AN ABSTRACT OF THE THESIS OF LISA SCHWEICKHARDT HOLDEN for the degree of Master of Science in Foods and Nutrition Title: presented on March 1 8, 1976 LECITHIN SUPPLEMENTATION AND FREE AND ESTERIFIED CHOLESTEROL IN PLASMA Abstract approved: Dr. Elisabeth/S. Yearick The effect of oral lecithin supplementation on plasma lipids was investigated. Thirty-one healthy middle-aged men received 7.2 gm soy lecithin daily for two months. Blood was drawn both before and after supplementation and analyzed for total and esterified cholesterol, total and individual phospholipids, total lipids, triglycerides, and lecithin:cholesterol acyltransferase (LCAT) activity. Information on dietary intake, physical activity, smoking habits and relative weight was also obtained. The proportion of cholesterol ester increased significantly (p < 0. 01) with supplementation, as did LCAT activity (p < 0. 025). None of the other lipid parameters changed during the experimental period. A positive correlation was found between LCAT activity and the ratio of lysophosphatidyl choline to phosphatidyl choline. Triglycerides were positively correlated with smoking and percent of desirable weight, and negatively correlated with activity level. Higher proportions of cholesterol ester were associated with more vigorous physical activity and with lower relative weights. Lecithin Supplementation and Free and Esterified Cholesterol in Plasma by Lisa Schweickhardt Holden A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed March 18, 1976 Commencement June 1976 APPROVED: Professor of Foods and Nutrition in charge of major Head of ©qpartment of Foods and Nutrition Dean of Graduate School Date thesis is presented March 1 8, 1976 Typed by A & S Bookkeeping/Typing for Lisa Schweickhardt Holden ACKNOWLEDGEMENTS As nay advisor, Dr. Elisabeth Yearick has offered me an invaluable combination of challenge and encouragenaent. Her untiring support of my research, especially during the manuscript preparation, is deeply appreciated. Many thanks are also due to Dr. Margy Woodburn and Dr. Lorraine Miller for their encouragement and interest in my studies. I appreciate very much the cooperation of Joan Halvorson in organizing and initiating the experiment and for the use of her phospholipid data. Thanks also to Gil Won Song for her LCAT data. Mr. Robert Lowry was very helpful in giving insights into the difficulties of cholesterol ester separation, and generously offered the results of his personal experimentation with column chromatography. Spencer Chester and Jason Johnston both helped greatly in the statistical analysis of my data. Mary Yocom has spent many hours critiquing and typing this manuscript and many mo re hours being a loving and stabilizing friend. Special thanks to Robert Holden who has laughed and cried and prayed with me, listened and suggested. He has offered scientific criticism and his bounding enthusiasm. Without his support, this research could not have been accomplished. TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW Plasma Lipids Plasma Lipids and Heart Disease Normal Plasma Lipid Levels Lecithin Lecithin Supplementation Interactions Between Lecithin and Plasma Lipids 3 3 3 5 12 13 15 MATERIALS AND METHODS Experimental Design Subjects Dietary Analysis Additional Information Collected Blood Lipid Determinations Collection of Blood Samples Analysis of Total Cholesterol Analysis of Cholesterol Ester Determination of Total Lipids Phospholipid Determination Analysis of Triglycerides Assay of Lecithin:Cholesterol Acyltransferase Statistical Treatment 19 19 20 20 21 22 22 23 23 25 25 26 26 26 RESULTS AND DISCUSSION Plasma Lipid Concentrations Mean Plasma Lipids Changes in Plasma Lipids Correlations Between Lipid Parameters Dietary Information Mean Dietary Intakes Correlations Between Dietary and Lipid Parameters Plasma Lipids and Additional Variables Conclusions 27 27 27 31 .34 38 38 39 40 42 Pase_ SUMMARY 44 BIBLIOGRAPHY 46 APPENDIX 55 LIST OF TABLES Table Page 1. Plasma lipids in healthy populations. 2. .Description of subjects. 28 3. Plasma lipids and lipid ratios before and after lecithin supplementation 29 Significant correlations among plasma lipids and between plasma lipids and other factors. 35 Significant correlations between lipids and percent change in plasma lipids. 37 Dietary factors most pertinent to lipid metabolism. 39 4. 5. 6. 6 LIST OF APPENDIX TABLES i. ii. iii. iv. v. vi. Complete description of subjects. 55 Plasma lipids and lipid ratios of individual subjects, before and after supplementation (mg/100 ml). 57 Correlation coefficients among plasma lipids and between plasma lipids and other factors. 59 Correlation coefficients between lipids and other factors, and percent change in plasma lipids. 61 Paired_t-statistics to evaluate changes with supplementation. 62 Dietary intake of subjects compared to National Research Council Recommended Dietary Allowances, 1974. 63 LECITHIN SUPPLEMENTATION AND FREE AND ESTERIFIED CHOLESTEROL IN PLASMA INTRODUCTION The epidemiological association between hyperlipemia and cardiovascular disease has prompted a search for means to lower plasma lipids. One agent which has received a great deal of attention recently is lecithin. Popular writers have claimed that soy lecithin, when used as a food supplement, is effective in reducing plasma lipids and even in dissolving cholesterol deposits (Williams, 1971). Recommended dosages in the popular literature vary from 6 gm/day (Davis, 1965) to 24 gm/day (Morrison, 1971). Results of the scientific research done on lecithin supplementation in humans have been contradictory. Morrison (1958) obtained a significant reduction in total cholesterol when he fed 36 gm of lecithin daily to hypercholesteremic patients. Other investigators, using 0.6-2. 