lillllllllllll Mil : ' ”w. 3 1293 10065 8719 ‘1 1 £33551 RY IJ‘,/v jgflhflbfiganU5t3€C U {L‘W'E'ISAW {7“ This is to certify that the thesis entitled Modification of Plasma Cholesterol Parameters, Lipoproteins, Lecithin: Cholesterol Acyltrans— ferase Activity and Tissue Lipids by Diet and Exercise in Pigs and Chickens presented by William Albert Forsythe III has been accepted towards fulfillment of the requirements for Ph.D. degree in Human Nutrition Z .33 -1: Major professor Date 2-21-80 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: "7'0: .» . " Place in book return to remove ‘t ° ‘Vfl’K ' charge from circulation records W ‘ T [mm C MODIFICATION OF PLASMA CHOLESTEROL PARAMETERS, LIPOPROTEINS, LECITHIN: CHOLESTEROL ACYL TRANSFERASE ACTIVITY AND TISSUE LIPIDS BY DIET AND EXERCISE IN PIGS AND CHICKENS By William Albert Forsythe III A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1980 ABSTRACT MODIFICATION OF PLASMA CHOLESTEROL PARAMETERS, LIPOPROTEINS, LECITHIN: CHOLESTEROL ACYL TRANSFERASE ACTIVITY AND TISSUE LIPIDS BY DIET AND EXERCISE IN PIGS AND CHICKENS By William Albert Forsythe III While elevated plasma cholesterol levels are one of the major risk factors associated with the development of atherosclerosis, the effects of diet and exercise on plasma cholesterol levels are still controversial. Experiments were undertaken, in pigs and chickens, to investigate how different dietary components (type of protein, type of fat, and type of fiber) or an aerobic exercise program affect Cholesterol parameters. Two experiments, utilizing young male pigs, tested the effects of varying the polyunsaturated (Puf) to saturated (Sat) fat ratio (P:S) and substituting plant (Pl) for animal (An) protein. The diets provided l6% of the energy as protein and 42% as fat with a P:S ratio of 3.0 in high-Puf diets and 0.3 in the high-Sat diets. In the first experiment, changing the P:S ratio from 0.3 to 3.0 reduced plasma cholesterol levels by 44 mg/dl. The high density lipoprotein (HDL) to low-density lipoprotein (LDL) ratio was 1.84 in the high-Puf diet and 1.42 in the high-Sat diet. A second experiment tested the effects substituting plant (50% from soybean and 25% each from corn and wheat) for animal (90% casein and l0% lactalbumin) protein on these same parameters. William Albert Forsythe III Increasing the P:S ratio reduced plasma cholesterol levels by 30% and also reduced the molar LCAT activity. Plant protein (compared to animal protein) reduced plasma total cholesterol levels by 23%. Other changes induced by substituting plant protein for animal protein were: increased fractional and molar LCAT rate; increased HDLzLDL ratio; and decreased plasma amino acid levels of lysine, threonine, valine, isoleucine, and leucine. Another important finding in this experiment was the ability of the dietary protein source to affect plasma cholesterol levels independent of the dietary P:S ratio. In another set of experiments, both pigs and chickens were used as experimental models to study the effect two different sources of dietary fiber have on body composition, plasma cholesterol parameters and LCAT activity. Wheat bran (WB) or rolled oats (R0) were fed as 6.5% neutral detergent fiber (NDF) in diets for l7 weeks to weanling castrated pigs and for 5 weeks to mature "broiler type" roosters. Diet composition was (% energy): l8% protein, 42% fat (P:S = 0.3), and 40% carbohydrate with 0.05% added cholesterol. Feeding R0 reduced body fat in both pigs and chickens compared to feeding WB or a control diet (CN, < l% NDF). While not affecting total cholesterol levels, in pigs, feeding WB increased HDL cholesterol levels, the percentage of cholesterol in the HDL fraction, and the HDL:LDL ratio. Feeding R0 did not reduce plasma total cholesterol levels in pigs but did reduce levels in chickens (220 mg/dl) compared to CN (266 mg/dl). In both pigs and chickens R0 increased William Albert Forsythe III HDL cholesterol levels and the molar LCAT activity compared to the other two treatments. In another experiment, young male castrated pigs were exercised aerobically; alternately 3 mph for 20 minutes per day or 3.3 mph for 9 minutes per day, seven days a week. During the first five weeks of the experiment, which consisted of 3 weeks training and 2 weeks of maximum exercise, the pigs were fed a corn-soy grower ration. The relatively short exercise program had no effect on any plasma cholesterol parameter. After 5 weeks the pigs were fed the high-fat (P:S = 0.3), high cholesterol diet fed as the control diet in the fiber experiments. Ten weeks of exercise significantly reduced plasma total and unesterified cholesterol levels. HDL cholesterol levels were not changed by exercise but the percentage of total cholesterol in the HDL fraction was significantly greater in the exercised compared to non-exercised pigs. Exercise also altered the plasma lipoprotein profile; increasing the proportion of HDL and decreasing the percentage of LDL. In total, these experiments have shown that dietary changes or an aerobic exercise program can significantly affect body- composition, plasma cholesterol parameters, including total and HDL cholesterol concentrations, and plasma LCAT activity. ACKNOWLEDGEMENTS The author wishes to express his appreciation to members of his doctoral committee, Dr. D. Romsos, Dr. E. Miller, Dr. W. VanHuss and Dr. G. Leveille. The author would especially like to thank his major advisor, Dr. M. Bennink, who provided both guidance and insight during his doctoral training. Appreciation is also extended to Gretchen Hill, Matt Parsons and Brian Curry for invaluable expertise in handling and exercising pigs. Loving gratitude is extended to his wife, Eileen, and children, Lara and William without whose dedication and encouragement the research would not have been possible. 11' TABLE OF CONTENTS Page LIST OF TABLES ........................ vi LIST OF FIGURES ....................... ix INTRODUCTION ......................... 1 PART I REVIEW OF LITERATURE CARDIOVASCULAR DISEASE .................... 3 Occurrence ........................ 3 Risk factors ....................... 4 LIPOPROTEIN METABOLISM .................... 7 CELLULAR CHOLESTEROL UPTAKE ................. 12 FACTORS AFFECTING PLASMA CHOLESTEROL ............. 14 Cholesterol ........................ 14 Protein .......................... 19 Dietary fiber ....................... 23 Exercise ......................... 25 PART II EFFECTS OF DIETARY PROTEIN AND FAT SOURCES ON PLASMA CHOLESTEROL PARAMETERS, LCAT ACTIVITY, AMINO ACID LEVELS AND TISSUE LIPID CONTENT OF GROWING PIGS INTRODUCTION ......................... 29 MATERIALS AND METHODS .................... 30 Animals .......................... 30 Diets . . ......................... 31 Plasma and tissue analyses ................ 34 Statistical analyses ................... 36 RESULTS ............................ 37 Experiment I ........................ 37 Experiment 2 ........................ 40 DISCUSSION .......................... 49 PART III EFFECT OF FEEDING ROLLED OATS OR WHEAT BRAN ON PLASMA LIPIDS, LIPOPROTEINS, LCAT AND TISSUE LIPIDS IN PIGS AND CHICKENS INTRODUCTION ......................... 58 MATERIALS AND METHODS ..................... 59 Animals .......................... 59 Diets ........................... 60 Plasma and tissue analyses ................. 62 Hepatic sterol synthesis .................. 64 Statistical analyses .................... 65 RESULTS ............................ 65 DISCUSSION .......................... 76 PART IV EFFECT OF AN AEROBIC EXERCISE PROGRAM ON BODY COMPOSITION, PLASMA CHOLESTEROL PARAMETERS. LCAT ACTIVITY. LIPOPROTEINS AND TISSUE LIPIDS IN YOUNG PIGS INTRODUCTION ......................... 82 MATERIALS AND METHODS ..................... 83 Animals and diets ..................... 83 Exercise program ...................... 84 Plasma and tissue analyses ................. 86 Statistical analyses .................... 87 RESULTS ............................ 87 DISCUSSION .......................... 94 iv PART V SUMMARY AND CONCLUSIONS SUMMARY AND CONCLUSIONS ................... 99 LITERATURE CITED LITERATURE CITED ....................... 104 Table LIST OF TABLES PART I REVIEW OF LITERATURE Percent revalence of selected "risk factors" in the United States .................. Composition and properties of human plasma lip0proteins ...................... PART II EFFECTS OF DIETARY PROTEIN AND FAT SOURCES ON PLASMA CHOLESTEROL PARAMETERS, LCAT ACTIVITY, AMINO ACID LEVELS AND TISSIE LIPID CONTENT OF GROWING PIGS Diet formulation (Experiment l) ............ Diet formulation (Experiment 2) ............ Analysis of selected nutrients in diets (Experiment 2) ..................... Effects of dietary fat on body weights and plasma lipids in pigs fed plant protein diets (Experiment 1) ..................... Final body weights, plasma cholesterol parameters and LCAT activity in pigs fed different protein or fat for 14 weeks (Experiment 2) ........... Effect of dietary protein and fat sources on plasma lipoprotein distributions (Experiment 2) vi Page 32 33 35 39 41 47 LIST OF TABLES (cont'd) Table 7. Page Plasma amino acid levels in pigs fed different protein and fat sources (Experiment 2) .......... 48 Effect of dietary protein and fat sources on liver and aorta lipid composition (Experiment 2) ..... 50 PART III EFFECT OF FEEDING ROLLED OATS 0R WHEAT BRAN ON PLASMA LIPIDS, LIPOPROTEINS, LCAT AND TISSUE LIPIDS IN PIGS AND CHICKENS Composition of diets, % ................. 6l Fatty acid composition of diets, % ............ 63 Dietary fiber effects on body, heart and aorta composition in pigs ................... 66 Effect of dietary fiber on plasma cholesterol and triglyceride levels and LCAT activity in pigs ........................... 68 Plasma lip0proteins in pigs as affected by dietary fiber ...................... 72 Effect of dietary fiber on final body weight, body water, plasma cholesterol parameters and plasma LCAT activity in chickens ........... 73 Effect of dietary fiber on plasma lipoprotein percentages in chickens ................. _ 74 PART IV EFFECT OF AN AEROBIC EXERCISE PROGRAM 0N BODY COMPOSITION, PLASMA CHOLESTEROL PARAMETERS, LCAT ACTIVITY, LIPOPROTEINS AND TISSUE LIPIDS IN YOUNG PIGS Composition of diets, % ................. 85 Final body weights, feed consumption and body and heart lipids in non-exercised and exercised pigs ........................... 88 vii LIST OF TABLES (cont'd) Table Page 3. Diet and exercise effects on plasma cholesterol levels, triglyceride levels and LCAT activity in pigs ......................... 90 4. Effect of exercise on plasma cholesterol levels, lipoprotein profile and LCAT activity in pigs ...... 93 viii LIST OF FIGURES Figure Page PART II EFFECTS OF DIETARY PROTEIN AND FAT SOURCES ON PLASMA CHOLESTEROL PARAMETERS, LCAT ACTIVITY, AMINO ACID LEVELS AND TISSUE LIPID CONTENT OF GROWING PIGS I. Effects of varying P:S ratio on bi-weekly plasma cholesterol levels in pigs (Experiment I) ........ 38 2. Effects of animal or plant protein and varying P:S ratio on bi—weekly plasma total cholesterol levels in pigs (Experiment 2) ........ 42 PART III EFFECT OF FEEDING ROLLED OATS OR WHEAT BRAN ON PLASMA LIPIDS, LIPOPROTEINS, LCAT AND TISSUE LIPIDS IN PIGS AND CHICKENS l. Effects of dietary fiber on plasma lipoprotein separation ........................ 69 ix INTRODUCTION Cardiovascular disease is one the the major health problems in the United States and the etiology of the disease has been intensively investigated in recent years (l). Most investigators will agree that plasma cholesterol levels are perhaps the most important single parameter involved in the development of atherosclerosis. Zilversmit (2) has recently reviewed this area, concluding that evidence supporting cholesterol involvement in atherogenesis are: l) accumulation of free and esterified cholesterol in human atherosclerotic plaques; 2) increased incidence of coronary heart disease (CHD) earlier in life in persons with genetic hypercholesterolemia; 3) epidemiological studies have consistently shown high correlations between CHD and plasma cholesterol levels; and 4) animal studies have shown that cholesterol accumulation into arteries follows induced hypercholesterolemia. What is still debatable is whether dietary changes can alter plasma cholesterol levels sufficiently to affect atherogenesis. The Senate Select Committee on Nutrition and Human Needs (3) after reviewing data linking diet to plasma cholesterol levels suggested these changes in the American diet as regarding atherosclerosis: 1) reduce caloric consumption; 2) reduce cholesterol consumption to 300 mg/day; 3) reduce overall fat consumption from 40 to 30 percent of dietary calories; and 4) reduce saturated fat consumption to ID percent of dietary calories and balance with 10 percent of the calories from monounsaturated and l0 percent of the calories from polyunsaturated fats. While prominent scientists (Hegsted (4); Glueck and Connors (5)) support these changes, other, equally as. prominent scientists (Ahrens (6); Reisser (7)), suggest that such changes are unwarranted. Clearly whether the diet has the capacity to affect the development of atherosclerosis needs further investigation. The primary objective of these experiments was to investigate whether altering dietary components in the presence of high-fat, high-cholesterol diets could beneficially change cholesterol parameters in pigs or chickens. The diet changes which were investigated were: I) substituting plant protein for animal protein; 2) altering the polyunsaturated fat:saturated fat (P:S) ratio from 0.3 to 3.0; and 3) feeding 6.5% neutral detergent fiber (NDF) from either wheat bran or rolled oats in the diet. Also investigated was the effect a moderate exercise program had on these same parameters in pigs. PART I REVIEW OF LITERATURE CARDIOVASCULAR DISEASE OCCURRENCE Cardiovascular disease (CVD) is the leading canse of death in the United States. While death rates (death/100,000 persons) have been falling slightly since 1968 (l), cardiovascular disease was still responsible for 1,037,000 deaths or 52.9% of total deaths in l974 (8)(latest year for complete figures). By comparison the next two leading causes of death combined claimed fewer lives; malignancies (351,000 deaths) and accidents (115,000 deaths)(8). Another indication of the severity of CVD is the death rate in 45-64 year old males, the major wage earners in the United States. The incidence of coronary heart disease (CHD) deaths in males 25-44 years old is 48 per 100,000 persons. But in males 45-64 years the incidence jumps to 621 deaths per 100,000 persons. Although CHD is a degenerative disease, this increase in the incidence of deaths in the major wage earners in the United States has tremendous economic impact. Kolata has estimated the economic loss approaches 40 billion dollars annually (9). The United States is not the only country with a high incidence of CHD. Keys (10) in an analysis of a World Health Organization (WHO) report on death in 34 countries, listed six countries with a high incidence of CHD: Australia, Canada, Finland, New Zealand, Scotland, and United States. Eight counties reported a low incidence of CHD deaths: Bulgaria, Greece, Italy, Japan, Poland, Portugal, Romania, and Taiwan. Countires were eliminated where deaths from ill-defined or unknown causes were high. Keys suggests that the data on the above countries is accurate and reflects actual CHD deaths. Data from Keys' seven country survey supports the WHO report (11). For instance, while both Japan and Italy have total mortality rates similar to the United States and Finland, they have CHD death rates of 30% or less than that of the United States or Finland. These and other reports have led to many epidemiological studies to elucidate differences responsible for the differences in CHD mortality between countries. While results from epidemiological studies cannot prove cause and effect, they can provide evidence that the incidence of CHD correlates with the occurrence of various parameters, which have been called risk factors. RISK FACTORS The earliest epidemiological studies were primarily retrospective in nature. Keys (10) has recently reviewed epidemiological evidence linking CHD to diet. In 1916, C.D. DeLangens, a Dutch physician, reported that natives of Java had a lower incidence of atherosclerosis than the Dutch. He related this decreased morbidity to lower blood cholesterol levels. Similarly, retrospective studies were undertaken to investigate the reasons a decreased incidence in CHD deaths occurred after World War II in both Finland and Norway. While there were many changes in lifestyle, Nalmros (12) concluded that the factor that correlated most highly with the decline in CHD was the decrease in saturated fat consumption. In 1948, the first major prospective study was undertaken to study CHD in Framingham, Massachusetts (13). This study initially surveyed over 5200 persons free from CHD and is still ongoing. Results from this study indicated three major risk factors in the development of atherosclerosis (14): elevated serum cholesterol levels, hypertension, and cigarette smoking. Other minor risk factors identified have been obesity, inactivity and diabetes mellitus. Kannel et al. (14) has reported on the relative incidence of the above risk factors in the United States population (Table 1). Reports from Gordon et a1. (15) have indicated that the morbidity increases geometrically when two or more risk factors are present in the same person. Men aged 30-59, with all three risk factors present have eight times the probability of developing CHD than similar aged men with no risk factors present (16). On the average, 18% of the men and women in the United States are at risk for the three major risk factors. While all three risk factors are important, this review of literature concentrates on hypercholesterolemia. Epidemiological studies have reported that, in addition to the amount of plasma cholesterol, the lipoprotein fraction that carries cholesterol is also important in the development of atherosclerosis. An increased low density cholesterol lipoprotein (LDL) cholesterol level correlates with an increased incidence of CHD (l7, l8) and an .Amo-omm_v memmgm Lo PE oo_\ms com mcowpmgucmucou pogmpmmroco Eagmm mw memFogwpmmpocugqu: .Aouopv maven; pcmcgzu op memwmg mcwxosm mppmgmmwu .Amouomopv om\om_ pmmm— pm mgzmmmga coop; m mw cowmcmugmaxz .Amouommpv cmwume m>onm mgos Lo ucmugma ow usmwmz m? xuwmmno .Ammuemmpv cws\gmpwp om.o cusp mmwp cowgaszmcou cmmxxo mmmgm>m m? auw>PpumcH .Aepv aHMImm _Pm==m¥ twpc< r—NMVLOO o.