either HC with Soy Protein C or HC with casein diet for 4 weeks and switching the diets for 2 weeks. 1. 1. Neither of the soy protein products, A or B, affected serum cholesterol levels when added to mash. Similarly, no changes were noted when casein was added to mash. 2. 2. Total hepatic microsomal HMG-CoA reductase activities were not altered by the addition of either Soy Protein A or casein to mash. The activities of the enzyme were reduced by 70% in the group in which soy protein was used in an HC diet as compared to the activities of the enzyme with the groups fed mash alone or mash plus Soy Protein A. 3. 3. All three soy protein products were hypocholesterolemic when added to HC diet. 4. 4. The effect of soy protein on lowering serum cholesterol levels as compared with casein in swine fed high-fat, high-cholesterol diet appears to be due to the increases in fecal steroid excretions not counter-balanced by a concomitant increase in cholesterol synthesis. However, the mechanism of such increases in steroid excretions is not known.

Dietary fish oil added to a hyperlipidemic diet for swine results in reduction in the excessive number of monocytes attached to arterial endothelium

Modest numbers of blood monocytes become attached at least temporarily to the endothelium of large arteries in normal swine fed low fat, low cholesterol diets. These numbers are increased several fold when the swine are fed a high saturated fat, high cholesterol atherogenic diet (BT). The main objective of this portion of a broader study was to see if the addition of fish oil (30 ml) to a BT diet (BT + FO) could prevent the increase in attached monocytes induced over arterial endothelium in BT fed swine. Six BT, 6 BT + FO and 5 control mash (MA) swine fed the respective diets for 4 months before killing were available for the current study. Other aspects of this experiment have been presented previously which in brief are that BT + FO resulted in retardation of atherosclerotic lesion development and a shift in lipoprotein components from predominantly apolipoprotein B,E containing with the BT diet to predominantly apo B only with BT + FO. There was a significant positive correlation between lesion development and apo B,E lipoproteins. In the current study we determined by scanning electron microscopy on the first portion of the left anterior descending coronary artery after perfusion fixation under pressure the number of monocytes per mm2 attached over or not over visible lesions. We also determined monocyte percentages in the circulating blood and analyzed the correlation of the numbers of attached monocytes and blood monocyte percentages with various lipoprotein components reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)

Population dynamics of arterial cells during atherogenesis: XIII. Mitogenic and cytotoxic effects of a hyperlipidemic (HL) diet on cells in advanced lesions in the abdominal aortas of swine fed an HL diet for 270–345 days

The abdominal aortas of five groups of young male Yorkshire swine were studied: (1) 0-day baseline group; (2) hyperlipidemic (HL) group with ballooning; (3) mash group with ballooning; (4) mash group without ballooning; and (5) HL group without ballooning. The last four groups were injected with [3H]thymidine at 270 days and sacrificed subsequently in subsets at intervals up to 75 days in order to study births and deaths (or loss by migration) among cells over the period 270-345 days. However, only in the HL-ballooned group were there enough swine for the isotopic data to be useful for most purposes. In the 0-day baseline group there were 6 +/- 2 X 10(6) cells in intimal cell masses (ICM); in the 270- to 345-day mash group without ballooning the number was 10 +/- 2 X 10(6), which is not a statistically significant increase over 0-day. This supports the hypothesis that in the normal state births and deaths (or loss by migration) among cells in ICM are nearly balanced at least up to 1 year of age. In the 270- to 345-day mash group with ballooning there were 61 +/- 12 X 10(6) cells in the ICM. Thus a single episode of deendothelialization results in tremendous hyperplasia of ICM. However, even the largest ICM (atherosclerotic lesion) in this group showed essentially no necrosis. In the 270- to 345-day HL group with ballooning there were 108 +/- 17 X 10(6) cells in the ICM turned atherosclerotic lesions. In addition an average of one-third of the lesion volume was occupied by lipid-rich, calcific necrotic debris. Thus the HL diet appears to have associated with it both mitogenic and cytotoxic influences on ICM cells. In the 270- to 345-day HL group not ballooned there were 130 +/- 30 X 10(6) lesion cells and lesions were somewhat more extensive and necrotic than in the HL-ballooned group, probably because the former group included by chance more hyperresponders (as regards serum cholesterol values) to the HL diet than the latter. Regardless of this, the data suggest that in this particular model of advanced atherosclerosis the balloon-injury stimulus to proliferation and the HL-diet stimulus are neither additive nor synergistic.(ABSTRACT TRUNCATED AT 400 WORDS)