4 gm/day, were unable to observe any changes in the plasma lipids of coronary patients (Butler et al. , I960; Enticknap, 1962) and type II hyperlipoproteinemic patients (ter Welle et al. , 1974). Takeuchi and Yamamura (1973) have suggested that supplemental lecithin might stimulate the enzyme responsible for plasma cholesterol esterification, lecithinrcholesterol acyltransferase (LCAT). Higher proportions of cholesterol ester and increased LCAT activity have been observed in healthy subjects compared to coronary patients (Soloff, Rutenberg and Lacko, 1973). Therefore, this study was undertaken to examine the effect of lecithin supplementation on total cholesterol concentrations and on cholesterol esterification. Healthy subjects were used and a lecithin dosage was chosen that avoided the extremes of previous studies and was more typical of the dosages recommended to the lay public. This was part of a larger study in which total lipids, total phospholipids, individual phospholipids, triglycerides and LCAT activity were also measured. In addition, information on dietary intake, activity patterns, smoking habits and body weight was obtained. LITERATURE REVIEW Plasma Lipids Plasma Lipids and Heart Disease Despite recent advances made in determining the pathogenesis of cardiovascular disease, the underlying causes of the disease remain poorly defined. For this reason, epidemiological studies which can help to identify risk factors are of great importance. Elevated plasma lipids have been frequently correlated with heart disease. Plasma cholesterol is the lipid fraction most often associated with heart disease. Doyle (1966) found that the probability of developing cardiovascular disease after the age of 40 increased as the plasma cholesterol increased over 275 mg/100 ml. In an analysis of the Framingham study, cholesterol concentrations of over 260 mg/100 ml were associated with an increasing risk of heart disease (Dawber, Moore and Mann, 1957). There is evidence that even these limits may be too high. Fredrickson et al. (1973) suggested that patients under age 55 with cholesterol values that exceed 220 mg/100 ml warrant a physician's closer scrutiny; such patients may be predisposed to hyperlipoproteinemia, and hence, to heart disease. 4 Some investigators have found triglycerides to be a more important indicator of heart disease than cholesterol. Albrink, Meigs and Man (1961) reported that the incidence of heart disease was much higher with elevated plasma triglycerides than with elevated cholesterol. Hatch et al„ (1966) also found high triglyceride levels associated with heart disease. It has been pointed out, however, that persons with the highest concentrations of individual lipids often have multiple lipid elevations. When triglycerides, cholesterol and lipoprotein levels were examined in a twelve-year follow-up of the Framingham study, no single lipid fraction appeared to increase the risk of heart disease. The highest morbidity was found with multiple lipid abnormalities (Kannel, Castelli, and McNamara, 1967). Abnormalities of lipid ratios have also been observed in heart disease. Morrison, in 1952, found that the phospholipid to choles- terol ratio was higher in normal subjects than in coronary patients. The '. lysophosphatidyl choline (LPC) to phosphatidyl choline (PC) ratio was lower than normal in the plasma of patients with type IV hyperlipoproteinemia (Kunz, Matt and Hackl, 1970) and atherosclerosis (Gillett and Besterman, 1975). Many other risk factors for cardiovascular disease have been identified. Smoking more than 20 cigarettes a day (Hammond, 1966; Kahn, 1966), being more than 20% overweight, having an abnormal electrocardiogram, having increased blood pressure, or having a family history of heart disease are all factors associated with an increased probability of developing heart disease (Doyle, 1966; Kannel et al. , 1967). Normal Plasma Lipid Levels Plasma lipid concentrations vary a great deal between individuals comprising a healthy population. Table 1 summarizes values that have been reported by several different investigators. Some of the observed variability can be attributed to differences in methodology and technique, or to day-to-day fluctuations. However, factors such as sex, age, exercise, smoking habits, stress, obesity and diet have also been found to affect the lipid levels of normal people. Before the age of 20, the cholesterol concentrations of boys and girls are similar. Men between the ages of 20 and 50 tend to have higher cholesterol values than women. surpass men. After 50, however, women This is apparently due to the hyperlipemia associated with menopause (Lopez-S, Krehl and Hodges, 1967). Adlersberg et al. (1956) reported a similar pattern in phospholipid values. In contrast, Bdttiger (1973) found no significant difference between total phospholipid or PC values of men and women. LPC, however, was considerably lower in women at all ages than in men. Table 1. Plasma lipids in healthy populations. Total Cholesterol (mg/100 ml) Reference Ahrens and Kunkel, 1949 Cholesterol Esters (% of total) Phospholipids (mg/100 ml) Triglycerides (mg/100 ml) Total Lipids (mg/100 ml) 25 men and women 185 70 219 636 Deuell, 1955 118 men and women 152 70 165 530 Lindholm, 1956 men, aged 40-59 180 63 250 1101 Adlersberg et al., 1956 230 men, aged 38-57 240 Okey et al., 1960 30 men. aged 50f 201 71 252 Svanborg and Svennerholm, 1961 62 men, aged 16-35 192 67 208 84 609 Bleileret al., 1963 11 men, aged 39-59 272 72 126 men, aged 40-55 (low exercise) 224 201 114 660 Marumoto, 1970 19 men, aged 18-19 138 163 73 464 MacDonald, 1972 10 men, aged 18-21 181 185 104 Bottiger, 1973 30 men, aged 38-63 263 278 122 Miller, 1973 11 men and women 186 73 Billimoria et al., 1975 58 men and women aged 30-70 (non-smokers) 215 80 215 82 Hoffman, Nelson and Goss, 1967 285 64 788 O 7 The plasma lipids of healthy men tend to increase with age. Cholesterol and phospholipids begin to increase dramatically at age 20 and may stabilize by age 33 (Adlersberg et al. , 1956). Lindholm (1956) reported that the rise in total lipids, phospholipids and cholesterol continued into the middle years. This was confirmed by Lopez-S et aL (1967) who found that cholesterol may continue to rise until age 45 or 50. Lipid levels have stabilized by 50 years and may decrease after 60 (Lopez-S et al. , 1967). This decrease probably reflects the greater longevity of individuals with lower lipid levels. There is some disagreement concerning the response of the cholesterol ester fraction to age. The general consensus is that the proportion of cholesterol ester is quite stable in healthy people, ranging between 66 and 75% of the total (Cook, 195 8; Oser, 1965). The results of Adlersberg et al. (1956) agree with this position; no variation was found in cholesterol esters with age. On the other hand, Lopez-S et al. (1967) observed a trend towards a decreased proportion of esterified cholesterol with increasing age. Since total cholesterol tends to increase with age, it is possible that the phenomenon which Lopez-S attributed