~m N.o— m.n¢ N.NN o.mm cuimm N.m¢ N.¢N N.Pm m.om m.om ecimm o.mN p.©m N.m— N.¢N m.op ¢m|m¢ m.NF m.wm m.m F.0N m.mp eclmm cmeoz m.PN w.NN F.NN N.NP F.NN enumm m.mm ¢.nm m.NN m.N~ o.—N ecumm “.mm P.m¢ m.mp N.¢F m.mp emim¢ N.om m.m¢ m.m— m.N— F.N~ ecumm cm: m mavemwmmmwnmpocu WQHWHMMMQ ecowmcmucmax: mapwmmno prw>wpumcH Ang mm< _.mmpmpm ump_c= mzu cw =mcouomw xmwg= umpomemm mo mucmpm>mcq ucmugwm .P mpnmh increased high density lip0protein (HDL) cholesterol level, correlates with a decreased incidence of CHD (17, 18). Thus any changes which can increase the ratio of HDL/LDL cholesterol could be beneficial in the prevention of CHD. This review first discusses lipoprotein and cholesterol metabolism. Then discussed are the effects diet (cholesterol, protein and fiber) and exercise exert on plasma cholesterol and lipoprotein metabolism. LIPOPROTEIN METABOLISM The composition of the four major classes of lip0proteins, chylomicra, very low density lipoprotein (VLDL), LDL and HDL are presented in Table 2. The lip0proteins are generally classified according to their flotation in salt gradients (19). Because of their importance in cholesterol metabolism and the development of atherosclerosis, intensive investigations into their structure, composition and metabolism have been undertaken in recent years. Nascent chylomicra, the lowest density lipoprotein class, are synthesized in the intestine to transport exogenous triglycerides from the small intestine. The composition of chylomicrons is 90% triglycerides, 5-7% phospholipids and 1-2% of cholesterol and protein (20). Apoprotein B (apoB), the major apoprotein of chylomicra, is also synthesized in the intestine (21) and is necessary for binding and transport of lipids (22). In individuals with abetalipoproteinemia there is an absence of apoB and triglycerides, while synthesized by intestinal cells, are not transported from the intestinal cells to the blood (22). Once nascent chylomicra .AGNV ._m pm pummPtLoz EOLL empame< F Focmummpocu umwwpcmpmmca mwuwgmuapmwgu mgmpwm mgmpmm PocmpmmPocu pocmpmmpocu Fogmumwpozo vmvmwgmummcz mnwnwpogamoca mv_awposamo;a mvPQWF Locwz mcrappogquLQ mgmpmm mvwaP—ogqmoca Pogwpmmpocu mmu_gmux_mwcu mmuPLmUAmecu mvwawp gown: moan comm . HHHuuoam comm HHuuoam HHn acctuweopxsu mmPmeQOLa F .mcwmuogaoawp mammpq amass to mmwpgmaoga use cowpwmoasou .N mpnmp reach the blood, Havel et a1. (23) have shown that apoprotein C (apoC) is transferred from HDL to complete the chylomicron particle. Smith et al. (24) have demonstrated that apoC-II binds to and activates lipoprotein lipase (LPL) associated with endothelial membranes. LPL then hydrolyzes the triglycerides in chylomicra to produce a chylomicron remnant. ApoC-II then returns to the HDL particle and the chylomicron particle is catabolized by the liver (19). Anderson and Dietschy (25) suggest that the cholesterol in the remnant is important in the regulation of hepatic cholesterolgenesis. Zilversmit (20) also suggests that the remnant is a major factor in the production of atherosclerotic plaques. VLDL is synthesized primarily in the liver to transport endogenously synthesized triglycerides (27). The composition of VLDL is similar to chylomicra with 90% of its mass as triglycerides. A small amount however, designated iVLDL, is synthesized in the intestine (27). While both sources contain apoB, only hepatic VLDL contains apoC-II.(29). iVLDL, in a manner similar to chylomicra, receives its apoC-II from HDL particles (28). After removal of triglycerides, intermediate density lipoproteins (IDL), precursors to LDL, are formed (28). IDL exchanges apoC with HDL and either in the liver or the periphery, is further delipidated to form LDL. LDL carries cholesterol to extrahepatic tissues and cholesterol comprises approximately 60% of the lipid and over 40% of the total mass of LDL; mostly in the form of cholesterol esters (l9). ApoB, which constitute more than 98% of the total apoproteins in LDL, has 10 been shown to interact specifically with cell surface receptors in many types of cultured cells, including human fibroblasts, lymphocytes, aortic smooth muscle cells and endothelial cells (29). The LDL particle is then degraded by internalization and catabolized by a mechanism elucidated by Brown and Goldstein (29-31). The significance of the LDL receptor system on cholesterol metabolism will be discussed in the next section. LDL is not extensively metabolized by the liver (32). In fact, Sniderman et a1. (33) reported an increase in the catabolism of LDL after removal of livers in pigs. The most dense lipoprotein fraction, HDL, contains about 50% protein, 30% phospholipid, and 20% cholesterol (32). ApoA constitutes approximately 88% of the apoproteins associated with HDL (34). ApoC is the other major apoprotein class. Although apoC accounts for only 5-10% of the total apoproteins in HDL, this amount is about one-half of the total apoC content in the blood (34). As previously described HDL contributes its apoC to chylomicra and iVLDL. The major function of apoA-I is to activate plasma LCAT (35). Brunzell et a1. (36) suggest that LCAT in association with HDL catalyzes the conversion of IDL to LDL. The VLDL particle consists of a hydrophobic core of triglycerides and cholesterol esters surrounded by a "membrane" of phospholipids, protein and unesterified cholesterol (36). Brunzell et a1. (36) hypothesize that as triglycerides are removed from the core the VLDL particle becomes smaller. Thus an excess membrane exists. HDL in conjunction with LCAT accepts lecithin and unesterified cholesterol from the IDL molecule. The net result is conversion ll of LDL to LDL and an increase in cholesterol esters in the HDL particle. Glomset (37) proposes that a major function of HDL is transportation of cholesterol, in the form of cholesterol esters, to the liver. Swartz et a1. (38) have recently provided evidence that, in vivo, the liver utilizes HDL cholesterol for bile acid synthesis. Unesterified cholesterol has the capacity to exchange freely between lipoproteins and between lipoproteins and cell surfaces (19). After esterification by LCAT the resulting cholesterol ester becomes relatively fixed in the polar core of HDL (37). Both Stein et al. (39) and Jackson (40) have reported that HDL facilitates the net removal of cholesterol from cells in culture presumably also by esterifying the unesterified cholesterol. Clearly a tremendous interaction exists between the lipoprotein classes. HDL exerts a most important influence on the metabolism of other lip0proteins. It is involved in the transfer of triglycerides to cells by contributing apoC-II to chylomicra and iVLDL. HDL may also facilitate the conversion of IDL and LDL in two ways: 1) activating plasma LCAT and 2) accepting phospholipids and unesterified cholesterol from the IDL membrane. Finally HDL, again interacting with LCAT, may transport cholesterol ester from the cells to the liver for excretion. 12 CELLULAR CHOLESTEROL UPTAKE As previously discussed (Lipoprotein Metabolism) cholesterol is carried in the blood primarily in the LDL fraction. Brown and Goldstein (29, 30, 31) in the past 5 years have elucidated a mechanism by which LDL-cholesterol can regulate extrahepatic cellular cholesterol metabolism. Under normal conditions the cholesterol content of the cell is balanced between cholesterol uptake or cholesterol synthesis of the cell and cellular losses of cholesterol. Cholesterol may be effectively lost when it is used in membrane synthesis or it may be lost in cellular secretions. Some cholesterol can be lost from the cell when transferred to HDL as previously described. Brown and Goldstein have shown that LDL plays a major role in regulating cellular cholesterol balance through a cell surface receptor mediated system. When the cellular cholesterol requirement is high, the number of LDL receptors increase on the cell surface. LDL binds to the surface receptors, probably facilitated by apoB (31). The lipoprotein is internalized by endocytosis and undergoes degradation in lysosomes. The lip0proteins are degraded to unesterified cholesterol and fatty acid. An accumulation of unesterified cholesterol causes a depression in the activity of 3-hydroxy-3-methy1glutaryl coenzyme A (HMG CoA) reductase, an enzyme that catalyzes the conversion of HMG CoA to mevalonate; the first committed step in the synthesis of cholesterol (41). While unesterified cholesterol effectively decreases cellular cholesterol synthesis, it increases acyl cholesterol acyl transferase (ACAT) 13 activity; an enzyme which esterifies cholesterol within the cell. ACAT produces a cholesterol ester containing predominantly oleate as the fatty acid (31). As cellular cholesterol storage increases the number of cell surface receptors for LDL are reduced through an unknown mechanism. Thus the cell by increasing the number of cell surface receptors has the ability to regulate LDL uptake and hence cholesterol uptake in response to cellular cholesterol needs. Dietschy and Wilson (42) hypothesize that plasma LDL levels can be regulated by extrahepatic tissue. If low levels of LDL are circulating in the blood, the cells have the capacity to synthesize their own cholesterol. But since extrahepatic cells usually synthesize only a small amount of cholesterol, if there are high levels of circulating LDL, the cells have only limited capacity to decrease these levels by decreasing cellular synthesis and increasing the uptake of LDL. Goldstein and Brown (43) hypothesize that LDL contributes to atherosclerosis through a mechanism similar to the receptor mediated process. Essentially aortic endothelial damage allows an increased infiltration of LDL into the intima of the aorta. There the LDL saturates the intimacytes' LDL receptors and undergoes internalization and degradation in the previously described manner. But because smooth muscle cells normally are not in contact with high LDL levels, metabolic feedback to reduce the number of cell surface receptors is not as rigorous as in other cell types (44). Thus the cholesterol esters continue to accumulate, resulting in foam cell formation. The fact that the major cholesterol ester found in atherosclerotic plaques is cholesterol oleate is cited 14 as evidence for this pathway (31, 45). If LDL simply deposited its cholesterol esters in the cells, one would expect cholesterol linoleate to be the predominate cholesterol ester within the cell. FACTORS AFFECTING PLASMA CHOLESTEROL CHOLESTEROL A controversy is growing as to whether feeding dietary cholesterol causes any changes in plasma cholesterol levels in humans. Those who support the hypothesis that dietary cholesterol does not affect plasma cholesterol levels, such as Mann (46) basically argue that: l) epidemiological data from America does not show any correlation between dietary cholesterol and plasma cholesterol values and 2) clinical studies, particularly those in which cholesterol has been fed with egg yolks, have failed to show that feeding cholesterol to free living subjects causes any appreciable increase in plasma cholesterol levels. A recent study by Flynn et al. (52) in which 1 or 2 eggs were fed daily to free living men is representative. In a cross-over design men were fed either 1 egg, 2 eggs or an egg-free diet; each period was for 12 weeks. The egg-free diet contained approximately 260 mg of cholesterol so that the diets with eggs contained either 580 or 800 mg of cholesterol when supplemented with l or 2 eggs respectively. No statistical difference in plasma cholesterol levels were observed between treatments in either experiment. Thus the authors argue that the recommendations of the Senate Select Committee on Nutrition 15 and Human Needs (3) to limit daily cholesterol intake to less than 300 mg will not significantly affect plasma cholesterol levels. Experiments where dietary cholesterol has been shown to affect plasma cholesterol levels have been well controlled, clinical studies where dietary cholesterol is either very restricted or totally removed for a period of time before the readdition of cholesterol to the diet (48). From these types of experiments, Keys (49) has predicted that the change in serum cholesterol levels follows the equation: Aserum cholesterol, mg/dl = 1.5(Adiet cholesterol, mg/1000 kcal)0°5. Generally this equation overestimates the change in plasma cholesterol that occurs when large amounts of dietary cholesterol are fed. The effects of dietary cholesterol feeding in humans on other aspects of cholesterol metabolism, such as lipoprotein or apoprotein alterations, has not been extensively investigated. Mahley (50) recently reported that supplementation of diets of healthy young men with 4 to 6 eggs per day for 4 weeks resulted in the generation of an HDLC fraction. This change occurred in subjects with unchanged plasma total cholesterol levels. Mistry et a1. (51) also reported an increase in the HDLC fraction when humans were fed 1500 mg of cholesterol per day. In this study there was an increase in B-VLDL, a cholesterol rich VLDL fraction also. However, Applebaum-Bowden et a1. (52) reported that feeding a liquid formula diet containing 5000 mg of egg yolk cholesterol to subjects for 30 days caused no increase in the HDLc fraction. These diets caused 16 a plasma cholesterol increase of 33 mg/dl (less than one-half of the increase predicted by Keys' equation) which was mainly associated with an increase in LDL cholesterol. The significance of increased HDLC blood levels relates to its apoprotein content. HDLC contains significant quantities of arginine rich ap0protein (ARP) which can compete with apoB for cell surface receptors (50). Thus, HDLc is internalized and contributes cholesterol to the cell in the same manner as LDL (50). Incubating aortic smooth muscle cells for 24 hours with HDLc resulted in similar amounts of unesterified cholesterol and esterified cholesterol in the cells as when the cells were incubated with LDL. But when the cells were incubated with normal HDL, unesterified cholesterol levels were reduced by two-thirds and cholesterol esters in the cell were decreased by 90% compared to the cell incubated with either LDL or HDLc (50). Thus, if consumption of cholesterol in humans causes an increase in HDLC levels, this may not be beneficial even though this results in an increase in HDL cholesterol levels. Moreover what is the significance of small changes in plasma cholesterol levels when massive and unphysiological amounts of cholesterol are fed to humans? Perhaps it would be more important to investigate the lipoprotein changes that might occur if plasma cholesterol levels were reduced from "normal" levels of 220 mg/dl to 150 mg/dl or less. The effects of cholesterol feeding in animal experiments have been researched more extensively. As the changes that occur depend on the species, I will review alterations that occur with cholesterol l7 feeding in pigs and chickens as these are the two species that I have used in most of my experiments. The lip0protein pattern of the pig is similar to man with approximately 50-60% of the cholesterol in the LDL fraction. Mahley et a1. (53) have extensively studied the effect of cholesterol feeding on cholesterol and lipoprotein changes in the pig. Upon cholesterol feeding the percentage of cholesterol in the LDL or lower density fractions rises from 50% to approximately 80-90%. Cholesterol feeding in pigs, as in man generates both a B-VLDL and HDLC fraction, both containing significant amounts of ARP. An IDL fraction, normally not seen in swine, is also generated containing a large amount of cholesterol. The mechanism behind these changes is still speculative. Ross and Zilversmit (54) hypothesize that B-VLDL and IDL are remnants from cholesterol induced intestinal lipoprotein synthesis. The increase in IDL and B-VLDL then arise from incomplete lipolysis of these intestinal chylomicra. Mahley (50) suggests that HDLC is produced when normal HDL is overloaded with cholesterol. Feeding cholesterol to chickens causes a greater elevation in plasma cholesterol than does feeding the same amount of cholesterol to pigs. A 20% protein, low fat diet with 0.25% cholesterol will elevate plasma cholesterol levels to approximately 225 mg/dl in chickens (55). In the pig feeding a similar diet would cause cholesterol levels to reach only 125 mg/dl. The difference in response may be due to ability of the chicken to absorb up to 80% of dietary cholesterol compared to 40% absorption in the pigs (56). 