Dietary lipids and thrombosis: Factors in blood that account for increased “binding power” in rats fed thrombogenic diets☆

Abstract In previous studies with rats we observed that certain thrombogenic diets resulted in a stronger clot (greater binding power as measured in the thrombelastograph) and prolonged clot-lysis times as compared to simple stock diets. Furthermore, there was a close correlation between the plasma fibrinogen concentration and the binding power. In an attempt to investigate further the previously observed relationship between plasma fibrinogen and binding power, varying amounts of purified fibrinogen were added to plasma with normal fibrinogen levels, and the binding powers were measured in the thrombelastograph. It was found that the binding power was directly proportional to the fibrinogen concentration in the plasma. The experiment was carried out further to determine how early changes in various coagulation factors begin to take place after initiation of high-fat diets. Fibrinogen and prothrombin levels began to rise as early as within 1 week after the administration of high-fat diets. The changes were accompanied by a significant increase of serum cholesterol levels. By the 18th day prothrombin and fibrinogen concentrations doubled their control values, and the increase in serum cholesterol was more than tenfold.

Fish Oil, Atherogenesis, and Thrombogenesisa

Marine fish consumption is known to reduce mortality from ischemic heart disease. The use of fish oil as a dietary supplement, however, is not universally recommended. In large doses, fish oil reduces plasma cholesterol and triacylglycerol but increases low density lipoprotein (LDL) levels and the potential for free radical generation and bleeding. Moderate marine fish consumption is known to reduce mortality without altering commonly measured variables, i.e., plasma cholesterol levels, in vitro platelet aggregation, and bleeding times. In swine, we observed that monocyte adhesions and platelet clumps over the lesion surface of proximal left anterior descending (LAD) coronary arteries are markedly reduced when an atherogenic diet was supplemented with cod-liver oil, even when the cholesterol levels were equalized with the untreated group. These findings suggest that fish oil is hypothrombogenic. We developed an in vitro assay to delineate the mechanism whereby fish oil reduced monocyte-endothelial cell interactions in vivo. The effects of supplementing the culture medium with different fatty acids on adhesions between lipopolysaccharide (LPS) stimulated swine aortic endothelial cells (SAEC) and the human monocyte-like cell line, U937, was investigated in a 10 minute adhesion assay at 37 degrees C. Exposure of SAEC for 6 hours to media containing 50-200 microMs eicosapentaenoic (EPA), stearic, oleic, linoleic, and arachidonic acid, respectively, revealed that only EPA reduced U937-SAEC adhesion. Exposure of U937 to EPA also reduced adhesions. EPA was not effective when added to the SAEC more than 2 hours after they were stimulated with LPS. Exposure of human umbilical vein endothelial cells (HUVEC) to EPA reduced the expression of VCAM-1, ELAM-1, and ICAM-1 after 5 hours of stimulation with LPS. These results suggest that EPA may functionally impair the induction/expression of adhesion molecules.

Intimal Cell Mass-Derived Atherosclerotic Lesions in The Abdominal Aorta of Hyperlipidemic Swine Part 1. Cell of Origin, Cell Divisions and Cell Losses in First 90 Days on Diet

Atherosclerotic lesions may originate and develop in a variety of ways. In this study we are focusing our attention on atherosclerotic lesions arising in normally occurring intimal cell masses (ICM) in the abdominal aortas of hyperlipidemic (HL) swine. Times chosen for study were 0, 14, 49 and 90 days on HL diet; mash-fed swine were used as controls. Total numbers of cells in the ICM of HL and mash swine were similar at 14 and 49 days; by 90 days the number of cells had increased dramatically in the HL swine to 8-fold greater than control values. Changes present at 49 days and thus preceding increase in cell numbers included extensive intracellular lipid accumulation with by count nearly half of the ICM cells involved and elevated tritiated thymidine labeling indices (LI) 4-fold greater than control. Differential cell counts by transmission electron microscopy were made on the ICM lesions in the HL swine at 49 and 90 days. More than 95% of all cells were smooth muscle cells (SMC), with relatively few monocytes being present. Calculations from the LI and total cell counts showed that the entire increase in cell numbers could be accounted for by divisions among the resident SMC in the ICM. Further calculations suggested that cell losses (deaths) from the ICM were minimal. Scanning electron microscopy studies reported elsewhere revealed no loss of endothelial integrity. The results suggest: (1) that the lesions arise by stimulation of the resident SMC in the ICM to hyperplastic activity, (2) that the role of monocytes in the early development of these lesions is minimal if any, (3) that in view of the intact endothelium platelets are not likely to play an important role, (4) that ICM cell death is not a major factor, (5) that the most likely candidate for the cell growth stimulatory role (? mitogen) is some component(s) of the excess lipid that accumulates in the ICM.