to age could really be a result of the increased total cholesterol which often accompanies aging. This was in fact the case in a study done on African subjects whose total cholesterol varied "widely. As the total cholesterol increased, 8 the proportion of esterified cholesterol decreased. Total cholesterol had a much greater influence on the percent ester than did age (Leonard, Shaper and Jones, 1965). Exercise has also been found to affect lipid levels. In a study on age-matched men, in which activity was classified by occupation, a highly significant inverse relationship was found between the intensity of exercise and serum cholesterol (Stulb et al. , 1965). Similar results were reported when a dynamic exercise program was introduced into a sedentary life-style. Campbell (1965) found that exercise involving fast, continuous muscular movement was effective in lowering cholesterol in male freshmen participating in a ten-week program. Medical students experienced a significant decrease in triglycerides and pre- (3-lipoproteins and a moderate decrease in cholesterol on a seven-week exercise program (Lopez-S, 1974). Air Force officers, 40 to 55 years old, who exercised daily for a year had lowered triglyceride, cholesterol and total lipids (Hoffman, 1967). Cigarette smoking does not appear to have an immediate effect on blood cholesterol. Page et al. (1959) had 20 adults smoke two cigarettes, inhaling deeply. There were no differences in cholesterol, triglyceride or phospholipid concentrations in blood drawn before and after inhalation. There was also no difference between smokers and non-smokers in their response to the trial. Very different results were found when the lipid levels of chronic smokers and non-smokers were compared. Significant elevations in phospholipids, triglycerides and cholesterol were found in men who smoked over 20 cigarettes per day. These differences were not observed between smoking and non-smoking women (Billimoria et al. , 1975). Dietary composition has been found to profoundly affect plasma lipid levels. Dietary fat increases the amount of exogenous cholesterol that will be absorbed. This is because the secretion of bile acids, necessary for cholesterol absorption, is stimulated by fat in the diet (Goodhart and Shils, 1973). In one study, serum choles- terol increased in rats when fat was included in the diet, despite a constant cholesterol intake (Swell et al. , 1955). The nature of the dietary fat is also important. When saturated fats (S) in synthetic diets were replaced with polyunsaturated fats (P), significant decreases in total lipids, total cholesterol, triglycerides and phospholipids were found (Okey et al. , I960; MacDonald, 1972). The proportion of cholesterol ester was largely unaffected by manipulation of the P/S ratio (Okey et al. , I960). Some inves- tigators have been able to quantify changes in lipid levels. Changes in serum cholesterol were correlated with the square root of the iodine value of the new fat (Keys, Anderson and Grande, 1965). Ahrens et al. , (1957) found that serum levels of cholesterol and phospholipids varied inversely with the iodine value of the dietary 10 fat. Results like these, obtained using synthetic diets, must be interpreted with caution, but studies using natural foods confirm these general trends. People who adhered to the American Heart Association fat-controlled diet experienced a significant decrease in cholesterol, triglycerides and p-lipoproteins. Although the total fat content of the new diet was the same as that of previous diets1 (41% of calories), the change to a diet with polyunsaturated fat resulted in a change of P/S ratio from 0. 4 to 1.6 (Wilson et al. , 1971). Some investigators have been concerned about the fate of the cholesterol which disappears from the serum as a result of a polyunsaturated diet. Connor et al. (1969), using a sterol balance technique, found that the fecal sterols increased when the iodine value of formula diets was increased. These results were confirmed by Nestel et al. (1975) who reported that the increased sterol excretion occurred during the first three weeks after the change to a polyunsaturated diet; after three weeks, a steady state was reached. Hellstrom and Lindstedt (1966) found no increased sterol excretion when they examined the bile of subjects on a polyunsaturated diet. However, they waited three weeks before collecting bile and may therefore have missed the excretion stage. There is general agreement that dietary cholesterol affects plasma cholesterol, but there is some disagreement about whether 11 this is a significant phenomenon. Keys, Anderson and Grande (1965) found that plasma cholesterol responded minimally to changes in cholesterol levels in synthetic diets. In contrast, Connor, Stone and Hodges (1964) found a significant decrease in cholesterol when the cholesterol content of a natural diet was reduced. Adherents to the American Heart Association diet experienced a decrease in serum cholesterol proportional to the decrease in dietary cholesterol (Wilson et al. , 1971). The more dramatic results found by Connor and Wilson and their colleagues may be due to their use of natural diets. The cholesterol of egg yolk was found to be more hyper- cholesteremic than equal amounts of pure cholesterol dissolved in oil, even when the quantity and quality of fat was controlled in the diets (Connor, Stone and Hodges, 1964). Although the effect of obesity on the incidence of heart disease is well documented (Doyle, 1966; Kannel et al. , 1967), the effect on lipid concentrations is less clear. Subscapular skin-fold thickness was highly correlated to plasma cholesterol in young men (Tanner, 1951). However, Thomas (I960) found no difference in serum cholesterol levels of young males when compared with weight, weight corrected for build, or radiographic outer-fat shadow measurements. An additional factor which may affect lipid levels is stress. Thomas and Murphy (1958) examined first-year medical students 12 immediately after an examination considered to be the most stressful one in medical school, and again during a less stressful period. They found the mean cholesterol level at exam time to be 10% higher than when less academic pressure was present. Some workers have suggested that seasonal change may be responsible for some of the variability of blood lipids. Thomas, Holljes and Eisenberg (1961) studied cholesterol concentrations