18 When a 1% cholesterol diet was fed to chickens with 20%, 15% and 10% dietary protein plasma cholesterol levels were 900 mg/dl, 1836 mg/dl, and 2332 mg/dl, respectively (55). The effect of cholesterol feeding on lipoprotein metabolism in chickens has not been as extensively investigated as it has been in pigs. Unlike the pig or human, chicken serum generally separates into only two classes of lip0proteins corresponding to LDL and HDL, when separated by agarose or polyacrylamide gel electrophoresis (57, 58). Similarly, pigeons also have been shown to have only two lipoprotein fractions when separated by electrophoresis (59). However, ultracentrifugation of chicken serum yields three lipoprotein classes; a small amount of VLDL and normal levels of LDL and HDL (60). Dangerfield et a1. (58) have reported that occasionally electrophoretic separation of lip0proteins yields two bands in the LDL area which may correspond to the VLDL and LDL fractions. The major change that occurs with cholesterol feeding is the increase in the LDL lipoprotein fraction. The ratio of HDL/LDL cholesterol was 11.3 in chickens fed high-fat diets without cholesterol but dropped to 2.3 when the same diets with 0.2% cholesterol were fed (Forsythe and Bennink, unpublished observations). Kurski and Narayan (60) found an increase in the VLDL fraction upon cholesterol feeding in chickens. They hypothesized that this increase was due to a decreased degradation of VLDL. 19 PROTEIN Kritchevsky (61) cites the experiments of Ignatowski in 1909 as providing the first evidence that the type of dietary protein fed could influence plasma cholesterol levels. Ignatowski, feeding meat and eggs to rabbits, suggested that the animal protein portion was responsible for the increased plasma cholesterol levels. But because the ingredients he fed also contained cholesterol, his hypothesis that the hypercholesterolemic response was due to protein was not accepted. While there have been a few other reports on the effect of protein source on plasma cholesterol levels, most of these results were considered to be due to other dietary components, such as essential fatty acid deficiency, and not due to the protein source (58, 59). Recent reports by Carroll et al. (64-66) and Kritchevsky et al. (68) have reawakened interest in the effects of protein source on plasma cholesterol levels. Hamilton and Carroll (65) reported that rabbits fed semi-purified low-fat, low-cholesterol diets developed significant hypercholesterolemia when animal protein was fed compared to feeding vegetable protein. Plasma cholesterol levels in rabbits fed animal proteins ranged from 105 mg/dl when raw egg whites were fed to 235 mg/dl when lipid extracted whole egg was the protein source. Feeding casein resulted in plasma cholesterol levels of 200 mg/dl. When vegetable protein was substituted for the animal protein in the same diet plasma cholesterol levels ranged from 25 mg/dl for soy protein to 80 mg/dl for wheat gluten. 20 Supplementation of the diet with choline and/or methionine (animal proteins have a high level of methionine relative to vegetable proteins) produced similar response as without supplementation, although rabbits receiving both the choline and methionine supplement showed slightly higher levels than those not receiving supplements. While these experiments showed that animal protein could raise plasma cholesterol levels relative to vegetable protein, questions were raised concerning the dietary ingredient responsible for this difference. That is, is it the protein component of the diet or an ingredient associated with the protein, such as fiber in vegetable proteins that causes the difference in response? Huff et a1. (66) tested the effect of feeding mixtures of soy protein and casein, feeding casein or soy protein hydrolysate and feeding amino acid mixtures of either casein or soy protein. A 75-25% mixture (casein/soy isolate) reduced plasma cholesterol levels to approximately one-half those occurring when 100% casein was fed. A 50-50% mixture further reduced cholesterol levels to concentrations observed when 100% soy protein was fed. Feeding the enzymatic hydrolysate of either protein source caused a small decrease in plasma levels compared to feeding the original source. Finally, feeding the amino acid mixture of casein did not change cholesterol levels compared to whole casein but the amino acid mixture of soy protein produced cholesterol levels approximately twice that of the intact soy isolate. Still, the levels that occurred when the soy amino acid mixture was fed were about one-half those occurring in the casein fed rabbits. Yadav and Leiner (67) showed that 21 feeding whole protein or amino acid mixtures mmPaEmm uooFm N .pcmswgmaxm msu do vcm mg“ as umcwmpno mew: .mxmmz NF Low mpmwu mcp cm» vcm xP—mwuwcw ox m.o A ¢.m mcwzmwmz warn xwm Low mcmmz — F.o A m.~ n m.~ h m.o H m n m n OF A m h mm umpooa acm._ no.mo a_.¢m mm.m mmm mmm mmo— mmm wag Fm nm~¢.P mm.om amp.o¢ m—.m mum mmmm neep 8mm paw _a mom._ mm.mm m~.¢¢ mm.m mmm new moop mmwm Pogpcou 4o4\4oz 4o: DOA 4o4> a» Fogmpmmpozu Pogmpmmpogu 4o: Payee ox .pcmwmz wp Axv mcwmpogqonwp «swarm Anon chwm p .o A_a\mev ma_aa_ mama_a F.AF acmewgmaxmv mumwu awmpoga use—a umw morn mo mvwawp mammpa new mpcmwmz Xuon go any xgmuopu mo mpumwew .5 open» 4O LDL and HDL and the HDL/LDL ratio were intermediate in pigs fed the plant protein, saturated fat diet. In summary, these results show the typical hypocholesterolemic response to an increased dietary P:S fat ratio which others have also observed (127, 128). A second experiment was designed to test whether protein source could affect changes in plasma cholesterol parameters and to examine possible interactions of protein source and fat source. EXPERIMENT 2 The removal of meat and the addition of dried skim milk to the animal protein diets markedly improved their consumption relative to results obtained in Experiment 1. No effect of dietary treatments on final body weights (Table 5) was observed as all groups of animals gained approximately 0.7 kg daily. Food intake was also similar among treatments. Pigs fed the experimental diets consumed approximately 0.5 kg diet (1 g of cholesterol) per day initially and 2.3 kg diet (4.6 g of cholesterol) per day by the end of the experiment. Cholesterol content of the diet was 0.6 g per 1000 kcal. Since food intake and growth of the pigs were similar among treatments, changes in plasma cholesterol levels of pigs fed the experimental diets do not reflect differences in growth or cholesterol consumption. Bi-weekly plasma total cholesterol values are plotted in Figure 2. Levels of circulating cholesterol tended to increase during the first 4 weeks in pigs fed the experimental diets. 41 .pcaawawcawm uoz .pme cmumgzummczxpoa mzmgm> um; umpcgzpmm use mewmpogq Fmswcm mzmem> cwmpoea u=a_a ”poucmswgmaxm mzmgw> yawn Poeucou was mcomwemaeou m ¢ .cowuumge 4o: cw Fogmummpozu Page» we mmmpcmucmam .POLmHmmPocu umwwwLmumw OH UwTZLGQmmc: we ovumm u MQ\QD N .mxmwz «F pm exact mm: voopm .mx m.o A o.NF xppmwuwcw mcwsmwmz pcmEummLa can mqu unnam— umu pom pom pad .852a .8525 .SOLQ .SOLa .SOLa Amo.ovav .uogm .me mz mz .axu .axm .axm .qu mmz ¢.:mwm N a N.o a F a mo.o H N n m n m n m h N h mm umpoom mN m.N V mN _¢.o om FN— ma amp mm Lam c< ma o.m Nm ~¢.o no pep em mON Nm pmm c< _m 0.0 ow ¢¢.o ma nu mm FFP mm wag Fa mm 8.8 mm mm.o ma moF mm mm_ em pom Fa «m N.“ Nm m¢.o mN Po mm mm _m —ogucoo Lao;\4\z: Lao;\w cow“ mx 1 N 40: cmwwwgmpmm umwewcmpmmc: ~muo» . apat.tapoz aquaLapmu _oaapmapo;u m0\u= Suwwmz pawn sua>_aa< Hwuum p<04 use .mcmumEMLmq Fogmummpozu osmmpa .ucmwwz auoa Pmcwm .m mpnmp Figure 2. 42 Effect of animal or plant protein and varying P:S ratio on bi-weekly plasma total cholesterol levels in pigs (Experiment 2). Pooled SE ranged from 5 to 10 mg cholesterol/d1 plasma for each collection point. Increasing the P:S ratio significantly decreased (p<0.05) plasma cholesterol values at weeks 2 to 14. Plant protein significantly increased plasma cholesterol levels compared with animal protein at weeks 2 and 4 but significantly decreased cholesterol levels at weeks 10, 12 and 14. The experimental diets increased plasma cholesterol levels compared with the control diet at weeks 2, 4, 10, 12, and 14. PLASMA CHOLESTEROL mg/dl 220 180 140 100 180 140 100 SATURATED FAT DIETS H " An Sat " Pl Sat - L Control { T 1 1 l J I 1 1 T 2 4 6 8 10 12 14 POLYUNSATURATED FAT DIETS II Ill An Puf - '1 Pl Puf ban-7 - A L Control 10‘ I: 1 1 1 J 1 1 1 3‘ 2 4 6 8 10 12 14 WEEKS 44 A similar response was noted in the first experiment. At both 2 and 4 weeks of the experiment plasma cholesterol levels were unexpectedly higher in pigs fed the plant protein experimental diets than in pigs fed animal protein. There was a transient decrease in cholesterol levels in both protein groups at 6 and 8 weeks. The reason for the initial elevation in plasma cholesterol levels of pigs fed the plant protein diets and for the subsequent drop in plasma cholesterol levels of all groups at 6 and 8 weeks is not clear. After 10 weeks pigs fed the animal protein diets had higher plasma cholesterol levels than pigs fed plant protein. Values in the animal protein groups remained approximately 50 mg/dl higher than values in the corresponding plant protein group until the end of the experiment. Increasing the dietary P:S ratio from 0.3 to 3.0 lowered plasma cholesterol values in both the animal and plant protein groups by approximately 40 mg/dl (Figure 2). This effect was evident by the second week of the study. As in Experiment 1, pigs that consumed the plant protein, polyunsaturated fat diet had plasma cholesterol levels similar to the control, low-fat, low-cholesterol group; despite consuming approximately 4.5 g of cholesterol per day. Blood lipid values from the final collection period (14 weeks) are presented in Table 5. Pigs fed the experimental diets had higher plasma cholesterol (total, unesterified, esterified and HDL-cholesterol) levels than did pigs fed the low-fat, low-cholesterol. control diet. Plasma total, unesterified and esterified cholesterol 45 levels were significantly higher in pigs fed the animal protein diets than in pigs fed the plant protein diets; likewise, values were higher in pigs fed the saturated fat diets than in pigs fed the polyunsaturated fat diets. Thus, the highest total cholesterol levels were in the animal protein, saturated fat group (205 mg/dl) and the lowest values in the plant protein, polyunsaturated fat group (111 mg/dl). The ratio of plasma unesterified to esterified cholesterol was not significantly affected by dietary protein or fat. Plasma HDL cholesterol (Table 5) was elevated significantly in pigs fed animal protein; this was a reflection of total cholesterol levels as neither the type of protein nor fat fed caused a difference in the percentage of total cholesterol in the HDL fraction. As in Experiment 1, plasma triglyceride levels were unaffected by dietary treatments. Lecithin:cholesterol acyl transferase esterifies unesterified cholesterol in plasma which presumably facilitates transport of cholesterol from tissue pools to the liver (129). The fractional esterification rate, which reflects the percentage of cholesterol esterified per hour, was negatively correlated (r = -O.75) with free cholesterol levels (Table 5). This observation is in agreement with previously reported results (130) and may explain part of the decrease in fractional esterification rate that occurred when animal protein was fed; these animals had higher unesterified cholesterol levels and thus, on a percantage basis, esterified less cholesterol than animals with lower cholesterol levels. However, the molar LCAT rate, which is an absolute rate that takes into account the level of unesterified cholesterol, was also significantly 46 reduced in pigs fed the animal protein diets and in the pigs fed polyunsaturated fat diets. Thus, the decrease in the fractional LCAT rate in pigs fed animal protein is greater than that accounted for by an increase in unesterified cholesterol levels. The effect of polyunsaturated fat on molar LCAT activity in our experiment agrees with other reports (131, 132) but to our knowledge this is the first report that dietary protein can affect LCAT activity. The distribution of plasma lip0proteins (Table 6) was unaffected by dietary treatment. The plasma HDL/LDL ratio was significantly lower in pigs fed the animal protein diets than in pigs fed plant protein. As in the first experiment, increasing the P:S ratio tended to elevate the HDL/LDL ratio. Kritchevsky (112) has postulated that the amount of arginine and lysine in diets may influence plasma cholesterol levels. The plant protein diets fed contained 1.10% arginine and 0.85% lysine (calculated values) while the animal protein diets contained 0.63% arginine and 1.36% lysine. The amino acid content of the control diet was similar to that of the plant protein diets. The ratio of arginine to lysine in the plant protein diets was almost three times that in the animal protein diets (1.29 to 0.46, respectively). Although plasma lysine levels were significantly higher in pigs fed the animal protein, no differences were found in plasma arginine levels or in the ratio of arginine to lysine between treatments. Plasma levels of branched-chain amino acids, leucine, isoleucine and valine, and of threonine were higher in pigs fed experimental diets than in pigs fed the control diet (Table 7). Feeding pigs animal protein also resulted in higher 47 Table 6. Effect of dietary protein and fat sources on plasma lipoprotein distributions (Experiment 2). Percentage Diet VLDL LDL HDL HDL/LDL Control 17.3 39.4 44.3 1.13 Pl Sat 11.6 40.1 49.3 1.26 P1 Puf 12.1 38.1 49.8 1.37 An Sat 12.2 42.5 45.3 1.11 An Puf 11.7 42.4 46.9 1.15 Pooled SE 0.3 1.0 0.9 0.06 Sign.2 NS3 NS NS Prot. (p<0.05) 1Eight pigs per treatment. Blood drawn at 14 weeks of the experiment. 2See Table 5 for explanation of statistical comparisons. 3Not significant. .pcauaewcmwm poz m .mcomwgmqeou quwpmvpmpm mo :owpmcmpaxm co» m mpamh mmmN .pcmspmmgp awn mqu Asmwmp pm; .poea .Aoga .Aoea “an .Aoea .pogm Amo.ovav .axm .nxm .axm .axm mmz N.:mmm w A m A «N A o A N A m A m A mm ompooa opN mFP owe map mo_ om Fm— Nam c< mON NmF mmm mN_ mop Nm Nmp Amm c< mm? mo— «Fm Nm— cmF mm o__ Nam Fa Nmp mm PoN om oa— Nu moF “mm P; cap AN NMN mo~ NPF om omp Fogpcou mcwusz mcwuampomH mcppm> mcwgm mcvcomgck wcwcwmg< mcwme Amwo Are\mm_oscv mku< ocwe< mammpa F.AN Acmswgmaxmv mmugaom paw tam :wmpoga pcmcmmmwc wok mmwa :_ mpm>mp upon ocw5m mammpa .N mpnm» 49 levels in the plasma of threonine and the branched chain amino acids, leucine, isoleucine and valine. Pigs fed the polyunsaturated fats had higher levels of serine and valine than pigs fed the saturated fat diets. Feeding the high-fat, high-cholesterol experimental diets increased liver cholesterol and total lipid and cholesterol in aortas of pigs (Table 8). Neither of the experimental treatments, fat or protein, caused any change in liver or aorta lipid content. Both the liver and aorta had slightly higher (p<0.1) cholesterol contents in animals fed the polyunsaturated fat diets than pigs fed the saturated fat diets. This would tend to support the hypothesis that one mechanism by which polyunsaturated fats lower plasma cholesterol levels is by redistribution to the tissue pool (133). Neither protein treatment affected the amount of cholesterol in either tissue. Longer term feeding experiments than those employed in the present study may be necessary to produce changes in tissue lipid. DISCUSSION Increasing the dietary P:S ratio from 0.3 to 3.0 decreased plasma cholesterol concentrations of the pigs in two different experiments. In the first experiment the decrease was 42 mg/dl and in the second experiment levels decreased by 40 mg/dl. Polyunsaturated fats decrease plasma cholesterol levels in humans as well (127, 128, 134) and in pigs, Greer et al. (135) reported that plasma cholesterol levels decreased by 25 mg/dl when 50 .pcaaatwcmwm “oz m .mcomwgmasou quwpmwpmum No cowumcmpnxm to; m mpnm» mmmN .AcmEummLu emu mmwa Acmwm_ Amo.ovav .axm .axm .axm mz .cmwm m N P.o ¢.o P.o N mm umFooa NN.¢ mN ¢¢.¢ um wag c< mm.¢ PN N¢.¢ we pom c< mw.¢ MN mN.¢ me man Fa AN.¢ NN NN.m Na pom Pa mo.¢ mp No.m Pa Pogpcoo Focwpmmpoco mcwawd Fmpoh Fogmpmmpogu muwan Papa» Azmwmz ace m\me .mpco< uzmwmz um; m\me .gm>w4 _.AN Acme_cmaxmv cowpwmoasou v_qw_ mpeom was cm>w~ co mmugzom umN ncm :wmuogq xgmpmwu mo pummwm .m mFDAH 51 soybean oil replaced tallow as the fat source in high-fat, cholesterol-containing diets similar to the diets in our experiments. Feeding the pigs plant protein decreased plasma cholesterol levels by 50 mg/dl compared with feeding animal protein. Kim et a1. (71) reported that in pigs fed high-fat diets containing cholesterol, replacement of casein with soy isolate reduced plasma cholesterol levels by 107 mg/dl. One reason for the greater treatment response in the experiment of Kim et a1. (71) than observed in our experiments may have been the sources of plant and animal proteins. Whereas we fed a mixture of plant proteins (soy, 50%; wheat, 25%; and corn, 25%) and milk proteins (casein, 90% and lactalbumin, 10%), Kim et a1. (71) fed either 100% casein or 100% soy protein. At least in rabbits, wheat protein was found to be less hypocholesterolemic than soy protein (111). A second possibility may relate to growth responses of the pigs. In their experiment, pigs fed casein gained considerably less weight than those fed soy isolate (71) whereas treatment did not affect weight gain of our pigs. The lower weight gain of pigs fed casein and consequently increased intake of cholesterol per kg body weight may have contributed to the larger response of plasma cholesterol levels to protein source seen in their study (107 mg/dl) compared with that seen in our study (50 mg/dl). Carroll et al. (113) reported that plasma cholesterol levels of humans decreased by 16 mg/dl when soy protein replaced a mixture of animal proteins (meat and casein) in high-fat diets. Sirtori et al. (73) also reported that consumption of a mixture of plant proteins (63% soy protein) in diets containing 25% of energy as fat reduced 52 plasma cholesterol levels an average of 50 mg/dl in hypercholesterolemic patients compared to feeding a mixture of plant and animal proteins. When a mixture of soy protein and casein (half and half) was fed to pigs plasma cholesterol levels were intermediate, but not equal distant, between levels observed when soy and casein were fed separately (71); levels were closer to those observed when soy was fed alone than when casein was fed as the sole source of protein. Although consumption of plant proteins, as a group. generally produce lower plasma cholesterol levels than does consumption of animal proteins, it is evident that not all plant proteins are equally effective in this regard (lll). Additionally it appears that responses of plasma cholesterol levels to mixtures of plant and animal proteins may not be directly proportional to levels of the two protein sources in the diet. We observed no interaction between dietary fat source and protein source on plasma cholesterol levels in pigs. Others have suggested that the