Effects of cholestyramine on cholesterol balance parameters and hepatic HMG-CoA reductase and cholesterol-7-α-hydroxylase activities in swine

Abstract Effects of cholestyramine treatment for 75 days on whole body cholesterol balance and hepatic HMG-CoA reductase and cholesterol-7-α-hydroxylase activities were studied in hypercholesterolemic swine. Sixteen male Yorkshire swine (10 kg) were divided into four groups; three groups were fed a high cholesterol (HC) diet for 50 days. One group was then switched to mash, the second was given cholestyramine, 12 gm daily, and the third was left on the high cholesterol diet, all for an additional 75 days. The fourth group was maintained on mash throughout the 125 days. Data for the cholesterol balance parameters, retention, excretion, and synthesis, were obtained during the terminal week. Hepatic HMG-CoA reductase and cholesterol-7-α-hydroxylase activities were assayed terminally. Cholestyramine reduced serum cholesterol concentrations in hypercholesterolemic swine very effectively although the reduction was not as complete as in those swine switched to mash diet. The drug also reduced whole body cholesterol retention. These changes appeared to be due to increases in both acidic and neutral steroids in the feces. Accompanying increases in whole body cholesterol synthesis probably partially offset the beneficial effect of increased steroid excretions. In vitro hepatic HMG-CoA reductase activities correlated well with whole body cholesterol synthesis determined by the balance method as well as with fecal steroid excretions. Cholesterol-7-α-hydroxylase activities of the liver microsomes correlated well with the amount of fecal bile acid excretion.

Atherosclerotic lesions in the coronary arteries of hyperlipidernic swine Part 1. Cell increases, divisions, losses and cells of origin in first 90 days on diet

The intima of the proximal portion of the coronary arteries of young swine is normally thickened by accumulations of cells about 90% of which are smooth muscle cells (SMC) and about 10% are of probable monocyte origin. Extracellular components such as collagen and elastic tissue are also present but we have chosen to emphasize their cellular nature by calling the regions of thickened intima, intimal cell masses (ICM). We have previously shown that atherosclerotic lesions produced in the coronary arteries of swine by 90 days of feeding a hyperlipidemic (HL) diet arise almost exclusively in the normally occurring ICM. We are reporting here a study of the pathogenesis of these lesions following killing at 0, 14, 49 and 90 HL diet days with comparisons between ICM in control mash-fed swine and ICM-lesions in the HL swine. We found that in the ICM: lipid accumulation was present by 14 days and increased thereafter; the lipid was mostly in SMC but percentage wise the monocyte-macrophages were involved as much or more, cell division activity was increased 3-4-fold by 49 days, cell numbers in ICM were similar in HL and control swine at 49 days but were about 6-fold greater in the HL swine at 90 days, (now in ICM-lesions), at 90 days, circa 90% of the cells appeared to be of SMC and circa 10% of monocyte origin both in the ICM-lesions of the HL swine and in the normal ICM of the controls. The data suggest but do not prove that early lipid accumulation precedes increased cell divisions especially among the SMC component and this in turn precedes increased numbers of cells in the ICM. Although SMC constitute the major cell component of the ICM-lesion at 90 days, the monocyte-macrophage-like cells also increase in number as a result of the HL diet and constitute a small but definite minor component. One possible explanation for the increased cell division activity is that one of the lipid constituents is acting as mitogen; another possibility is that the effect of a well known mitogen such as platelet-derived growth factor is enhanced by the lipid; another is that the monocytes are being stimulated to produce monocyte-derived growth factor. In any event in the very early stage of atherogenesis in the coronary arteries in these experiments excessive proliferation of resident SMC in the ICM appears to be the predominant feature.