in young male prisoners over a year's time. Cholesterol tended to be higher in November, December and January and lower in the warmer months. No differences in activity patterns or stress seemed to be responsible. However, the possibility that fat intake had increased during the winter months was suggested. In another study, the cholestdrol levels of men were highest from March to May (Bleiler et al. , 1963). Possible changes in dietary intake with seasonal change were not examined in the study. No significant differences were found when total lipids, cholesterol, phospholipids and triglycerides were measured in middle aged men at different times of year (Hoffman et al. , 1967). It seems likely that seasonal differences which have been observed are secondary to other changes. Lecithin The term "lecithin" is commonly used in two different ways. It is used to refer to the specific phospholipid, phosphatidyl choline, 13 and also to the general class of phospholipids which are soluble in most organic solvents except acetone. In the following pages, lecithin will refer to the extractable class of phospholipids, while phosphatidyl choline (PC) will be referred to by name. It is interesting to note that the most abundant phospholipid in lecithin is PC, composing 29-38% (Erdahl, Stolyhwo and Privett, 1973; Halvorson, 1976). Lecithin is found naturally in many foodstuffs and is extracted commercially from soybeans. Many claims have been made about its effectiveness as a dietary supplement. It is purported to dissolve cholesterol deposits (Williams, 1971), to lower cholesterol by enhancing fat metabolism and transport, and to promote feelings of well-being (Morrison, 1971). Adelle Davis (1965) describes lecithin as helpful in dealing with such varied conditions as obesity, multiple sclerosis and nephritis. Dosages recommended to the lay public vary from 6 gm/day (Davis, 1965) to 24 gm/day (Morrison, 1971). Lecithin Supplementation When lecithin has been used to treat animals and humans, varied results have been reported in the scientific literature. Intravenous infusions of lecithin given to rats caused an increase in both plasma cholesterol and plasma phospholipids (Friedman and Byers, 1956). The increase in cholesterol occurred even when the 14 liver was absent, implying that cholesterol was diffusing out of other parts of the body (Friedman, Byers and Rosenman, 1957). However, Howard and Patelski (1974) examined the aortas of atherosclerotic baboons after an infusion of lecithin and found no regression of aortic lesions as judged by cholesterol content. Since these studies were all done on animals, their direct applicability to human beings is questionable. One of the early lecithin studies using humans was done by Morrison in 1958. Twenty-one hypercholesteremic patients were given 36 gm lecithin daily for three months. They had all been on a low fat (less than 25 gm/day) diet for a year or more and had been treated unsuccessfully with other hypocholesteremic agents. Six of the patients quit the study because of intolerance to the lecithin dosage. The 15 who remained experienced significant decreases in plasma cholesterol and total lipids and an increase in phospholipids. On the other hand, a commercial phospholipid preparation (lipostabil ) was unsuccessful in changing 10 blood parameters when given to 114 coronary patients for a period of more than two years (Enticknap, 1962). However, the dosage was considerably lower than that given by Morrison (1958), 0. 6-1. 8 gm/day. Butler et ah (I960) also reported no change in plasma cholesterol or phospholipids Aspro-Nicholas, Slough, Bucks, England. 15 when lipostabil was given to 50 coronary patients. Unfortunately these investigators did not report the amount of phospholipid given. Most recently, ter Welle et al. (1974) have given lecithin supplements to 12 persons with type II hyperlipoproteinemia. After 8 months of supplementation with 1. 2-2.4 grams lecithin per day, no changes in blood lipids were found. different studies. day. It is difficult to draw conclusions from these The dosages varied from 0.6 gm to 36 gm per The subjects' previous medical history and diets are additional variables. Since these studies were done on persons with abnormal lipid conditions, it is impossible to know what effect lecithin supplementation would have on normal subjects. Interactions Between Lecithin and Plasma Lipids Other research has suggested possible mechanisms by which lecithin might affect lipid metabolism. One of the earliest theories relating to lecithin supplementation was based on the phospholipid/ cholesterol ratio in serum. It was found that serum turbidity was related to the low phospholipid content of serum and not to total lipid content (Boyd, 1937). Coronary patients had a greater frequency of serum turbidity than did controls (Horlick, 1954) and a lower phospholipid/cholesterol ratio (Morrison, 1952). Morrison (1958) was able to significantly increase the phospholipid/cholesterol ratio of 15 hypercholesteremic patients by feeding them 36 gm of lecithin 16 daily. Studies using smaller doses, however, have been unsuccessful in modifying the phospholipid/cholesterol ratio (Butler et al. , I960; Enticknap, 1962; ter "Welle et al. , 1974). It has been theorized that lecithin may stimulate the lecithin: cholesterol acyltransferase (LCAT) reaction in plasma. LCAT is an enzyme which catalyzes the transfer of a fatty acid from the C-2 position of phosphatidyl choline (PC) to the hydroxyl group of free cholesterol, forming lysophosphatidyl choline (LPC) and esterified cholesterol. Cholesterol is esterified preferentially from high density lipoproteins compared with other lipoproteins (Glomset, 1968). The LCAT reaction is responsible for the formation of almost all the cholesterol ester in plasma (Glomset, 1970). Coronary patients have been found to have a lesser ability to esterify cholesterol than controls. This was shown by a lower rate of serum esterification during in vitro incubation and a lower fraction of cholesterol ester in the serum (Soloff, Rutenberg and Lacko, 197 3). Animal studies have indicated that feeding lecithin causes an increase in serum cholesterol esterification (Takeuchi and Yamamura, 1973). One theory, therefore, is that supplemental lecithin could protect against heart disease by increasing the serura LCAT activity. Another theory is that LCAT may promote clearance of free