hypocholesterolemic effect of soy is reduced when fed with saturated fat (72, 73). Feeding diets with low P:S fat ratios did not reduce the effectiveness of plant protein in lowering plasma cholesterol levels in pigs. Plasma cholesterol levels decreased by 50 mg/dl in pigs fed the polyunsaturated fat diets when plant protein replaced animal protein. Plant protein was equally effective when pigs were fed saturated fat diets; plasma cholesterol levels were also 50 mg/dl lower in pigs fed plant protein than in those fed animal protein. Carroll and Hamilton (111) fed casein or soy isolate protein diets with either 15% butter or 53 15% corn oil to rabbits. In rabbits receiving 15% butter, consumption of soy protein resulted in plasma cholesterol levels approximately 75 mg/dl lower than when casein was consumed (estimated from Figure 3 of reference 111). Similarly in rabbits fed corn oil, switching from casein to soy isolate caused an approximate 60 mg/dl dr0p in plasma cholesterol levels. Thus their results show, like our results, that there is little interaction between dietary fat source and protein source in the regulation of plasma cholesterol levels. Shorey and Davis (72), in an experiment with mildly hypercholesterolemic young men, concluded that saturated fat negated the hypocholesterolemic effect of soy protein. They, however, did not compare protein sources in diets high in polyunsaturated fat. Thus, it is possible that soy protein may not have exhibited a hypocholesterolemic effect in their subjects under these latter conditions, either. Sirtori et a1. (73) also suggested a decreased effectiveness in the ability of soy protein to lower plasma cholesterol levels in hypercholesterolemic humans when soy protein was fed with saturated fats. Their results clearly show the expected hypocholesterolemic action of polyunsaturated fat, but they did not make direct comparisons between animal and plant proteins. Until direct comparisons of protein and fat sources are made in the same experiment, it is not possible to state unequivocally that saturated fat negates the hypocholesterolemic effect of soy protein in humans. Since both fat and protein can affect plasma cholesterol levels, conditions could exist where their effects on plasma cholesterol offset each other, however, this was not observed in our study with pigs. 54 The mechanism by which dietary protein sources alter plasma cholesterol values is unclear. Plasma LCAT activity has been suggested to be involved in the turnover and clearance of plasma cholesterol (130). Effects of dietary fat and protein source on LCAT activity were observed in our study. When polyunsaturated fats were fed to pigs, both molar LCAT activity and plasma cholesterol levels were decreased compared to when saturated fats were fed. Feeding animal protein, compared to plant protein, also decreased plasma molar LCAT activity but increased plasma cholesterol levels. Decreases in molar LCAT activity by polyunsaturated fats have been observed in humans (131, 132) and rats (136). Larking and Sutherland (136) reported decreased levels of unesterified cholesterol when polyunsaturated fats were fed to rats. They suggested that decreased unesterified cholesterol levels were responsible for decreased molar LCAT activity. This is possible since the method used to assay molar LCAT activity depends on endogenous substrate availability, and does not differentiate between changes in enzyme activity and substrate availability (129, 130). Changes in molar LCAT activity in humans positively correlates with changes in unesterified cholesterol, triglyceride, and phospholipid blood levels (35). Feeding polyunsaturated fat diets decreased plasma unesterified cholesterol levels in pigs but did not alter plasma triglyceride levels. Thus in this experiment, the effect of feeding polyunsaturated fats on molar LCAT activity and unesterified cholesterol levels are consistent 55 with decreases observed in other experiments in which polyunsaturated fats have been fed (129, 130, 134). The reason plasma molar LCAT activity decreased when animal protein was fed is also unclear. Animal protein caused an increase in unesterified cholesterol levels compared to plant protein. Based on results obtained when polyunsaturated fats were fed (low LCAT activity and low plasma unesterified cholesterol levels), one would expect that pigs fed animal protein to exhibit higher molar LCAT rates than pigs fed plant protein. Since the opposite effect occurred (low LCAT activity and high unesterified cholesterol levels) it is apparent that LCAT activity is influenced by additional factors besides the concentration of unesterified cholesterol. Feeding animal protein reduced the ratio of HDL to LDL compared to values in pigs fed plant protein. While the absolute level of HDL cholesterol in the plasma was higher in the animal protein fed pigs the percentage of total cholesterol was increased in the animal protein fed pigs. Wallentin (107, 130) has reported little correlation between HDL lipid changes and molar LCAT activity so that these changes in the pig may not be responsible for the decreased LCAT activity. Possibly feeding animal protein altered the metabolism of apoprotein A,an activator of LCAT (129), but this was not investigated in this experiment. Finally, it must be emphasized that while the differences in LCAT activity between treatments is statistically significant, the physiological significance, particularly since there was no difference in the percentage of esterified cholesterol, is unknown. 56 One other mechanism proposed to explain the varying effect of dietary protein source on plasma cholesterol levels is the amino aicd composition of the proteins (66, 67). Kritchevsky has suggested that the dietary ratio of arginine to lysine, high in plant proteins but low in animal proteins is important in the development of atherosclerosis (112). He hypothesizes that high lysine levels inhibit hepatic arginase activity, perhaps resulting in greater production of arginine-rich lip0proteins (112). Kritchevsky et a1. (69) reported an increased incidence of atherosclerosis in rabbits when lysine was added to soy protein diets. But the additional lysine did not significantly increase plasma cholesterol levels in their study compared to groups fed soy protein diets without lysine. Our animal protein diets contained more lysine than the plant protein diets. While this increase was reflected in plasma lysine levels in pigs fed animal protein, there was no dietary effect on plasma arginine levels or on the ratio of arginine to lysine in plasma. The lower methionine content of soy protein, relative to animal proteins, has also been hypothesized to be a factor in the hypocholesterolemic action of soy protein (65, 137). Kim et al. (111) reported no effect of supplementation of soy diets with methionine in pigs on plasma cholesterol levels compared to pigs fed soy protein alone. No difference in plasma methionine levels were observed in our pigs with any of the treatments. It must be noted that the amino acids were determined from samples obtained from the peripheral circulation. These samples may not reflect the flux 57 of amino acids from the intestine to the liver. This initial liver influx of amino acids into the liver could influence plasma cholesterol metabolism. In conclusion, our studies clearly show that feeding plant protein, compared to animal protein, reduces plasma cholesterol levels when fed in conjunction with high—fat, high-cholesterol diets. Furthermore, we observed no interaction between the type of dietary fat fed and the type of dietary protein fed. The hypocholesterolemic effect of plant protein was significant even though the protein source was mixed (soy, wheat and corn) so that soy isolate provided only 50% of the diet protein. This study while confirming previous reports that polyunsaturated fat lowers plasma molar LCAT activity and offers the first evidence that molar LCAT activity is reduced in the plasma of pigs fed animal protein. PART III EFFECT OF FEEDING ROLLED OATS OR WHEAT BRAN ON PLASMA LIPIDS, LIPOPROTEINS, LCAT AND TISSUE LIPIDS IN PIGS AND CHICKENS INTRODUCTION Burkitt (74) and Trowell (75) have hypothesized that the lower incidence of coronary heart disease (CHD) in developing countries, compared to Western nations, is due to the higher intake of dietary fiber in the developing countries. They also suggest that the high fiber intake in developing countries contributes to the lower plasma lipid levels compared to the developed countries. Experimental studies in both animals and humans have shown that individual fiber types uniquely affect plasma cholesterol levels. Wheat bran has been shown to be ineffective in lowering plasma cholesterol levels in many studies (65, 68, 138-140), while other fibers, such as pectin or rolled oats, have been shown to lower plasma total cholesterol levels (86, 89). In the present experiments wheat bran or rolled oats were chosen, because of their seemingly different effects on plasma cholesterol levels, to be fed at levels of 6.5% neutral detergent fiber (NDF) to either pigs or chickens. Pigs were chosen as an experimental model because they metabolize cholesterol in a manner similar to man, have similar lipoprotein characteristics as man (53) and have been shown to develop atherosclerotic lesions similar to man (141). Since pigs generally exhibit low levels of plasma cholesterol the diets were also fed to chickens. Plasma cholesterol concentrations increase to much higher levels in chickens than pigs when fed the same diet (55). 58 59 While the major objective of these experiments was to investigate the effect wheat bran or rolled oats have on plasma cholesterol levels, other parameters, such as body composition, plasma HDL cholesterol and plasma lecithinzcholesterol acyl transferase (LCAT) activity were also investigated. MATERIALS AND METHODS ANIMALS Two experiments were undertaken to investigate the effects feeding wheat bran or rolled oats have on plasma cholesterol and lipoprotein parameters and tissue lipid deposition. In Experiment 1 crossbred (Yorkshire x Duroc or Yorkshire x Hampshire pigs) castrated male pigs were assigned at seven weeks of age to the dietary treatments. Ten pigs in each treatment were housed together in an environmentally controlled building with slatted floors. They had free access to diet and water. Pigs were weighed and blood samples obtained by vena cava puncture at weeks 4, 7, 10, and 13, after a 24 hour fast. At slaughter (17 weeks after the start of dietary feeding) aorta, liver, heart, and hams were obtained and stored for subsequent analysis. In the second experiment broiler strain roosters were assigned to the same dietary treatments as in Experiment 1. Prior to dietary treatments, the chickens were fed a low-fat, low-cholesterol semi-purified diet for 8 weeks. Average weight of the chickens at the beginning of the experiment was 2.9 i 0.1 kg. As in the first experiment the chickens had free access to food and water. 60 Blood samples were taken via cardiac puncture at 3 and 5 weeks of the experiment after an overnight fast. DIETS Composition of the diets fed in both experiments are shown in Table 1. Both fiber diets were formulated to contain 6.5% neutral detergent fiber (NDF) while the control diet contained less than 1% NDF. Neutral detergent fiber was determined as described by Goering and Van Soest (115). The diets were plant protein based, and high fat, with a P:S ratio of 0.3. Crystalline cholesterol was added (0.05%) to each diet. Dietary fatty acids were extracted in chloroformzmethanol (2:1, v/v). The non-saponifiable lipids were extracted as described in the section on hepatic sterol synthesis. Fatty acids were extracted with petroleum ether after acidifying with 3N HCl. After solvent evaporation the fatty acids were converted to their methyl esters by adding excess etheryl diazo-methane. The fatty acids were determined by gas-liquid chromatography (GLC) equipped with a flame ionization detector and an electronic integrator. GLC conditions were: injector temperature, 135°C; detector temperature, 175°C; column temperature was initially held at 130°C for 5 minutes after injection and programmed from 130-160°C at 2°/min. Temperature was held at 160°C until the last methyl ester was eluted; N2 carrier gas flow 30 ml/min; H2 flow, 15 ml/min; and air flow, 240 ml/min. Fatty acids were separated on a 1 meter long, 2 mm ID glass Supelco column packed with 61 Table 1. Composition of diets, %. Diet Ingredients Control Wheat Bran Rolled Oats Wheat bran1 - 16.0 -— Rolled oats2 - -— 50.0 Soybean meal 37.0 32.0 22.0 Corn starch 39.6 30.1 7.1 Mineral and vitamin supplement 1.3 1.3 1.3 Tallow 18.0 17.0 16.0 Corn oil 1.0 1.0 1.0 Cholesterol 0.05 0.05 0.05 1Standard wheat bran from American Association of Cereal Chemists (AACC), St. Paul, MN. 2Rolled oats from Quaker Oats, Chicago, IL. 3Supplying the following per kilogram of diet: 3300 IU; vitamin D, 660 IU; 11 IU of vitamin E; menadione sodium bisulfite, 2.2 mg; riboflavin, 3.3 mg; niacin, 17.6 mg d-pantothenic acid, 13.2 mg; choline, 110 mg; vitamin 812, 19.8 pg; zinc, 75 mg; iron, 60 mg; manganese, 37 mg; copper, 10 mg; iodine, 2.8 mg, and selenium, 0.1 mg. vitamin A, 62 10% SP 2340 coated on chromosorb WAW (100/120 mesh). Fatty acids were identified based on retention times of standard fatty methyl esters. Values are expressed as percentage of fatty acid, by weight, of total fatty acids in the diet (Table 2). Dietary cholesterol and plant sterol concentrations were determined by gas-liquid chromatography. Cholesterol composition of the diets was (mg/100 g diet): control, 40; wheat bran, 39; and rolled oats, 65. Plants sterol content was (mg/100 g diet): 2.3; wheat bran, 4.8; and rolled oats. 5.1. PLASMA AND TISSUE ANALYSES Plasma total and unesterified cholesterol were determined 1 Esterified cholesterol was calculated as the enzymatically. difference between total and unesterified cholesterol values. Plasma high density lipoprotein (HDL) cholesterol was also determined enzymatically after the low density (LDL) and very low density (VLDL) lipoproteins were precipitated with heparin-manganese solution (120). Plasma 1ecithin:cholesterol acyl transferase (LCAT, EC 2.3.1.43) activity was assayed by the method of Stokke and Norum (123) with modifications suggested by Wallentin and Vilkrot (124). Plasma lip0proteins were separated by electrophoresis on polyacrylamide gel,2 quantitated by densitometry at 610 nm, and percentages of VLDL, LDL 1Cholesterol reagent set, BMC, Indianapolis, IN 46250. 2Redi-Disc. Ames Company, Elkhart, IN 46514. 63 Table 2. Fatty acid composition of diets, %.1 Diet Fatty acid Control Wheat Bran Rolled Oats C14 3.13 2.15 3.06 C15 0.87 0.34 0.91 Cl6:0 19.56 18.19 18.66 C16:1 4.54 6.65 5.20 C17 1.54 0.26 1.78 C18:0 14.07 12.13 13.06 C18zl 40.76 36.24 35.43 Cl8:2 9 19 17.96 16 53 C18z3 l 12 1.46 l 76 C20 (or greater) 0 28 0.18 l 40 1Percent of total fatty acids, based on weight. 64 and HDL fractions were calculated (121). Plasma triglycerides were extracted in isopropanolzheptane (5.6:3.0, v/v) and determined gravimetrically. Hams were separated from the carcasses and trimmed according to commercial practices; the meat separated from bone and connective tissue, ground to pass a 5 mm screen, and vacuum dried at 60°C. The extracted lipid (chloroformzmethanol, 2:1) was used to determine total tissue fat (gravimetrically) and tissue cholesterol (colorimetrically)(125). HEPATIC STEROL SYNTHESIS In vivo liver sterol synthesis was determined in fed chickens in the 5th week of the experiment. One ml of saline containing 7.7 millicuries of tritiated water was injected into the heart of each chicken. Fifteen minutes after injection the chickens were killed by cervical dislocation and livers were removed quickly and frozen in a dry ice-acetone bath. Duplicate 3 gram samples were homogenized in 3 ml of distilled water. From each sample two 0.5 ml aliquots were added to 3 m1 of 30% KOH and brought up to 10 ml with 95% ethanol and heated in a water bath at 70°C for 3 hours. After cooling, 10 ml of distilled water was added and the nonsaponifiable lipids were extracted 3 times with petroleum ether. The pooled extracts were backwashed with distilled water to remove any tritiated water. Any traces of remaining water were removed with anhydrous sodium sulfate. After the petroleum ether was evaporated, 10 m1 of scintillation cocktail (4 g 2,5-diphenyl-oxazole and 200 mg 1-4 bis (2-(4-methyl-5-phenylxazdyl))-benzene in 667 ml toluene 65 and 333 ml of triton-X 100) was added and the samples were counted in a liquid scintillation counter. Fifteen minutes after injection of tritiated water a blood sample was taken to determine the specific activity of body water. This was used to calculate the moles of 3H20 incorporated into nonsaponifiable lipids per gram wet tissue per minute. Total body water was estimated by tritiated water dilution from the Specific activity of plasma at 50 min. STATISTICAL ANALYSES The values are reported as mean i standard error of mean (SEM). In both experiments data were analyzed by analysis of variance with means compared by Tukey's procedure (126). RESULTS Neither the final body weights nor the carcass weights were affected by feeding dietary fiber (Table 3). Average daily feed consumption, computed for the entire experimental period, was 1.88. 2.02 and 1.88 kg food consumed/day for the control, wheat bran and rolled oats diets, respectively. Ham composition was used to estimate carcass composition since ham composition is highly correlated with overall body composition (141). The percentage of fat in hams of pigs fed rolled oats was significantly reduced in comparison to either the control or wheat bran fed pigs. And the percentage fat in the heart and thoracic aorta tended to be lower in pigs receiving the rolled oats diet than in pigs fed 66 Table 3. Dietary fiber effects on body, heart and aorta composition in pigs. Diets Control Wheat Bran Rolled Oats Final weight, kg 121 s 21°2 118 s 3a 122 5 3° Carcass weight, kg 91 s 3° 90 s 4° 89 s 3° 2 Fat, ham3 48.2 5 2° 46.1 s 2° 41.7 5 2b Heart3 % fat 36.3 s 2° 37.7 e 2° 35.6 5 3a total cholesterol, a mg/g dry matter 15.2 a o.3° 15.5 s 1.1 15.8 s 1.3°' Aorta3 % fat 28.1 s 2° 33.2 5 6° 26.3 3 1° total cholesterol, a a mg/g dry matter 3.4 e 0.1 3.6 s 0.1 3.3 e o.1° 1Mean i SEM. Ten pigs per treatment. Blood drawn at 13th week of experiment. 