cholesterol from peripheral tissues. Murphy (1962) showed that when serum and erythrocytes were incubated together, there was a 17 flux of free cholesterol out of the red blood cells. The amount of free cholesterol lost from the cells was related to the amount of cholesterol ester that appeared in the serum. This flux is presumably an equilibrium response, the free cholesterol from erythrocytes replacing that which is esterified by the LCAT reaction. If a similar equilibrium existed between serum and arterial tissues, then the increased proportion of cholesterol esters formed by LCAT in serum could help remove free cholesterol from arteries. Ruten- berg and Soloff (1971) tested this hypothesis by incubating healthy arterial segments in serum. into the serum. They observed a flux of free cholesterol This theory could explain the studies in which intravenous infusions of lecithin caused an increase in plasma cholesterol and apparently a diffusion of cholesterol out of peripheral tissues (Friedman and Byers, 1956; Friedman, Byers and Rosenman, 1957). Increased LCAT activity in plasma would have the effect of increasing the lysophosphatidyl choline (LPC) and decreasing the phosphatidyl choline (PC). Some recent research has focused on changes in the LPC/PC ratio in atherosclerosis. Portman et al. (1970) found that the LPC/PC ratio and LCAT activity were higher in the plasma of monkeys with nutritionally-induced atherosclerosis than in the controls. However, Mohan and Chakravarti (1975) compared the phospholipid profiles of rhesus monkeys with 18 spontaneous and with diet-induced1 atherosclerosis. They found the LPC/PC ratio of monkeys with spontaneous atherosclerosis to be much lower than the non-diseased group, whereas induced atherosclerosis caused only a slight decrease. The work of Gillett and Besterman (I 975) confirms these findings. Their data indicate that the LPC/PC ratios of humans with chronic or acute ischemic heart disease, peripheral arterial disease or myocardial infarctions were considerably lower than normal controls. The differences between the Portman et al. (I 970) study and the ones done by Mohan and Chakravarti (1975) and Gillett and Besterman (I 975) seem to be the result of differences between natural and induced atherosclerosis. 19 MATERIALS AND METHODS Experimental Design Dietary supplements of lecithin were given to a group of middle-aged men for a two-month period beginning in January, 1975,, Each subject received 7. 2 gm of lecithin per day in the form of gelatin capsules. 2 Two capsules containing 1.2 gm soy lecithin in soybean oil were taken three times a day. This dosage was selected because it is typical of recommended supplemental dosages. Blood samples were drawn both before and after the period of supplementation to determine the effects of lecithin on blood lipids. Duplicate sampling was done to minimize the effects of normal daily fluctuations in plasma lipids; two blood samples were taken within three days of each other before supplementation began and two more were taken at the end of the study. Each sample was analyzed separately, but values were reported as averages of the two samples obtained in the same week. The experimental plan was approved by the Committee for the Protection of Human Subjects in accordance with the DHEW guidelines (DHEW, 1971). 2 "Natural Needs1' Soya Lecithin, Western Wholesalers, Portland, Oregon. 20 Subjects Thirty-one men between the ages of 38 and 56 participated i n the study. By limiting the subjects to men of one age group, it was hoped that differences in lipid patterns due to age and sex would not mask possible changes attributable to lecithin. The men were healthy, with no history of heart disease and no known abnormality of lipid metabolism. lipid metabolism. They were not taking any drugs which alter The subjects agreed to maintain constant patterns of dietary intake and physical activity. Participants were not chosen by random selection; instead, men volunteered who were interested in the research topic. Dietary Analysis To obtain information about their usual dietary habits, the subjects were asked to record three-day dietary intakes, both at the beginning and at the end of the study. Each three-day period consisted of two weekdays and one weekend day, since food consumption is often very different on weekends. The diets were analyzed by computer, using a nutrient data bank compiled at Ohio State University, essentially from information in Watt and Merrill (1963). The following nutrients were computed in the diets: Calories, protein, total fat, saturated fat, linoleic and oleic acids, calcium, 21 Vitamin A, thiamin, riboflavin, niacin, ascorbic acid, and iron. In addition, the cholesterol content of the diets^was calculated from the table compiled by Feeley, Criner and Watt (1972). Dietary information was reported as an average of the two three-day records. Additional Information Collected Activity levels were determined from information supplied by the subjects about the nature of their work, their planned exercise and sports, and their mode of travel to and from work. The subjects were assigned to one of the activity groupings defined by Bogert, Briggs and Galloway (1973): 1) sedentary, 2) light activity, 3) moderate activity, and 4) strenuous activity. The energy demands of a subject's occupation determined the group into which he was initially placed. In addition, if he undertook 45 minutes of strenuous exercise at least three times a week, he was placed in the next higher category. Percent of ideal weight was calculated from the subjects' height and weight according to the Metropolitan Life Insurance Company tables (Keys and Grande, 1973). assumed to be of medium frame. The subjects were If a subject was overweight, the percentage of ideal weight was calculated from the highest number of the suggested range. used. If he was underweight, the lowest number was 22 Subjects were classified according to their smoking habits. If a person smoked 20 cigarettes or more per day, he was called a "smoker". Those who smoked infrequently or not at all were "non- smokers". Blood Lipid Determinations Collection of Blood Samples Blood samples were collected in the morning after an overnight fast. Twenty milliliters of venous blood were drawn into Vacutainers containing ethylenediaminetetraacetic acid (EDTA) as anti-coagulant. The blood was stored on ice until it could be returned to the laboratory. Hemoglobin and hematocrit were determined once at the beginning of the study and once at the end to give an indication of overall nutritional status. Hemoglobin was measured by the cyanomethemoglobin method (Oser, 1965), and hematocrit was measured according to the method described by Richterich (1969). The blood was centrifuged at 35, 000 RPM for 35-40 minutes. The plasma was removed and stored in capped vials at -10 C until further analyses could be carried out. 23 Analysis of Total Cholesterol Total cholesterol was determined on the Technicon AutoAnalyzer according to the method of Block, Jarrett and Levine (1966). This method is based on the reaction of steroids having the 5-ene, 3(3-ol grouping with concentrated sulfuric acid and ferric chloride in glacial acetic acid (color reagent). (1:20 in isopropanol) were prepared in advance. Plasma extracts The extracts were mixed on the AutoAnalyzer with preheated color reagent (95 C) and the absorbance measured at 550 nm in a tubular flow cell. Total cholesterol was calculated from the regression equation determined from standards. Analysis of Cholesterol Ester Esterified cholesterol was first separated chromatographically from free cholesterol and then measured on the AutoAnalyzer. In preparation for chromatographic separation, 0. 5 ml of plasma was extracted into 10 ml of isopropanol. Two 3-ml aliquots of the extract ■were dried under purified nitrogen gas in a 50 C water bath. were used for duplicate cholesterol ester analyses. These In addition, 2-ml of the same extract were stored in an AutoAnalyzer sampling cup for total cholesterol analysis. 24 Lowry's 3 adaption of the column chromatography method of Fillerup and Mead (1953) was modified for use in our laboratory. A glass blower was asked to construct glass columns having a twenty milliliter reservoir above a 10x70 mm tube. The columns were dried thoroughly and filled with silicic acid (100-200 meshBio-Sil A) which had been heated overnight at 120 C The columns were tapped and tamped to insure uniform packing and then rinsed with hexane. The dried isopropanol extracts were dissolved in hexane and transferred quantitatively to the column. The cholesterol ester was eluted, first with 10 ml of 2. 5% diethyl ether in hexane, followed by 5 ml of 10% diethyl ether in hexane. New solvent was added only after the previous solvent had passed through the column. Eluates were collected in tubes and dried under nitrogen gas in a 50 C water bath. For analysis on the AutoAnalyzer, the cholesterol ester was redissolved in two milliliters of isopropanol and transferred to sampler cups. Each cholesterol ester sample (in duplicate) was run on the AutoAnalyzer in juxtaposition to the corresponding total cholesterol sample. This eliminated any slight variability that would arise from differences in standards or in instrument behavior. Concentrations were calculated in the same way as total cholesterol, and the cholesterol ester was calculated as percent of total. 3 Robert Lowry, personal communication. 25 Precision and accuracy were examined by running several consecutive assays of Hyland control serum. The assigned choles- terol ester value of this lot of Hyland serum was 78% of total cholesterol. The modified method described above yielded a mean of 78% + 1.6% (p < 0.05). Determination of Total Lipids Total lipids were extracted with chloroform and methanol (2:1 v/v) according to the method of Chiu (1969). The extract was reduced under purified nitrogen gas in a 50 C water bath, transferred quantitatively to a tared one-milliliter volumetric flask, and dried under nitrogen. Total lipids were determined gravimetrically. The lipid extracts were stored at -10 C. Phospholipid Determination The chloroform/methanol lipid extract was used for phospholipid analysis. Total phosphorus was measured according to the method of Lowry et al. (1954) as modified by Hawthorne, Smith and Pescadore (1963). Individual phospholipids were separated by thin-layer chromatography, the spots removed and the phospholipid fractions quantified by the total phosphorus method. 4 Analysis done by Ms. Joan Halvorson. 4 26 Analysis of Triglycerides Triglycerides were analyzed by the micromethod of Van Handel 5 and Zilversmit (1957), using the chloroform/methanol lipid extract. Assay of LecithimCholesterol Acyltransferase Plasma LCAT activity was determined according to the method of Stokke and Norum (1971). Statistical Treatment The paired t-test was used to compare the means of plasma lipid concentrations before and after supplementation. A one-tailed, unpaired_t-test was used to determine if the change in LCAT activity was significantly greater than zero. In addition, correlation coefficients were determined for all pertinent combinations of variables (Snedecor and Cochran, 1973). 5 Analysis done by Dr. Elisabeth Yearick. Analysis done by Ms. Gil Won Song. 27 RESULTS AND DISCUSSION A description of the participants is summarized in Table 2. In addition, the data for individual subjects appears in Appendix Table i. The subjects were, on the average, slightly overweight (4% above ideal weight). Three individuals were less than 100% of their ideal weight for height while fourteen were more than 100%; only one subject was 25% above ideal weight and could be considered obese. Twenty-one of the thirty-one subjects had light physical activity patterns. Four were sedentary, three were moderately active, and three engaged in strenuous activity. The predominance of low activity levels is undoubtedly due to the fact that 26 of the subjects were university professors and therefore confined to an office most of the day. All of the subjects had hemoglobin and hematocrit values within the ranges found in healthy men (Oser, 1965; Wintrobe, 1961). Plasma Lipid Concentrations Mean Plasma Lipids The means and ranges for plasma lipids are found in Table 3, while individual data are detailed in Appendix Table ii. Total cholesterol averaged 206 mg/100 ml before and 210 mg/100 ml 28 Table 2. Description of subjects. Age Relative Weight13 Activity Level a Mean Range 46 38-56 104% 2 Hemoglobin gm/100 ml 15.9 Hematocrit % 45 94-129% 1-4 14.1-17.3 40-48 a. Four out of thirty-one subjects were smokers; the rest, nonsmokers. b. Percent of desirable weight for height from the Metropolitan Life Insurance Company tables (Keys and Grande, 1973). c. Classification of activity levels: 1 = sedentary, 2 = light activity, 3 = moderate activity, 4 = strenuous (Bogert, Briggs and Galloway, 1973). 