2Values with different superscripts are different (p<0.05). 3Expressed as percent of dry matter. 67 either of the other diets. There was no effect of dietary fiber on the cholesterol content of either the heart or thoracic aorta. Feeding dietary fiber did not cause any changes in plasma total or unesterified cholesterol levels or in the ratio of unesterified cholesterol to esterified cholesterol (Table 4). While ‘ the values reported in Table 4 are for the 13 week of the experiment, similar results were observed at the 7th and 10th weeks. Plasma triglyceride levels were unaffected by dietary fiber. Feeding wheat bran or rolled oats caused an increase in HDL cholesterol levels compared to the control diet. Since there was no difference in total cholesterol, the percentage of cholesterol in the HDL fraction was also significantly greater in pigs fed wheat bran or rolled oats. Plasma fractional LCAT rate, which estimates the fraction of unesterified cholesterol converted to cholesterol esters, was not significantly affected by dietary treatment. Fractional LCAT activity is highly correlated with unesterified cholesterol levels (130). Since there was no difference in unesterified cholesterol levels between dietary treatments it was not surprising that the fractional rate~wasalso similar among treatments. Molar LCAT rate, an absolute rate which takes into account the levels of unesterified cholesterol, was affected by diet. Feeding rolled oats increased molar LCAT activity compared to feeding either the control or wheat bran diets. Feeding wheat bran caused a significant increase in the HDL fraction and a significant decrease in the LDL fraction (Figure 1). 68 Table 4. Effect of dietary fiber on plasma cholesterol and triglyceride levels and LCAT activity in pigs. Control Wheat Bran Rolled Oats Plasma cholesterol, mg/dl total 134 3 52°3 132 5 6° 133 5 3° unesterified 26 s 2° 28 1 2° 30 s 2° HDL 42 3 3° 58 s 3° 55 3 4° 00/054 0.19 s 0.01° 0.21 s 0.01° 0.22 s 0.01° HDL cholesterol, %5 31.3 3 1a 43.9 s 3° 41.2 s 2b Triglycerides, mg/dl 54 s 3° 46 s 5a 42 s 4a Plasma LCAT activity fractional, %CE/ a hour 4.7 s 0.4 4.4 s 0.4° 5.3 s 0.6° molar uM/L/hour 31.4 s 2.7a 29.4 s 1.6a 41.8 s 6.8b 1Ten pigs per treatment. Blood drawn at 13th week of the experiment. Mean i SEM. Values with different superscripts are different (p<0.05). UC/CE = unesterified cholesterol/esterified cholesterol. 5Percentage of total cholesterol in the HDL fraction. 6Percentage of tritiated cholesterol converted to cholesterol ester/hour. 2 3 4 Figure l. 69 Effects of dietary fiber on plasma lipoprotein separations. Representative samples separated by polyacrylamide gel electrophoresis. CN = control; WB = wheat bran; and DB = rolled oats diets. HDLzLDL ratios are: CN, 1.27 i 0.1; WB, 1.52 i 0.1; and 0B, 1.27 i 0.1. I... 110 .8111 110 1011—‘-—-- — Till—- m 71 This same pattern was observed at the 7th and 10th weeks of the experiment as well. The ratio of HDL/LDL was also significantly greater in plasma of pigs fed wheat bran than in pigs fed either the control or rolled oats diets (1.52 and 1.27, Table 5). A second experiment was undertaken to test the effect of feeding dietary fiber on plasma cholesterol levels in chickens. Usually plasma cholesterol levels are mich higher in chickens than in pigs if they are both fed a high fat diet which contains cholesterol. Other plasma cholesterol and lipoprotein parameters were investigated to determine if they were altered in chickens in the same manner as they were in pigs. As occurred in the pig study, the experimental diets had no affect on the final body weights of the chickens (Table 6). Chickens fed the rolled oats diet had a higher percentage of body water than chickens fed the control diet. Since body water and fat are inversely related, Experiment 2 confirms the results in Experiment 1; namely feeding rolled oats tends to decrease body fat compared to feeding the control, fiber-free diet. Feeding wheat bran had no effect on body composition. In this experiment feeding dietary fiber to chickens did alter plasma total cholesterol levels (Table 6). Chickens fed the rolled oats diet had an 18% decrease in plasma total cholesterol levels compared to chickens fed the control diet (220 mg/dl vs. 266 mg/dl, respectively). Feeding wheat bran resulted in intermediate cholesterol levels (246 mg/dl) in comparison to chickens fed control and rolled oats diets. There was no significant effect of feeding 72 Table 5. Plasma lipoproteins in pigs as affected by dietary fiber. Diets Control Wheat Bran Rolled Oats Lipoproteins, %1 VLDL 16 3 1° 14 s 0.3° 16 s 1° LDL . 38 s 2° 35 5 1° 38 5 2° HDL 46 5 2a 52 s 1° 47 s 2° HDL/LDL 1.27 s 0.1° 1.52 s 0.1° 1.27 s 0.1° 1Relative percentage of total lipoprotein. 2Mean 2 SEM. Ten pigs per treatment. Blood drawn at 13th week of experiment. 3Values with different superscripts are different (p<0.05). 73 Table 6. Effect of dietary fiber on final body weight, body water, plasma cholesterol parameters and plasma LCAT activity in chickens. Diet Control Wheat Bran Rolled Oats Final body weight, kg 4.0 : 0.2°3 4.1 s 0.3° 4.0 s 0.2° Body water. % 49.9 e 2.8° 53.5 s 2.3°° 62.1 s 4.9° Plasma cholesterol, mg/dl total 266 s 8° 246 s 15°° 220 s 15° unesterified 83 5 3° 75 5 4° 69 s 5° HDL 104 3 1° 103 s 2° 98 5 5° HDL cholesterol, %5 39 3 1° 43 3 3°° 46 s 4° 00/054 0.46 s 0.02° 0.44 s 0 02° 0.47 s 0.02° Liver sterol synthesis pmoles 3H20/g/min6 7.9 i 1a 8.1 i 2a 10.4 i la Plasma LCAT activity fractional rate, b % CE/hour7 4.7 s 0.6° 4.7 s 0.6° 7.6 s 0.9 molar rate, a b uM/L/hour 106 s 6 95 : 8° 143 s 19 1Eight chickens per treatment with an initial weight of 2.9 i 0.1 kg. Blood was drawn after 5 weeks of dietary treatment. Mean i SEM. Values with different superscripts are different (p<0.05). UC/CE = unesterified cholesterol/esterified cholesterol. Percentage of total cholesterol in the HDL fraction. Micromoles of 3H20 incorporated into unsaponifiable lipids/g of liver/min. 7Percent of tritiated cholesterol converted to cholesterol ester/ hour. 0501-500“) 74 Table 7. Effect of dietary fiber on plasma lipoprotein percentages in chickens. Diets Control Wheat Bran Rolled Oats Lipoproteins, %2 LDL, 1 37 s 43’4° 30 s 5°° 21 s 4° LDL, 2 10 (1)5 14 (3) 21 (4) HDL 62 5 4° 65 5 4° 69 s 3° HDL/LDL 2.0 s 0.4° 3.2 s 0.9° 4.1 s 0.6° 1Eight chickens per group. Blood was drawn at 5 weeks of experiment. Relative percent of each lipoprotein. Mean t SEM. Values with different superscripts are different (p<0.05). (II-DOOM Number in parentheses indicate number of chickens with 2 bands in the LDL fraction. The value is the average for the number showing a fraction. 75 dietary fiber on either unesterified cholesterol levels or the ratio of unesterified to esterified cholesterol. The percentage of cholesterol carried by the HDL fraction tended to be higher in both groups of chickens fed fiber. However, only the chickens receiving rolled oats had HDL cholesterol values which were significantly different from the control values. In the first experiment with pigs, both fibers caused an increased percentage of cholesterol in the HDL fraction. Liver sterol synthesis was unaffected by dietary fiber treatment. Plasma LCAT activity, both the fractional rate and molar rate were higher in chickens fed the rolled oats diet than in chickens fed either of the other diet treatments. In Experiment 1, feeding the rolled oats diet to pigs caused an increase in the molar, but not the fractional, LCAT rate. The effect of dietary fiber on electrOphoretic lipoprotein separations in chickens is shown in Table 7. The relative percentage of LDL was reduced in the Rolled oats diet compared to the control diet. Furthermore feeding fiber increased the number of bands observed in the LDL region compared to the control diet. Dangerfield et a1. (58) have previously shown that occassionally electrophoretic separations do produce two bands in the LDL region although the significance of these bands is unknown. 76 DISCUSSION The effect of dietary fiber on body composition has not been extensively investigated. In both experiments feeding rolled oats reduced body fat compared to feeding either the control or wheat bran diets. In pigs fed rolled oats the fat percentage in hams was 41.7% as compared to 46.1% or 48.2% fat in the hams of pig fed wheat bran or control diets, respectively. The fat content of the aorta and heart also tended to be lower in pigs fed rolled oats. In chickens, feeding rolled oats resulted in an increase in body water (62%) compared to either the control (50%) or wheat bran diet (53.3%). Since body water and fat are inversely related, chickens fed rolled oats would have the lowest percentage of body fat. Tsai et al. (143) reported that carragheenan and gum arabic reduced the relative dry body weight, which indicates an increase in body water and a relative decrease in body fat, compared to either a control (no fiber) or wheat bran diet. Sundaravalli et al. (144) reported that cellulose fed to rats did not affect body composition. Forsythe and Bennink (unpublished observations) have observed that liver total lipids were reduced when oat bran was fed to rats compared to a control diet (low fiber diet). The mechanism by which rolled oats lowers body fat is not clear. Southgate and Durin (145) reported that fecal lipids were increased when fiber was fed but whether this is a factor was not investigated in these experiments. 77 Wheat bran did not affect plasma total cholesterol levels in either pigs or chickens. Many other reports have shown that wheat bran does not decrease plasma cholesterol levels in either humans (138-140) or animals (65, 68, 143). In the pig, feeding wheat bran did increase plasma HDL cholesterol levels by 16 mg/dl over the control levels. The percentage of total cholesterol in the HDL fraction was increased from 31% in the pigs fed the control diet to 44% in the pigs fed the wheat bran diet. McDougall et al. (84) reported that feeding 50 g of wheat bran daily for 6 months, while not affecting total cholesterol levels, did increase HDL cholesterol levels compared to pre-treatment levels. Miettinen (146) also reported, again without any change in total cholesterol levels, that feeding wheat bran to humans resulted in a 36% increase in HDL cholesterol levels. In the second experiment, in chickens, wheat bran did not significantly increase the HDL cholesterol levels. In Experiment 1, in pigs, wheat bran also increased the percentage of HDL by 15% and decreased the percentage of LDL by 10% compared to the other two diets; and the ratio of HDL/LDL was 20% greater in the wheat bran fed pigs than in either the control or rolled oats fed pigs. Hill et al. (147) has shown in pigs that the relative percentages of lip0proteins obtained by electrophoretic separations are similar to the percentages of lip0proteins obtained with ultracentrifugation and Sclieren optics. In Experiment 2, in chickens, the HDL/LDL ratio tended to be increased in the wheat bran diets compared to the control diet (3.2 to 2.0, respectively) but this was not a significant increase (p<0.05). Brodribb et a1. 78 (148) reported feeding 24 g/day of wheat bran to free living humans for 6 months caused a 28% decrease in LDL fraction and a 68% increase in the HDL fraction, even though there were no changes in total cholesterol levels. The mechanism by which wheat bran causes these changes is not at all clear. McDougall et a1. (84) suggested that wheat bran causes an increased excretion of bile acids and neutral sterols resulting in liver cholesterol depletion; however they have no evidence for this contention. They contended that HDL cholesterol was utilized for bile acid synthesis and this caused a relative increase in HDL cholesterol levels. There are data in the literature which supports as well as refutes the hypothesis that wheat bran increases the excretion of fecal sterol. Neither Eastwood et a1. (138) nor Walters et al. (149) found an increase in excretion of either fecal bile acids or neutral sterol when humans were fed 39 g or 30 g of wheat bran per day, respectively. Both experiments, however, did report increases in fecal weight and in fecal fat excretion. Pomare and Heaton (150) reported that wheat bran caused an increased excretion of bile acids when fed to rats. In our laboratory (Forsythe and Bennink, unpublished observations) feeding wheat bran to male rats did not increase the excretion of fecal neutral sterol but did increase the excretion of fecal bile acids compared to rats fed a low-fiber, control diet. It appears, in the pig at least, that wheat bran has a beneficial effect on plasma cholesterol by increasing the HDL cholesterol levels and decreasing the LDL cholesterol levels even 79 though total cholesterol levels were unchanged. Furthermore the absolute levels of these lipoproteins were altered through as yet unknown mechanisms. Rolled oats affected plasma cholesterol parameters to a greater extent than did wheat bran. Like wheat bran, it did not lower plasma cholesterol levels in pigs but it did decrease plasma cholesterol levels by 18% in chickens compared to the control diet. In one of the first papers on the effects of rolled oats on plasma cholesterol levels Degroot et a1. (89) reported that rolled oats decreased plasma cholesterol levels to 20% of control values in rats fed hypercholesterolemic diets. In a study with hypercholesterolemic men, reported in this same paper, the addition of 140 g of rolled oats per day, as bread in the diet, decreased plasma cholesterol levels from 251 mg/dl to 223 mg/dl in 21 days. McNaughton (151) reported that feeding rolled oats (18%) in low-fat diets without cholesterol to chickens, decreased plasma cholesterol levels from 130 mg/dl (control) to 88 mg/dl. Degroot et a1. (89) suggest that some of the hypocholesterolemic effect of the rolled oats is due to its relatively high vegetable fat content resulting in an increased P:S ratio. The fatty acid composition of each diet fed in this experiment shown in Table 2 indicates no major differences in fatty acid composition. Rolled oats may bind more fecal sterols than does wheat bran. Balmer and Zilversmit (93) found that ground oats bound more sodium taurocholate than did ground wheat. Forsythe and Bennink (unpublished observation) have observed that feeding oat bran to rats increased fecal bile acid excretion 4 fold and neutral sterol 81 In conclusion, both of these experiments show beneficial affects due to feeding dietary fiber. Wheat bran, while not decreasing plasma total cholesterol, did increase plasma HDL cholesterol and HDL levels in pigs. Feeding rolled oats resulted in a decrease in body fat in both pigs and chickens. Rolled oats, also, decreased plasma total cholesterol in chickens and increased the percentage of cholesterol in the HDL fraction in both pigs and chickens. Finally, it increased the molar LCAT activity in both pigs and chickens. These changes should be beneficial in decreasing the severity of atherosclerosis. 80 excretion 3 fold as compared to control animals (low fiber diet). This resulted in a decrease in both total liver lipids and in liver sterols in rats fed oat bran compared to those fed the control diet. Plasma molar LCAT activity was increased in plasma of both pigs and chickens fed the rolled oats diet. LCAT, activated by apoprotein A-I from HDL, transfer linoleate from lecithin to unesterified cholesterol (37). This cholesterol ester is transported to the liver by HDL where Schwarz et a1. (38) have shown that HDL cholesterol is utilized for bile acid synthesis. While HDL levels are important in the reaction they have not been highly correlated with LCAT activity. Wallentin (107) has hypothesized that LCAT activity increases in response to an increase in the turnover of cholesterol esters. Lopez et al. (152) reported that intensive exercise, a treatment which has been shown to increase the turnover of body cholesterol (153), caused an increase in molar LCAT activity. This is consistent with the hypothesis that fiber (rolled oats) increases the turnover of cholesterol through an increased excretion of fecal sterols (154). The method used to assay LCAT activity is a self-substrate method. As such it is impossible to relate changes in activity to an actual change in enzyme activity or a change in substrate availability. Also unclear is the actual physiological significance of altered molar LCAT rates, especially when there was no change in the ratio of unesterified to esterified cholesterol as there were in these experiments. PART IV EFFECT OF AN AEROBIC EXERCISE PROGRAM 0N BODY COMPOSITION. PLASMA CHOLESTEROL PARAMETERS, LCAT ACTIVITY. LIPOPROTEINS AND TISSUE LIPIDS IN YOUNG PIGS 82 INTRODUCTION The ability of exercise to lower plasma total cholesterol levels in humans is still controversial. While Lopez et al. (152) reported a small decrease in plasma cholesterol levels after a seven week intensive exercise program, many other studies have failed to show that exercise can lower plasma t0tal cholesterol levels (100-102, 109). In animal stidies, neither Link at al. (155), exercising pigs, nor Kenealy et al. (156), exercising goats, were able to show that a moderate exercise program could reduce plasma total cholesterol levels. Fukuda et al. (103) has reported that a moderate exercise program in rats was able to significantly reduce plasma total cholesterol levels compared to pre-exercise levels. While plasma total cholesterol levels have not been conclusively shown to decrease after an exercise program, other parameters of cholesterol metabolism, have been shown to beneficially change after exercise. Many studies have reported increases in plasma HDL cholesterol levels after exercise, even when total cholesterol' concentrations have remained unchanged (101, 102, 109, 152). Lopez et al. (152) has reported that exercise increased plasma molar 1ecithin:cholesterol acyl transferase (LCAT) activity in humans and Simko reported that exercise in rats increased fractional LCAT activity. Plasma lipoprotein profiles have also been reported to be changed by exercise in humans, with an increase in HDL fraction and a decrease in LDL fractions after an exercise program. 