29 Table 3. Plasma lipids and lipid ratios before and after lecithin supplementation. Variables No. Mean S.D. Range Total Cholesterol (mg/100 ml) Before 31 206 36 122-266 After 31 210 37 102-288 Percent Cholesterol Ester Before 31 66 2 60-70 After 31 67 3 59-74 Total Lipids (mg/100 ml) Before 31 655 164 287-1282 After 31 645 133 372-1146 Phospholipids (mg/100 ml) Before 31 181 32 89-268 After 31 187 27 132-263 Triglycerides (mg/100 ml) Before 31 91 43 22-209 After 31 106 59 40-285 Phospholipid/Cholesterol Before 31 0.88 0.12 0.72-1.18 After 31 0.90 0.11 0.73-1.29 Before 12 0.14 0.05 0.06-0.23 After 12 0.12 0.03 0. 08-0. 18 (%) LPC/PC LCAT Activity b Change as percent of initial value. Data supplied by Gil Won Song. 12 Percent change3 2% 2% (p<.01) -2°/o 3% 16% 2% -9% 11% (p<0. 025) 30 after supplementation. Other investigators have obtained similar means for middle-aged men (Okey et al. , I960; Hoffman, Nelson and Goss, 1967). One subject had considerably lower cholesterol concentrations than the other subjects: 122 and 102 mg/100 ml. Eight of the subjects had concentrations greater than the 220 mg/100 ml limit proposed by Frederickson et al. (1973), although for only two of them were the concentrations consistently above the 260 mg/ 100 ml level associated with increased risk of heart disease (Dawber, Moore and Mann, 1957). Sixty-six percent of the cholesterol was in the ester fraction prior to the study and 67% at the conclusion. Although these values are at the lower end of the normal range given by Oser (1965), other studies report similar means (Lindholm, 1956; Svanborg and Svennerholm, 1961). The mean concentrations of total lipids, phospholipids and triglycerides are comparable to those reported in the literature (Hoffman, Nelson and Goss, 1967; Billimoria et al. , 1975). It is interesting to note that the subject with the lowest cholesterol concentrations also had extremely low levels of the other lipid fractions. The eight subjects who had high cholesterol also tended to have elevated concentrations of the other lipids. Pertinent ratios were calculated from the raw data. The LPC/PC ratio decreased from an average of 0. 137 to 0. 124 during 31 the experimental period (Table 3). Both of these ratios are similar to those found in healthy, middle-aged men (B'ottiger,-, 1973; Gillett and Besterman, 1975). averaged 0. 88 and 0. 90. Phospholipid/cholesterol ratios When phospholipid/cholesterol ratios are calculated from other studies, a ratio greater than one is almost always obtained (Okey et al. , I960; Marumoto, 1970; BSttiger,'. 1973). In addition, Morrison (1952) found that healthy controls had phospholipid/cholesterol ratios greater than one, whereas coronary patients had ratios less than one. However, a ratio of 0. 90 can be calculated from the data of Hoffman, Nelson and Goss (1967). Hoffman's study involved middle-aged Air Force officers with sedentary jobs in the Pentagon. These subjects may be more com- parable to the subjects in the present study, most of whom also have sedentary and prestigious positions. Since all the men, both in the Hoffman study and in the present study, are apparently healthy it is possible that the calculated phospholipid/cholesterol ratio is normal when variables of sex, age and occupation are held constant. Changes in Plasma Lipids The degree that each lipid fraction changed during the period of lecithin supplementation, expressed as the percent of the initial concentration, is also found in Table 3. Although most of the lipid fractions increased during the experimental period, only one 32 increased significantly (the _t-statistics are found in Appendix Table v). The 2% increase in the proportion of cholesterol ester was highly significant (p < 0.01). This finding suggested that lecithin supple mentation had stimulated the cholesterol esterifying mechanism. Soloff, Rutenberg and Lacko (1973) had demonstrated an association between cholesterol esterification and LCAT activity in men who had varying levels of these two factors. Data were available on LCAT activity in plasmas of 12 of the subjects in the present study. As shown in Table 3, there was a significant (p < 0.025) increase of 11% in LCAT activity. This suggested that the phosphatidyl choline contained in soy lecithin was absorbed in sufficient quantities to stimulate the enzyme for which it is a substrate. Takeuchi and Yamamura (1973) also found increased LCAT activity in rats after lecithin feeding. It is interesting to note in the present study that there is a range of 10% in cholesterol ester values before supplementation but a range of 15% afterwards. This increased variability implies that individuals respond differently to the stimulus of lecithin supplementation. Although most of them experienced an increase in cholesterol ester, eight showed a decided decrease. The concentrations of total cholesterol, total lipids, phospholipids and triglycerides did not change significantly with supplementation. Morrison (1958) was able to demonstrate significant changes 33 in cholesterol, phospholipids and total lipids by feeding his subjects 36 gm of lecithin daily. On the other hand, other investigators have found no change in these lipids after lew-level (< 2 gna/day) lecithin supplementation (Enticknap, 1962; ter Welle et al. , 1974). An intermediate dose of 7.2 gm/day was used in the present study. It seems likely, therefore, that the effect of lecithin on these lipids is dose-related and that 7. 