83 Interestingly, except for the report by Link et al. (155) with miniature pigs, there are no other reports on how exercise affects plasma cholesterol levels in pigs. The pig is an excellent model in which to study cholesterol metabolism and exercise. It is physiologically and cardiovascularally similar to humans (141). The pig also responds to cholesterol feeding, has similar lipoprotein patterns and develops atherosclerotic lesions as do humans (53). And because its weight is similar to humans the energy response to exercise should also be similar to humans. Thus, experiments were undertaken to study how a moderate exercise program would affect plasma cholesterol levels, HDL cholesterol levels and plasma LCAT activity in pigs fed high-fat, high cholesterol diets similar to those consumed by Americans. MATERIALS AND METHODS ANIMALS AND DIETS Twelve purebred (Yorkshire or Duroc) and four crossbred (Yorkshire x Hampshire) castrated male pigs were assigned to either an exercise or non-exercise group. The pigs were randomly assigned to treatments except where two pigs were from the same litter in which case one pig was assigned to each treatment. All pigs in each treatment were housed together in pens with slatted floors with free access to feed and water. The pigs weighed 23 kg at the start of the experiment. 84 Both treatments were fed the same diets (Table 1). For the first five weeks of the study, 16% protein corn-soybean meal grower diet was fed. After five weeks the pigs were fed a high fat-0.05% cholesterol diet for the remainder of the experiment. Food consumption was recorded while the pigs were fed the high-fat diet. Weights were recorded and blood samples were drawn from the vena cava at 5, 7 and 13 weeks of the experiment, 24 hours after the removal of feed. EXERCISE PROTOCOL The exercise program was primarily aerobic and an amount to which an average American could easily adapt. By the end of a three week training period, during which the time and distance the pigs were exercised was gradually increased, the pigs were accustomed to running on a treadmill. The exercise regimen was running 3.3 mph for 9 minutes one day and 3.0 mph for 20 minutes on alternate days at 0% grade. Except for two periods of two days each near the end of the experiment when the belt on the treadmill broke, the pigs were exercised every day throughout the experimental period. Initially the pigs received an electrical shock from a 9 volt prod if they did not run but after a few shocks the pigs adapted to the exercise and usually ran without shocks throughout the experiment. Prior to beginning the exercise program all pigs under went a stress test. The test consisted of three consecutive three minute runs at three mph at 0%, 2.5% and 5% grade. At the end of the test 85 Table 1. Composition of diets, %. Diet Ingredients Corn-soy High fat Corn 78.1 - Corn starch - 39.5 Soybean meal 17.5 37.5 Dicalcium phosphate 1.1 1.5 Calcium carbonate 1.3 1.5 Salt 0.5 0.3 Vitamin premix1 0.5 0.5 Vit E-Se premix2 0.5 0.5 Antibiotic premix 0.53 0.24 Corn oil - 1.0 Tallow -— 18.0 Cholesterol5 - __;;41_ 100% 100% 1Supplied the following nutrients per kg of diet: vitamin A. 3300 IU; vitamin D, 660 IU; menadione sodium bisulfite, 2.2 mg; riboflavin, 3.3 mg; niacin, 17.6 mg; d-pantothenic acid. 13.2 mg; choline, 110 mg; vitamin B12, 19.8 pg; zinc, 75 mg; gran, 60 mg; manganese, 37 mg; copper, 10 mg; and iodine, .8 mg. 2Supplied 11 IU of vitamin E and 0.1 mg of Se per kg of diet. 3Supplied 110 mg of chlortetracycline per kg of diet. 4Supplied 88 mg of chlortetracycline, 88 mg of sulfamethazine 5 and 44 mg of penicillin per kg of diet. As crystalline cholesterol. 86 maximal heart rate, obtained by palpitation, was 300-320 beats/ minute. Within the experiment the maximum exercise (20 minutes at 3 mph) produced heart rates of 220 beats/minute; or 70% of maximum heart rate. PLASMA AND TISSUE ANALYSES Plasma total and unesterified cholesterol were determined enzymatically.1 Esterified cholesterol was calculated as the difference between total and unesterified cholesterol values. Plasma high density lipoprotein (HDL) cholesterol was also determined enzymatically after the low density (LDL) and very low density (VLDL) lip0proteins were precipitated with heparin- manganese solution (120). Plasma LCAT (EC 2.3.1.43) activity was assayed by the method of Stokke and Norum (123) with modifications suggested by Wallentin and Vikrot (124). Plasma lipoproteins were separated by electrophoresis on polyacrylamide 2 The tubes were then scanned on a densitometer at 610 nm, gel. and the percentage of the alpha, pre-beta and beta lipoproteins were calculated. Triglycerides were extracted in is0propanol: heptane (5.6:3.9, v/v) and then determined colorimetrically (122). 1Cholesterol Reagent Set, BMC, Indianapolis, IN 46250. 2Redi-oisc, Ames Company, Elkhart, IN 46514. 87 Total heart lipids were extracted with chloroform:methanol (2:1, v/v) and determined gravimetrically. An aliquot of the extracted lipid was assayed for total cholesterol (125). Carcass fat and water was determined by densitometry (157). Specific gravity measurement were made on chilled carcasses (2°C) 24 hours after slaughter. Thoracic aortas were stained with Sudan IV to assess lipid infiltration (158). STATISTICAL ANALYSES All values are reported as mean i standard error of mean (SEM). Treatment means were compared by t-test (126). RESULTS Final body weights were not affected by the exercise treatments (Table 2). Since the pigs were allowed to eat ad libitum, the exercised pigs increased their food consumption to compensate for the energy expenditure of running. Because the pigs in each treatment were penned together no statistics can be computed for feed efficiency; however, the exercised pigs consumed more food per unit weight gain than did the non-exercised pigs (Table 2). Exercise did not affect the percentage of lipid in the carcass or heart or cholesterol content of the heart. Heart Weight was heavier in the exercised pigs but when expressed as weight per relative body weight there was no difference. Lipid infiltration in the thoracic aorta, as assessed by sudan staining, was minimal and similar for~both treatments. 88 Table 2. Final body weights, feed consumption, and body and heart lipids in non-exercised and exercised pigs. Non-exercised Exercised p value Final weight, kg 95 s 21 100 s 3 N32 Feed, 1b/gain, lb 2.65 2.86 -— Carcass 1ipid,3 2 23.6 s 0.9 24.3 s 0.7 NS Carcass water,3 % 55.9 i 0.6 55.4 i 0.5 NS Heart weight, g 310 i 10 350 i 9 p<0.05 Heart weight g/kg body weight 0.33 i 0.01 0.35 i 0.01 NS Heart lipid, % 5.0 i 0.5 5.9 t 0.01 NS Heart cholesterol, mg/100 g 1.5 i 0.2 1.6 i 0.2 NS Aortic sudanophilia4 <1 <1 Mean 1 SEM. Eight pigs per treatment. Not significant. #de Carcass lipid and water estimated from specific gravity. Lipid infiltration was graded on a relative scale; 0 to 4. 0 = no infiltration and 4 = maximal infiltration. 89 In the first five weeks of the experiment, which corresponds to 3 weeks of training and 2 weeks of full exercise, all pigs were fed a low-fat, corn soy protein diet. Because this diet was low-fat and did not contain cholesterol, blood lipid levels were low. Exercise did not produce any changes in plasma cholesterol, triglyceride or other parameters (Table 3). Two weeks later blood was again drawn. During this two weeks, and for the remainder of the experiment, the pigs were fed a high-fat (tallow) diet with added cholesterol. At this time, after 4 weeks of running, plasma total and HDL cholesterol levels were higher in the exercised pigs. The exercised pigs, however were consuming approximately 8% more food and consequently more cholesterol than the non-exercise group. Cholesterol consumption was approximately 0.68 g cholesterol/day for the exercised group and 0.60 g cholesterol/day for the non-exercised group at this time. The higher plasma cholesterol values seen in the exercised pigs probably reflects this increased consumption of cholesterol. The ratio of unesterified to esterified cholesterol was slightly reduced (p<0.1) in the exercised pigs but exercise had no effect on triglycerides, percentage of total cholesterol in the HDL fraction or plasma LCAT activity. Switching from a low-fat diet with no added cholesterol to a high-fat diet with cholesterol resulted in increases in most plasma lipid levels. Plasma total and HDL cholesterol and plasma triglycerides were higher in the high-fat diets. The percentage of total cholesterol carried in the HDL fraction was also increased. 90 Table 3. Diet and exercise effects on plasma cholesterol levels, triglyceride levels and LCAT activity in pigs. Non-exercised Exercised P value Corn-soy Diet (Week 51) Cholesterol, mg/dl total 95 s 52°3 99 s 4° N54 unesterified 27 5 3° 23 s 2a NS HDL 23 i la 26 : 3° NS UC/CE5 0.29 s 0.03a 0.23 + 0.02° NS HDL cholesterol, %6 25 i la 26 t 3a NS Triglycerides, mg/dl 26 i 5a 33 t 5a NS Plasma LCAT activity fractional rate, % CE/hour 6.5 s 0.5° 6.9 s 0.5a NS molar rate, uM/L/hour 46.4 i 7a 40.8 i 6a NS High-fat Diet (Week 78) Cholesterol, mg/dl total 126 1 6b 146 5 6° p<0.05 unesterified 32 i 2a 31 2 lb NS HDL 41 3 2b 50 5 3b p<0.05 UC/CE5 0.35 a 0 02° 0.29 s 0.02° NS HDL cholesterol, %6 33 : 2° 34 1 2b NS Triglycerides, mg/dl 45 i 4° 45 i Sb NS Plasma LCAT activity fractional rate, % cs/hour 3.6 s 0.2b 3.5 i 0.2b Ns molar rate, uM/L/hour . 38.3 s 3° 30.3 s 2° NS 91 Table 3 (cont'd.) 1Three week training period and 2 week exercise period. 2Mean i SEM. Eight pigs per treatment. 31(Ialues with different superscripts in each diet period are different p<0.05 . Not significant. Unesterified cholesterol/esterified cholesterol. Percentage of total cholesterol in the HDL fraction. Percentage of tritiated cholesterol converted to esterified cholesterol/ hour. \IOSUT-h 8Four weeks of exercise concluded. The high-fat diet was consumed for two weeks. 92 The high-fat diet caused a decrease in the fractional LCAT activity, but no change was observed in the molar LCAT rate. By the 13th week of the experiment (10th week of exercise) the exercise protocol produced a significant decrease in plasma total cholesterol levels (Table 4). The exercise group had plasma total cholesterol levels which were 30 mg/dl less than the non-exercised group. Again it should be noted that the exercised pigs were consuming more food and more cholesterol than the non-exercised pigs. The exercised pigs consumed approximately 2.4 kg of feed and 1.3 g of cholesterol per day while the non-exercised consumed approximately 2.2 kg of feed and 1.2 g of cholesterol per day. The unesterified cholesterol levels followed total cholesterol levels and were also reduced. No difference in the absolute amount of plasma HDL cholesterol was found but, the percentage of total cholesterol carried in the HDL fraction was significantly greater in the exercised pigs than the non-exercised pigs. Relative plasma lip0proteins, are reported in Table 4. Exercise caused an increase in percentage of HDL and a decrease in percentage of LDL compared to the non-exercised pigs. Thus the ratio of HDL to LDL was significantly increased (1.40 to 1.08) in the exercised pigs. Plasma fractional LCAT activity in the exercised pigs after 10 weeks of exercise, as was the case after 2 and 4 weeks of exercise, was not different from values of non-exercised pigs. The non-exercised pigs, however, did have higher molar LCAT activity compared to the exercised pigs after 10 weeks of exercise. 93 Table 4. Effect of exercise on plasma cholesterol levels, lip0protein profile and LCAT activity in pigs. Non-exercised Exercise p value Cholesterol, mg/dl total 194 i 111 163 i 7 p<0.05 unesterified 39 i 1 31 i 2 p<0.05 HDL 46 a 4 49 s 4 N52 00/053 0.26 s 0.01 0.23 s 0.01 NS HDL cholesterol, %4 22.2 s 2 29.5 s 2 p<0.05 Relative lip0proteins, %5 VLDL 12 i 1 11 i 1 NS LDL 43 t 38 s 2 p<0.05 HDL 45 i 52 i 3 p<0.05 HDL/LDL 1.1 i 0.08 1.4 s 0.13 p<0.05 Plasma LCAT rate fractional %CE/hour6 3.37 s 0.26 3.11 s 0.27 NS molar uM/L/hour 33.9 i 3.1 25.1 i 1.8 p<0.05 1Mean i SEM. Eight pigs per treatment. Blood drawn after 10 weeks of exercise. Not significant. Unesterified cholesterol/cholesterol ester. Percentage of total cholesterol in the HDL fraction. Relative percentage of each lipoprotein. Percentage of tritiated cholesterol converted to cholesterol ester/ hour. 030-1-wa 94 DISCUSSION While regular exercise in humans can contribute to weight reduction and increase lean body mass (95, 159), the pigs undergoing exercise in this study had similar final body weights and percentage of body fat and water as the non-exercised pigs. Both Fitts et al. (161) and Weiss et al. (160) have reported that moderate exercise did not alter body weight or body composition in pigs. Link et al. (155) also reproted no effect of exercise on body composition in pigs run on a treadmill for 10 minutes at 10 mph 5 days per week compared to non-exercised pigs. Similar to results obtained in this study, Link et al. (155) reported that the exercised pigs consumed more food per day than the non-exercised pigs. Holloszy (161) using rats and Barnard et al. (163) using guinea pigs, have reported that exercise in these animals decreased food consumption; resulting in lower body weights than in control animals. Both these investigators used mature animals and fairly intensive exercise programs (approximately 30 m/minute for up to one hour daily). Fitts et al. (161) has suggested that the energy requirement for growth in young animals, as for pigs in the present experiment, stimulates the appetite and overrides the appetite depression that occurs with exercise. Leiberman et al. (163) has shown that exercised young guinea pigs gained weight at the same _rate as control (sedentary) guinea pigs, but that exercised mature guinea pigs did not gain as much weight as non-exercised guinea pigs. It seems probably that the animal's age as well as the 95 exercise duration and intensity contribute to differences in body weight and composition. Plasma total cholesterol levels were 18% lower in the exercised pigs compared with the non-exercised pigs. Link et al. (155) reported that a moderate exercise program (1.75 miles run per day) for 22 months did not affect plasma cholesterol levels in miniature pigs. In exercising goats, Kenealy et al. (156) also reported no changes in plasma cholesterol levels compared to sedentary goats. Fukada et al. (103) reported that moderate exercise in rats (11 m/minute for one hour daily) did decrease plasma cholesterol levels. Simko and Kelley (108) also using rats, showed that exercise decreased red blood cell cholesterol but they did not report wheather total cholesterol levels were affected. In cross-sectional studies, plasma cholesterol levels are usually lower in trained men compared to matched controls (96, 98). Wood et a1. (96) reported that male runners (at least 15 miles per week) had lower plasma cholesterol levels than sedentary matched controls. However, while Wood et a1. (96) matched the groups for age and blood pressure, the sedentary group were almost 20% above their ideal weight, compared to the runners who were close to their ideal weights. This difference could contribute to the difference in plasma cholesterol levels between treatment groups. Clinical studies, in which humans have started an exercise program, have been inconclusive as to the response of plasma cholesterol levels to exercise. Some reports have indicated that exercise decreases plasma total cholesterol levels (152, 165) but the majority have failed to show a relationship between exercise 96 and plasma total cholesterol levels. The differences in response may relate to the exercise duration and intensity. In our study, as in the study in rats by Fukuda et al. (104) which also showed a decrease in cholesterol levels with exercise, the animals were exercised daily. While the effects of exercise on plasma total cholesterol levels is questionable, particularily in humans, it does appear that exercise increases HDL cholesterol levels (94, 109, 152). Gilliam et al. (109) reported that a moderate exercise program in girls (40 minutes per day of running and skipping rope for six weeks) increased HDL cholesterol levels and increased the percentage of cholesterol in the HDL fraction from 21.8% in the non-exercised girls to 28.8% in the exercised girls. These changes occurred even though plasma total cholesterol levels were not affected by exercise. Lopez et al. (152) also reported an increase in HDL cholesterol in young males after an intensive seven week exercise program. These results are similar to the results obtained in the present experiment. Although the absolute level of HDL cholesterol was not greater in the exercised group of pigs compared to the non-exercised pigs (49 mg/dl versus 46 mg/dl, respectively), the percentage of cholesterol in the HDL fraction was significantly greater (29.5% versus 22.2%). Exercise also increased the ratio of HDL to LDL in this study. Hill et al. (147) showed that electrophoretic plasma lipoprotein profiles obtained in pigs are highly correlated with the percentages of lip0proteins obtained by ultracentrifugation. Thus in the exercised pigs in this experiment, not only was the percentage of 97 cholesterol in the HDL fraction increased but the concentration of high density lipoproteins in the plasma were also increased. Similar increased in plasma HDL levels after exercise in humans have been previously reported (97, 166). Plasma molar LCAT activity was reduced in the exercised pigs compared to the non-exercised pigs after 10 weeks of exercise. Lopez et al. (152) reported that 7 weeks of intensive exercise by young males increased molar LCAT activity over pre-exercise values. Possibly the difference in molar LCAT activities between our experiment and that of L0pez et al. (152) relates to the difference in exercise intensity. Simko et al. (108) also reported an increase in fractional LCAT activity in rats undergoing one hour of swimming daily. Plasma LCAT activity is negatively correlated with unesterified cholesterol levels (107, 130). Since plasma unesterified or total cholesterol levels were not reported by Simko et al. (108), it is not possible to determine if molar LCAT activity was changed by exercise in their experiment. In our experiment both total and unesterified cholesterol levels were decreased by exercise. Wallentin (107, 130) has reported that changes in molar LCAT activity are positively correlated with changes in plasma unesterified cholesterol, phospholipid or triglyceride levels in the plasma. Since endogenous substrates are used to assay LCAT activity, changes in activity could be due to changes in enzyme activity or changes in substrate availability (37, 107). Possibly the decrease in unesterified cholesterol levels that was observed in our experiment was 98 responsible for the decrease in molar LCAT activity. While statistically significant changes in molar LCAT activity have been reported, the physiological significance of these changes remains to be determined. The mechanism by which exercise alters plasma cholesterol levels is still speculative. Fukuda et al. (l03) reported that bile acid excretion was greater in exercised rats than in sedentary, pair-fed, control rats. Bobek et al. (106) has reported that exercise increases the turnover of cholesterol in exercised rats. These parameters were not investigated in our experiment. The results obtained in this experiment indicate that an aerobic exercise program can beneficially modify plasma cholesterol parameters in pigs. After l0 weeks of exercise plasma cholesterol levels were reduced, HDL cholesterol percent was increased and levels of plasma HDL were increased compared to non-exercised pigs. These results show that changes occurring in the exercised pigs are similar to changes that occur in humans undergoing an exercise program. Utilization of the pig could provide an excellent model to investigate not only changes that occur with exercise but also the mechanism behind these changes. PART V SUMMARY AND CONCLUSIONS SUMMARY AND CONCLUSIONS These experiments were conducted to investigate factors which may influence body composition, plasma cholesterol levels, including total, unesterified and HDL cholesterol levels, and plasma LCAT activity. The dietary treatments investigated were: l) comparison of plant protein vs animal protein, 2) saturated fat vs polyunsaturated fat (P:S, 0.3 vs 3), and 3) presence or absence of dietary fiber. The effects of an aerobic exercise program on these same parameters in pigs were also studied. Two animal species were utilized in these experiments; the pig, because it is physiologically similar to humans and the chicken, because its plasma cholesterol levels increase to a much higher level than does the pig when similar diets are fed. Although specific dietary ingredients were varied, in general energy composition of the experimental diets was: 42% from fat, 40% from carbohydrate and l8% from protein. Depending on the experiment the diets contained between 0.05% to 0.1% cholesterol. The only exception was when a corn-soy, grower diet (low-fat, low-cholesterol) was fed as a reference in one experiment. Thus, the diets were similar in energy composition to those typically consumed in the United States. Body composition was investigated after two treatments: 1) after a moderate exercise program in pigs and 2) after feeding dietary fiber to pigs for l4 weeks and to chickens for 5 weeks. 99 100 Although exercise has been shown in some experiments to alter body composition (161, 162), in this experiment the moderately exercised pigs had similar carcass fat and water percentages as the non-exercised pigs. As both groups of pigs were allowed unlimited access to food, food consumption was approximately 8% greater in the exercised pigs compared to the non-exercised pigs. While rather intensive exercise has been shown to depress appetite and result in less weight gain in exercised animals compared to non-exercised animals (162, 163), in this experiment the rapid growth of the pigs plus the moderate exercise regimen may have stimulated appetite; at least to compensate for the energy expended in the exercise. A very interesting finding was that feeding rolled oats decreased the percentages of body fat in both pigs and chickens. Wheat bran, however, was without affect on body composition. The mechanism by which rolled oats lowers body fat is not clear. Wheat bran has been shown to increase the excretion of fecal lipids, but whether rolled oats also increases fecal lipids has not been investigated. Also, whether an increase in fecal lipids could alter body composition to the extent observed in this experiment is questionable; especially since the final body weight and food consumption were similar between treatments. Seemingly, if significant amounts of energy were being lost in the feces the body weights of the animals fed rolled oats would be less than those fed the other diets. Certainly an area that warrants further investigation is the mechanism by which rolled oats alter body composition. .F. 101 These experiments have also shown that dietary changes or an aerobic exercise program can alter plasma total cholesterol levels, even when high-fat, high-cholesterol diets are fed. In pigs, plasma total cholesterol levels were reduced, compared to appropriate controls, when: plant protein replaced animal protein, the P:S ratio was increased from 0.3 to 3.0 and when the pigs underwent an aerobic exercise program. In chickens feeding rolled oats, but not wheat bran, reduced plasma cholesterol levels. When polyunsaturated fats were fed in diets with plant proteins. although the pigs were consuming approximately 4.5 g of cholesterol per day, their plasma cholesterol levels were similar to pigs consuming low fat diets without cholesterol. Since plasma cholesterol levels are one the major risk factors in the development of atherosclerosis in humans, reduction in plasma cholesterol levels would be beneficial. These experiments show that, in animals, dietary modifications or a relatively moderate exercise program can reduce plasma total cholesterol levels. Recently, epidemiological studies have identified plasma HDL cholesterol concentrations as being important in the development of atherosclerosis (13-15). It is important to report both the absolute HDL cholesterol levels and the percentage of total cholesterol in the HDL fraction, as HDL cholesterol levels tend to correlate with total cholesterol levels (48, 53). In these experiments the percentage of total cholesterol in the HDL fraction was most significantly increased when dietary fiber was fed to either pigs or chickens, or when the pigs were exercised. While the protein source fed or the fat source fed both influenced the absolute 102 levels of HDL cholesterol neither treatment altered the percentage of cholesterol in the HDL fraction. Whether dietary fiber or exercise increase HDL cholesterol levels by similar mechanisms remains to be determined. Both the feeding of dietary fiber and exercise increase plasma cholesterol turnover (93, 106), and both treatments have been shown to increase the excretion of fecal steroids (93, 103). Since HDL levels have been reported to be a substrate for synthesis of hepatic bile acid, it has been hypothesized that HDL cholesterol levels increase in response to the increased hepatic synthesis of bile acids (105). At least in exercise a more conceivable hypothesis is that the increased HDL cholesterol levels arise due to an increased turnover of VLDL. As VLDL remnants are produced, HDL has been postulated to facilitate the conversion of VLDL remnants to LDL by accepting unesterified cholesterol from the VLDL remnant (31). Thus, through this mechanism HDL cholesterol levels could be increased by exercise. Whether a similar mechanism occurs when different sources of dietary fibers are fed is unknown. No consistent effects of treatments on plasma LCAT activity were observed. Molar LCAT activity was decreased in two treatments in which plasma unesterified cholesterol levels were decreased (increasing the P:S ratio and exercise). Wallentin (107) has reported that unesterified cholesterol levels are correlated with molar LCAT activity. But two other treatments, substituting plant protein for animal protein and feeding dietary fiber, resulted in decreases in plasma unesterified cholesterol levels and increases 103 in molar LCAT activity. The LCAT reaction is very complicated, involving many substrates. Since changes in LCAT activity could be due to either changes in actual enzyme activity or changes in the availability of endogenous substrates, it is unclear as to the actual physiological significance of changes in LCAT activity. In summary these experiments have demonstrated that changes in dietary ingredients and an aerobic exercise program can produce beneficial changes in plasma cholesterol parameters in species with similar cholesterol and lipoprotein metabolism as man. LITERATURE CITED 10. 11. 12. LITERATURE CITED McMillan, G.C. Atherosclerotic disease and vessel wall. Empt. Mbl. Pathol. 31:163-168. 1979. Zilversmit, D.B. Atherogenesis: Apostprandial phenomenon. Circulation 60:473-485. 1979. Dietary Goals for the United States. Washington, D.C. Superintendent of Documents, 1977. Hegsted, D.M. Dietary goals —-a progressive view. Am. J. Clin. Nutr. 31:1504-1509, 1978. Glueck, C.J. and W.E. Connors. Diet-coronary heart disease relationships reconnoitered. Am. J. Clin. Nutr. 31: 727-737. 1978. Ahren, Jr., E.H. The management of hyperlipidemia: whether, rather than how. Anns. Int. Med. 85:87-93, 1976. Reiser, R. Oversimilification of diet: Coronary heart disease relationships and exaggerated diet recommendations. Am. J. Clin. Nutr. 31:865-875, 1978. United States Department of Health, Education and Welfare: Monthly vital statistics reports: Final mortality statistics 1974. Death and death rates. 25: Suppl. 1-3, 1977. Kolata, G.B. Prevention of heart disease: Clinical trials at what cost? Science 190:764-765, 1975. Keys, A. Coronary heart disease —-The global picture. Atherosclerosis 22:149-192, 1975. Keys, A. Coronary heart disease in seven countries. Circulation 41:Supp1. 1, 1-210, 1970. Malmros, A. The relation of nutrition to health —-A statistical study of the effect of wartime on atherosclerosis, cardiosclerosis, tuberculosis and diabetes. ACTA Med. Scand. Suppl. 246:137-153, 1950. 104 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 105 Kannel, W.B. Recent highlights from the Framingham study. Aust. N.Z. J. Med. 6:373-386, 1976. Kannel, W.B., D. McGee and T. Gordon. A general cardiovascular risk profile. The Framingham study. Am. J. cardiol. 38: 46-51, 1976. Gordon, T., W.B. Kannel and D. McGee. Death and coronary attacks in men after giving up cigarette smoking. Lancet 2:1345-1348, l974. ’ Stamler, J. Epidemiology of coronary heart disease. Med. Clin. N. Amer. 57:5-46, 1973. Rhoads, G.G., C.L. Gulbrandsen and A. Kagan. Serum lipoproteins and coronary heart disease in a p0pulation study of Hawaii Japanese men. New Engl. J. Med. 294:293-298, 1976. Miller, G.J. and W.E. Miller. Plasma high-density lipoprotein concentration and development of ischaemic heart disease. Lancet 1:16-19, 1975. Mantulin, W.W. and A.M. Gotto, Jr. Human plasma lip0proteins: Structure and metabolism. In: International Conference on Atherosclerosis. (L.A. Carlson, ed.), Raven Press, New York, NY, p. 57-69, 1978. Zilversmit, D.B.A Assembly of chylomicrons in the intestinal cell. In: Disturbances in Lipid and Lipoprotein Metabolism. (J.M. Dietschy, A.M. Gotto, Jr. and J.A. Ontko, eds.), American Physiological Society, Bethesda, MD, p. 69-82, 1978. Windmeuller, H.G., P.N. Herbert and R.I. Levy. Biosynthesis lymph and plasma lip0proteins apoprotein by isolated perfused rat liver and intestine. J. Lipid Res. 14: 215-223. 1973. Gotto, A.M., Jr., R.I. Levy, K. John and D.S. Fredrickson. On the protein defect in abetalipoproteinemia. New Engl. J. Med. 284:813-818, 1971. Havel, R.J., J.P. Kane and M.L. Kashyap. Interchange of apolipoproteins between chylomicrons and high-density lip0proteins during alimentary lipemia in man. J. Clin. Invest. 52:32-38, 1973. Smith, L.C., P.K.J. Kinnvnen, R.L. Jackson, A.M. Gotto, Jr. and J.T. Sparrow. Activation of lipoprotein lipase by native ans synthetic peptide fractions of apolipoprotein C-II. In: International Conference on Atherosclerosis. (L.A. Carlson, ed.), Raven Press, New York, NY, p. 269- 273, 1978. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 106 Anderson, J.M. and J.M. Dietschy. Regulation of sterol synthesis in 15 tissues of the rat. II. Role of rat and human high and low density plasma lipoproteins and of rat chylomicrons remnants. J. Biol. Chem. 252: 3652-3659, 1977. Morrisett, J.D., R.L. Jackson and A.M. Gotto, Jr. Lipid- protein interactions in the plasma lip0proteins. Biochim. Biophys. ACTA 472:93-133, 1977. Schonfeld, G. Lipoproteins in atherogenesis. Artery 5: 305-329, 1979. Gotto, A.M., Jr. and R.L. Jackson. Plasma lipoproteins and atherosclerosis. In: Atherosclerosis Reviews, Vol. 3, (R. Paoletti and A.M. Gotto, Jr., eds.), Raven Press, New York, NY, p. 231-242, 1978. Brown, M.S. and J.L. Goldstein. General scheme for regulation of cholesterol metabolism in mammalian cells. In: Disturbances in Lipid and Lipoprotein Metabolism. (J.M. Dietschy, A.M. Gotta, Jr. and J.A. Ontko, eds.), American Physiological Society, Bethesda, MD, p. 173- 180, 1978. Brown, M.S. and J.L. Goldstein. Receptor-mediated control of cholesterol metabolism. Science 191:150-154, 1976. Goldstein, J.L. and M.S. Brown. The low-density lipoprotein pathway and its relation to atherosclerosis. Ann. Rev. Biochem. 46:897-930, 1977. Schaefer, E.J., S. Eisenberg and R.I. Levy. Lipoprotein apoprotein metabolism. J. Lipid Res. 19:667-687. 1978. Sniderman, A.D., T.E. Carew, J.G. Chandler and D. Steinberg. Paradoxical increase in rate of catabolism of low-density lip0proteins after hepatomectomy. Science 183:526-528, 1974. Cham, B.E. Inportance of apolipoproteins in lipid metabolism. Chem-Biol. Interactions 20:263-277, 1978. Fielding, C.J., V.G. Shore and P.E. Fielding. A protein co-factor of 1ecithin:cholesterol acyltransferase. Biochem. Biophys. Res. Commun. 46:1493-1498, 1972. Brunzell, J.G., A. Chait and E.L. Bierman. Pathophysiology of lipoprotein transport. Metabolism 27:1109-1127, 1978. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 107 Glomset, J.A. Plasma 1ecithin:cholesterol acyltransferase. In: Blood Lipids and Lipoproteins: Quantification, Composition and Metabolism. (G.J. Nelson, ed.), John Wiley and Sons, New York, NY, p. 745-787, 1972. Schwartz, C.C., L.G. Halloran, Z.R. Vlachcavic, D.H. Gregory and L. Swell. Preferential utilization of free cholesterol from high-density lip0proteins for biliary cholesterol secretion in man. Science 62:200-203, 1978. Stein, Y., M.C. Glangeaud, G. Fainaru and O. Stein. The removal of cholesterol from aortic smooth muscle cells in culture and Landshutz ascites cells by fractions of human high-density apolipoprotein. Biochim. Biophys. ACTA 380:106-118, 1975. Jackson, R.L., 0. Stein, A.M. Gotto, Jr. and Y. Stein. A comparative study on the removal of cellular lipids from Landschutz cells by human plasma apolipoproteins. J. Biol. Chem. 250:7204-7209, 1975. Rodwell, V.W., J.L. Nordstrom and J.J. Mitschelen. Regulation of HMG CoA reductase. Advan. Lipid Res. 14:1-74, 1976. Dietschy, J.M. and J.D. Wilson. Regulation of cholesterol metabolism. New Engl. J. Med. 282:1128-1138, 1179-1183, 1241-1249, 1970. Goldstein, J.L. and M.S. Brown. Lipoprotein receptors, cholesterol metabolism and atherosclerosis. Arch. Pathol. 99:181-184, 1975. Bierman. E.L. and J.J. Albers. Regulation of low-density lipoprotein receptor activity by cultured human arterial smooth muscle cells. Biochim. Biophys. ACTA 488:152-157, 1977. Smith, E.B. The influence of age and atherosclerosis on the chemistry of aortic intima. J. Atheroscler. Res. 5: 224-240, 1965. Mann, G.V. Diet heart: End of an era. New Engl. J. Med. 297:644-650, 1977. Flynn, M.A., G.B. Nolph, T.C. Flynn, R. Kahrs and G. Krause. Effect of dietary egg on human serum cholesterol and triglycerides. Am. J. Clin. Nutr. 32:1051-1057, 1979. Mattson, E.H., B.A. Erickson and A.M. Klingman. Effect of dietary cholesterol on serum cholesterol in man. Am. J. Clin. Nutr. 25:589-594. 1972. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 108 Keys, A., J.T. Anderson and F. Grande. Serum cholesterol response to changes in the diet. (the effect of cholesterol in the diet) Metabolism 14:759-765, 1965. Mahley, R.W. Alteration of plasma lip0proteins induced by cholesterol feeding in animals including man. In: Disturbances in Lipid and Lipoprotein Metabolism. (J.M. Dietschy, A.M. Gotto, Jr. and J.A. Ontko, eds.), American Physiological Society, Bethesda, MD, p. 181- l99, 1978. Mistry, P., A. Nicoll, C. Niehaus, 1. Christie, E. Janus and B. Lewis. Cholesterol feeding revisited. Circulation 54:11-178, 1976. (Abstract) Applebaum-Bowden, D., W.R. Hazzard, J. Cain, M.C. Cheung, R.S. Kushwaha and J.J. Albers. Short term egg yolk feeding in humans: Increase in apolipoprotein B and low density lipoprotein cholesterol. Atherosclerosis 33:385-396. 1979. Mahley, R.W., K.H. Weisgraber, T. Innerarity, H.B. Brewer, Jr. and G. Assman. Swine lipoproteins and atherosclerosis. Changes in plasma lip0proteins and apoproteins induced by cholesterol feeding. Biochemistry 14:2817—2823, 1975. Ross, A.C. and 0.8. Zilversmit. Chylomicron remnant cholesteryl esters as the major constituent of very low-density lipoproteins in plasma of cholesterol fed rabbits. J. Lipid.Res. 18:169-181, 1977. Kakika, C., P.J. Johnson, R. Pick and L.N. Katz. Relationship between plasma cholesterol level and coronary atherosclerosis in cholesterol fed cockerals. Atherosclerosis 15:17-29, 1972. Kruski, A.W. Influence of cholesterol in the concentration, composition and synthesis of chicken serum lip0proteins. Ph.D. Dissertation, University of Illinois, Urbana, IL, 1971. Hillyard, L.A. and H.M. White. Characterization of chicken serum lip0proteins. Fed. Proc. 28:447, 1969. (Abstract) Dangerfield, W.G., R. Finlayson, G.M. Yatt and M.G. Mead. Serum lipoproteins and atherosclerosis in animals. Atherosclerosis 25:95-106, 1976. Jensen, P.F., G.L. Jensen and S.C. Smith. Serum lipoprotein profiles of young atherosclerosis-susceptible white carneau and atherosclerosis-resistant show racer pigeons. Comp. Biochem. Physiol. 608:67-69, 1978. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 109 Kruski, A.W. and K.A. Narayan. Lipoprotein synthesis in chickens fed cholesterol. Atherosclerosis 15:141-145, 1975. Kritchevsky, D. Diet and atherosclerosis. Am. J. Pathol. 84:615-632. 1976. Newburgh, L.H. and T.L. Squier. High protein diets and arteriosclerosis in rabbits —-a preliminary report. Arch. Int. Med. 31:653-673. 1923. Meeker, D.R. and H.D. Kester. Effect of high protein diets on experimental atherosclerosis of rabbits. Arch. Pathol. 31:147-162, 1941. Carroll, K.K. Plasma cholesterol levels and liver cholesterol biosynthesis in rabbits fed commercial or semisynthetic diets with and without added fat or oils. Atherosclerosis 13:67-76, 1971. Hamilton, R.M.G. and K.K. Carroll. Plasma cholesterol levels in rabbits fed low fat, low cholesterol diets. Effect of dietary proteins, carbohydrates and fiber from different sources. Atherosclerosis 24:47-69, 1976. Huff, M.W., R.M.G. Hamilton and K.K. Carroll. Plasma cholesterol levels in rabbits fed low-fat, cholesterol-free, semi-purified diets: Effect of dietary proteins, protein hydrolysates and amino acid mixtures. Atherosclerosis 28: 187-195, 1977. Yadav, N.R. and I.E. Liener. Reduction of serum cholesterol in rats fed vegetable protein or an equivalent amino acid mixture. Nutr. Reps. Inter. 16:385-389, 1977. Kritchevsky, D., S.A. Tepper, D.E. Williams and J.A. Story. Experimental atherosclerosis in rabbits fed cholesterol- free diets. Part 7 -Interaction of animal or vegetable protein with fiber. Atherosclerosis 26:397-403. 1977. Kritchevsky, D., S.A. Tepper and J.A. Story. Influence of soy protein and casein on atherosclerosis in rabbits. Fed. Proc. 38:749, 1978. (Abstract) Fumagelli, R., R. Paoletti and A.N. Howard. Hypocholesterolemic effect of soya. Life Sciences 22:947-952, 1978. Kim, D.N., K.T. Lee, J.M. Reiner and W.A. Thomas. Effect of a soy protein product on serum and tissue cholesterol concentration in swine fed high-fat, high-cholesterol diets. Exptl. Mbl. Pathol. 29:385-399, 1978. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 110 Shorey, R.L. and J.L. Davis. Effects of substituting soy for animal protein in the diets of young mildly hypercholesterolemic males. Fed. Proc. 39:551, 1979. (Abstract) Sirtori, C.R., E. Gatti, 0. Mantero, F. Conti, E. Agradi, E. Tremoli, M. Sirtori, L. Fraterrigo, L. Tavazzi and D. Kritchevsky. Clinical experience with soybean protein diet in the treatment of hypercholesterolemia. Am. J. Clin. Nutr. 32:1645-1658. 1979. Burkitt, D.P., A.R.P. Walker and N.S. Painter. Dietary fiber and disease. J. Am. Med. Assoc. 229:1068-1074, 1974. Trowell, H.C. Ischemic heart disease and dietary fiber. Am. J. Clin. Nutr. 25:925-932. 1972. Kritchevsky, 0. Dietary fiber and other dietary factors in hypercholesterolemia. Am. J. Clin. Nutr. 30:979-984. 1977. Trowell, H. and D. Burkitt. Dietary fiber and cardiovascular disease. Artery 3:107-119, 1977. Van Soest. P.J. Dietary fibers: Their definition and nutritional properties. Am. J. Clin. Nutr. 31:512-520, 1978. Theander, 0. The chemistry of dietary fibers. In: Food and Fibre symposium, Marabou,Sundbyberg, Sweden, p. 23-30, 1976. Van Soest, P.J. and J.B. Robertson. What is fibre and fibre in food? In: Food and Fibre symposium, Marabou, Sundbyberg, Sweden, p. 12-22, 1976. Forsythe, W.A., W.L. Chenoweth and M.R. Bennink. Laxation and serum cholesterol in rats fed plant fibers. J. Food Sci. 43:1470-1476, 1978. Looney, M.A. and K.Y. Lei. Dietary fiber, zinc and copper: Effects on serum and liver cholesterol levels in the rat. Nutr. Reps. Inter. 17:329-337, 1978. Keys, A., F. Grande and J.T. Anderson. Fiber and pectin in the diet and serum cholesterol concentrations in man. Proc. Soc. Exp. Biol. Med. 206:555-558, 1961. McDougall, R.M., L. Yakymyshyn, K. Walker and 0.6. Thurston. Effect of wheat bran on serum lipoproteins and biliary lipids. Can. J. Surg. 21:433-435. 1978. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 111 Truswell, A.S. Food fibre and blood lipids. In: Food and Fibre Symposium, Marabou, Sundbyberg, Sweden, p. 51-54, 1976. Lopez, A., J. Hopson and W.A. Frehl. Effect of dietary pectin on plasma fecal lipids. Fed. Proc. 27:485. 1968. (Abstract) Cookson, F.B., R. Altschul and S. Federoff. Effect of alfalfa on serum cholesterol and in modifying or preventing cholesterol-induced atherosclerosis in rabbits. J. Atheroscler. Res. 7:69-81, 1967. Manilow, M.R., P. McLaughlin, H.K. Naito, L.A. Lewis and W. P. McNulty. Effect of alfalfa meal on shrinkage (regression) of atherosclerotic plaques during cholesterol feeding in monkeys. Atherosclerosis 30:27-43, 1978. Degroot, A.P., R. Luyken and N.A. Pikaar. Cholesterol lowering effect of rolled oats. Lancet 2:303-305, 1963. Fisher, H. and P. Griminger. Cholesterol-lowering effects of certain grains and of oat fractions in the chick. Proc. Soc. Emptl. Biol. Med. 126:108-111, 1967. Husseini, M.D., W.F. Krueger, R.C. Fanguy and J.W. Bradley. Blood serum and egg yolk cholesterol in hens as influenced by wheat middlings and oats in the diet. Poultry Sci. 55: 1595, 1976. (Abstract) Story, J.A. and D. Kritchevsky. Comparison of the binding of various bile acids and bile salts in vitro by several types of fiber. J. Nutr. 106:1292-1294, 1976. Balmer, J. and 0.8. Zilversmit. Effects of dietary roughage on cholesterol absorption, cholesterol turnover and steroid excretion in the rat. J. Nutr. 104:1319-1329, 1974. Kritchevsky, D. and J.A. Story. Binding of bile salts in vitro by non-nutritive fiber. J. Nutr. 104:458-462, 1974. Leon, A.S. and H. Blackburn. The relationship of physical activity to coronary heart disease and life expectancy. Ann. NY Acad. Sci. 301:561-578, 1977. Wood, P.D., W.L. Haskell, M.R. Stern, S. Lewis and C. Perry. Plasma lipoprotein distributions in male and female runners. Ann. NY Acad. Sci. 301:748-763, 1977. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 112 Wood, P.D., W. Haskell, H. Klein, S. Lewis, M.P. Stern and J.W. Farquhar. The distributions of plasma 1ipoproteins in middle-aged runners. Metabolism 25:1249-1257, 1976. Carlson, L.A. and F. Mossfeldt. Acute effects of prolonged, heavy exercise on the concentration of plasma lipids and lipoproteins in man. ACTA Physiol. Scand. 62:51-59, 1964. Lopez, A. Effects of exercise on serum lipids and 1ipoproteins. In: Low Density Lipoproteins. (C.E. Day and R.S. Levy, eds.), Plenum Press, New York, NY, p. 135- 148. 1976. Widham. K., E. Maxa and H. Zyman. Effect of diet and exercise upon cholesterol and triglyceride content of plasma lipoproteins in overweight children. Eur. J. Pediatr. 1127:121-126, 1978. Bonanno, J. and J.E. Lies. Effect of physical training on coronary risk factors. Am. J. cardiol. 33:760-764. 1974. Holloszy, J.0., J.S. Skinner, G. Tara and T.K. Cureton. The effects of a six month program of endurance exercise on serum lipids of middle-aged men. Am. J. cardiol. 14: 753-760, 1964. Fukuda. N., T. Ide, Y. Kida, K. Takamine and M. Sugana. Effects of exercise on plasma lipids and liver lipids of rats. IV. Effects of exercise on hepatic cholesterol- genesis and fecal steroid excretion in rats. Nutr. Metab. 23:256-265. 1979. Simko. V., R. Nemac and E. Grinter. Incorporation of acetate 1-14C into liver cholesterol of rats subjected to prolonged exercise. Ebperimentia 22:749-750. 1970. Manilow, M.R., P. McLaughlin and A. Perley. Cholesterol: treadmill activity accelerates oxidation in rats. Science 160:1239-1240, 1968. Bobek. P., E. Grinter. J. Cerven, V. Chorvathova and L. Mikus. Effect of infrequent feeding and increased physical activity on cholesterol kinetics in the rat. Physiol. Bohemoslov. 27:145-150. 1978. Wallentin, L. Lecithin:cholesterol acyltransferase rate in plasma and its relation to lipid and lipoprotein concentrations in primary hyperlipidemia. Atherosclerosis 26:233-248. 1977. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 113 Simko, V. and R.E. Kelley. Effect of chronic intermittant exercise on biliary lipids, plasma 1ecithin:cholesterol acyltransferase and red blood cell lipids in rats. Am. J. Clin. Nutr. 32:1376-1380, 1979. Gilliam, T.B. and H.B. Burke. Effect of exercise on serum lipids and liporpteins in girls, ages 8 to 10. Artery 4:203’2139 1978. Carroll, K.K. The role of dietary protein in hypercholesterolemia and atherosclerosis. Lipids 13:360-365. 1978. Carroll, K.K. and R.M.G. Hamilton. Effects of dietary protein and carbohydrate on plasma cholesterol levels in relation to atherosclerosis. J. Food Sci. 40:18-23, 1975. Kritchevsky, 0. Vegetable protein and atherosclerosis. J. Am. Oil Chemists Soc. 56:135-140. Carroll. K.K.. P.M. Grovannetti, M.W. Huff. 0. Moase, 0.C.K. Roberts and B.M. Wolfe. Hypocholesterolemic effect of substituting soybean protein for animal protein in the diets of healthy young women. Am. J. Clin. Nutr. 31:1312-1321. 1978. Carroll. K.K. Dietary protein in relation to plasma cholesterol levels and atherosclerosis. Nutr. Rev. 36:1-5. 1978. Goering. H.K. and P.J. Van Soest. Fiber analysis (apparatus. reagents, procedures and some applications). Agric. Handbook 379, USDA. 1970. Official Methods of'Analyses. (W. Horwitz, ed.) Association of Official Analytical Chemists, Washington. D.C., 1975. Yacowitz. H.. A.I. Fleischman and M.L. Bierenbaus. Effects of oral calcium upon serum lipids in man. Brit. Med. J. 1: 1352-1354. 1965. Klevay, L.M. The role of copper and zinc in cholesterol metabo1ism. In: Advances in Nutritional Research, V01. 1. (H.H. Draper, ed.) Plenum Pub. Co., New York. NY. 227- 252. 1977. Eisemann. J.H., W.G. Pond and Thonney. M. Effect of dietary zinc and copper on performance and tissue mineral and cholesterol concentrations in swine. J. Animal Sci. 48: 1123-1128. 1979. Lipids Research Clinics Program Manual of'Laboratory Operations, Department of Health Education and Welfare, Publication No. (NIH) 75-628, 1974. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 114 Muniz, N. Measurement of plasma 1ipoproteins by electrophoresis on polyacylamide gel. Clin. Chem. 23:1826-1833, 1977. Biggs. H.G., J.M. Erickson and W.R. Morehead. A manual colorimetric assay of triglycerides in serum. Clin. Chem. 21:437-441. 1975. Stokke. R.T. and K.R. Norum. Determination of lecithin: cholesterol acyl transferase rate in plasma. Scand. J. Clin. Lab Invest. 35:21-27, 1975. Wallentin, J. and 0. Vikrot. Evaluation of an in vitro assay of 1ecithin:cholesterol acyl transferase. Scand. J. Clin. Lab. Invest. 35:661-667. 1975. Kates. M. Techniques of Lipidology: Isolation, Analysis and Identification of Lipids. American Elsevier, New York, p. 360-361. 1975. Gill, J.L. Design and Analysis of’Emperiments in the Animal and Medical Sciences. Iowa State University Press, Ames, 1978. Jackson. R.L., 0.0. Taunton, J.D. Morrisett and A.M. Gotto. The role of dietary polyunsaturated fat in lowering blood cholesterol in man. Circ. Res. 42:447-453. 1978. Bronte-Stewart, B., A. Antonis, L. Eales and J.F. Brock. Effects of feeding different fats on the serum cholesterol level. Lancet 1:521-526, 1956. Glomset, J.A. and K.R. Norum. The metabolic role of lecithin: cholesterol acyl transferase: Perspectives from pathology. Adv. Lipid Res. 11:1-65. 1973. Wallentin, L. Lecithin:cholesterol acyl transferase rate and high density 1ipoproteins in plasma during dietary and clofibrate treatment of hypertriglyceridemic subjects. Atherosclerosis 31:41-52, 1978. Miller, J.P., A. Chair and B. Lewis. The relationship between dietary fat composition and plasma cholesterol esterification in man. Clin. Sci. Mblec. Med. 49: 617-620. Gjone. E.. A. Nordoy, J.F. Blomhoff and I. Wiencke. The effects of unsaturated and saturated dietary fats on plasma cholesterol, phospholipids and 1ecithin:cholesterol acyl transferase activity. ACTA Med. Scand. 191:481-484, 1972. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 115 Grundy. S.M. and E.H. Ahrens. The effects of unsaturated dietary fats on absorption, excretion. synthesis and distribution of cholesterol in man. J. Clin. Invest. 49:1135-1152. Ullman. M.G. and W.L. Chenoweth. Changes in serum cholesterol in response to decrease in dietary cholesterol and modification of dietary fat. Fed. Proc. 39:551. 1979 (Abstract) Greer. S.A.N.. V.W. Harp, V.C. Speer and J.T. McCall. Effects of dietary fat, protein and cholesterol on atherosclerosis in swine. J. Nutr. 90:183-190. 1966. Karking. P.W. and W.H.F. Sutherland. Lecithin:cholesterol acyl transferase activity in the serum of rats fed saturated and unsaturated fats. Atherosclerosis 26: 225-232. 1977. Gatti, E. and C.R. Sirtori. Soybean protein diet and plasma cholesterol (reply). Lancet 1:805-806. 1977. Eastwood. M.A., J.R. Kirkpatrick, W.D. Mitchell, A. Bone and T. Hamilton. Effects of dietary supplements of wheat bran and cellulose on feces and bowel function. Brit. Med. J. 4:392-395. 1973. Bremner. W.F., P.M. Brooks, J.L.H.C. Third and T.D.V. Laurie. Bran in triglyceridemia: a failure of response. Brit. Med. J. 3:574. 1975. Connell, A.M., C.L. Smith and M. Somsel. Absence of effect of bran on blood lipids. Lancet 1:496-497, 1975. Vesselinovitch, 0. Animal models of atherosclerosis, their contributions and pitfalls. Artery 5:193-206. 1979. Priee, J.F.. A.M. Pearson and E.J. Benne. Specific gravity and chemical composition of the untrimmed ham as related to leanness of pork carcasses. J. Animal Sci. 16: 85-92. 1957. Tsai, A.C.. J. Elias, J.J. Kelley, R.S.C. Lin and J.R.K. Robson. Influence of certain dietary fibers on serum and tissue cholesterol levels in rats. J. Nutr. 106: 118-123, 1976. Sundaravalli. D.E., K.S. Shurpalekar and M.N. Rao. Effects of dietary cellulose supplementation on the body composition and cholesterol metabolism of albino rats. J. Agr. Food Chem. 20:116-118. 1971. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 116 Southgate, D.A.T. and J.V.G.A. Durnin. Calorie conversion factors. An experimental reassessment of the factors used in the calculation of the energy value of human diets. Brit. J. Nutr. 24:517-535. 1970. Miettinen, T.A. Effects of dietary fibers on ion-exchange resins on cholesterol metabolism in man. In: International Conference on Atherosclerosis. (L.A. Carlson, ed.), Raven Press, New York, NY. p. 193- 198. 1978. Hill, C. L., C. L. Silbernick and F. T. Lindgren. Development of hyperbetalipoproteinemia in pigs fed atherogenic diet. Lipids 10:41-43,1975. Brodribb, A.J. M. and D.M. Humphreys. Diverticular disease: three studies. Brit. Med. J. 1:424-426, 1976. Walters, R.L.. I.M. Baird, P.S. Davies, M.J. Hill. B.S. Drasan, D.A.T. Southgate. J. Green and 8. Morgan. Effects of two types of dietary fiber on fecal steroid and lipid excretion. Brit. Med. J. 2: 536-538. 1975. Pomare, E.H. and Heaton, K.W. Alteration of bile salt metabolism by dietary fiber (bran). Brit. Med. J. 4:262, 1973. McNaughton. J.L. Effect-of dietary fiber on egg yolk, liver and plasma cholesterol concentrations of the laying hen. J. Nutr. 108:1842-1848. 1978. Lopez, A., R. Vial, L. Balart and G. Arroyava. Effect of exercise and physical fitness on serum lipids and 1ipoproteins. Atherosclerosis 20:1-9, 1974. Bobek. P., E. Ginter. J. Cerven, V. Chorvathova and L. Mikos. Effect of infrequent feeding and increased physical activity on cholesterol kinetics in the rat. Physiol. Bohemoslov. 27:145-150. 1978. Palmer, H.J. and M.R. Bennink. Oat bran decreases cholesterol synthesis and biological half-life in chicks. J. Nutr. 109:xix, 1979. (Abstract) Link, W.P., H.M. Pedersoli and A.H. Safanie. Effect of exercise on development of atherosclerosis in swine. Atherosclerosis 15:107-122. 1972. Keneally, M.D., N.L. Jacobson and K.D. Wiggers. The effect of supplemental dietary cholesterol and exercise on blood cholesterol and atherosclerosis in the goat. Atherosclerosis 27:65-69, 1977. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 117 Brown. C.J., J.C. Hiller and J.A. Whatley. Specific gravity as a measure of the fat content of the pork carcass. J. Animal Sci. 10:97-103, 1051. Holman. R.L., H.C. McGill, J.P. Strong and J.C. Geer. Technics for studying atherosclerotic lesions. Lab Invest. 7:42-47. 1958. Simko, V., W.H. Merrifeild and J.R. Stouffer. Mild exercise. Effect on body composition and metabolism. N.Y. State J. Med. 74:1563-1567, 1974. Weiss. G.M., E.R. Peo, Jr., R.W. Mandigo and 8.0. Moser. Influence of exercise on performance and carcass parameters of confinement reared swine. J. Animal Sci. 40:457-462. 1975. Fitts. R.H.. R.G. Cassens and R.G. Kauffman. Effect of exercise on porcine muscle and body composition. J. Animal Sci. 42:854-859. 1977. Holloszy, J. Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J. Biol. Chem. 242:2278-2282, 1967. Barnard, R.J., V.R. Edgerton and J.B. Peters. Effect of exercise on skeletal muscle. 1. Biochemical and histochemical properties. J. Appl. Physiol. 28: 762-766. 1970. Lieberman. D.A., L.C. Maxwell and J.A. Faulkner. Adaptation of guinea pig muscle to again and endurance training. Am. J. Physiol. 222:556-560, 1972. Altekruse. E.B. and J.H. Wilmore. Changes in blood chemistries following a control exercise program. J. Occup. Med. 15: 110-113. 1973. ‘ Oscai. L.B.. J.A. Patterson, D.L. Bogard, R.L. Beck and B.L. Rothermal. Normalization of serum triglycerides and lipoprotein electrophoretic patterns by exercise. Am. J. Cardiol. 30:775-780. 1972. MICHIGAN S TATE UNIV. LIBRARIES Ill 11111 W 11111111 ”11” 1111 11 “WI 931 0065871 9 312