2 gm/day was not sufficient to elicit the response Morrison obtained using 36 gm/day. Neither LCAT nor cholesterol ester, the two parameters which changed significantly with supplementation, have been measured in human lecithin studies (Morrison, 1958; Butler et al. , I960; Enticknap, 1962; ter Welle et al. , 1974). The results of the present study suggest that LCAT and cholesterol ester measurements should be included in future investigations. There are other factors which may account for the lack of significant change in total cholesterol, phospholipids and triglycerides. The hypercholesteremic effect of stress (Thomas and Murphy, 1958) may have counteracted the possible cholesterollowering effect of lecithin. The beginning of the study coincided with the beginning of the school term, a relatively calm time for university professors. The final blood samples were taken during the last two weeks of the term, when the academic staff is under a great deal of pressure. The stress factor was therefore increasing throughout the 34 course of the study. Although the effect of seasonal variation on lipids was inconclusive in previous studies (Thomas et al. , 1961; Bleiler et al., 1963; Hoffman et al. , 1967), the possibility that the change in season during this study affected the lipid patterns cannot be excluded. Correlations Between Lipid Parameters Significant correlations among plasma lipids are presented in Table 4. All of the correlation coefficients appear in Appendix Tables iii and iv. There were strong positive correlations between total lipids, total cholesterol, phospholipids and triglycerides, both before and after supplementation. Other investigators have also noted the tendency for elevations in one lipid to be associated with increases in other lipids (Kannel, Castelli and McNamara, 1967; Lindholm, 1956). The cholesterol ester fraction was negatively correlated with triglycerides and total lipids. This supports the work of Lopez-S et al. (1967) who observed that the percent cholesterol ester tended to decrease as other lipids increased with age. Another study indicated that the proportion of cholesterol ester varied inversely with total cholesterol concentration (Leonard, Shaper and Jones, 1965). Although high concentrations of cholesterol tended to be associated with lower proportions of cholesterol ester, this Table 4. Significant correlations among plasma lipids and between plasma lipids and other factors. Total cholesterol Before After Total Cholesterol ^Cholesterol Ester Before After Total Lipids Before After Phospholipids Before After Triglycerides Before After Phospholipid/ Cholesterol Before After Dietary Cholesterol Before After % Cholesterol Before Ester After Total Lipids Before After Phospholipids Before After Triglycerides Before Dietary P/S Activity Level % Desirable Weight Smoking Correlation coefficients were significant at the p <0.05 level and positive (+) or negative (-), or they were significant at the p < 0. 01 level and positive (++) or negative (—). 36 relationship was not statistically significant. Perhaps more distinct patterns would have emerged with a larger sample size. Significant correlations between the plasma lipids and the percent change following lecithin supplementation are shown in Table 5. Increases in triglycerides correlated with increases in total lipids and phospholipidso The change in phospholipids also cor- related with the change in total lipids. This further supports the observation that lipid fractions tend to rise and fall together. Higher initial concentrations of total lipids, triglycerides, total cholesterol and phospholipids were associated with a greater decline in total lipids as a result of supplementation. Initial phospholipid concen- trations were also negatively correlated with the change in phospholipids and in triglycerides. This indicates that the initial concen- tration of these lipids influenced the direction of change undergone by certain other lipids. The change in LCAT enzyme activity was positively correlated with the change in LPC/PC ratio (p < 0.05). This correlation occurred despite the fact that mean LCAT activity increased significantly with supplementation whereas the mean LPC/PC ratio decreased slightly. The direction of LPC/PC change was not con- sistent, perhaps because of the small number of samples used for this analysis (n = 12). Nevertheless, when a change did occur, it was associated with a change in LCAT activity in the same direction. Table 5. Significant correlations between plasma lipids and percent change in plasma lipids. Total Cholesterol Total Cholesterol % Cholesterol Ester Before After Percent Change Total Phospholipids lipids — ++ Total Lipids Before — Phospholipids Before ~ Triglycerides Before - LPC/PC Before LPC/PC - % Cholesterol Ester After Phospholipid / Before Cholesterol After Triglycerides ~ ++ - + ++ ++ - % Change in Phospholipids % Change in Triglycerides % Change in LPC/PC Correlation coefficients were significant at the p <0.05 level and positive (+) or negative (-), or they were significant at the p <0. 01 level and positive (++) or negative (--). LCAT 38 It is to be expected that LCAT activity and the LPC/PC ratio would be related since an increase in LCAT activity would be followed by an increase in LPC and a decrease in PC concentrations. Indeed, parallel changes in LCAT and LPC/PC ratios have been reported previously (Portman et al. , 1970). Dietary Information Mean Dietary Intakes Average dietary intakes for individual subjects are presented in Appendix Table vi. Twenty-two of the thirty-one subjects com- pleted dietary records both at the beginning and end of the study. Six participants returned only one diet record. The reported intakes were compared to the National Research Council Recommended Dietary Allowances (1974) for middle-aged men. All of the diets were adequate in Calories, protein, iron, niacin and ascorbic acid. Calcium and vitamin A intakes were low (less than 67% of the RDA) in three cases, thiamine was low in four cases and one subject had a low intake of riboflavin. A qualitative examination of the diets suggested that a limited consumption of milk and vegetables was responsible for these low-nutrient intake's. The means and ranges of the dietary factors most pertinent to lipid metabolism are found in Table 6. Fat as percent of Calories averaged 38% and 418 mg cholesterol were consumed per day. The average American eats slightly more fat and cholesterol than these subjects, with 40% of the Calories as fat and 533 mg dietary 39 cholesterol per day (National Diet-Heart Study Final Report, 1968; Friend, 1967). The P/S ratio in the typical American diet is 0.25 (National Diet-Heart Study, 1968) compared to a mean of 0. 37 in the present study. Because high cholesterol and saturated fat intakes have been linked with elevated plasma cholesterol, the Food and Nutrition Board (1972) has issued a statement advising that persons in coronary risk groups increase the P/S ratio and decrease the cholesterol content of their diets. It seems, therefore, that the subjects in this study had diets slightly less atherogenic than the American norm. Table 6. Dietary factors most pertinent to lipid metabolism. Mean Dietary fat as percent of Calories P/S ratio Dietary cholesterol (mg/day) Range 38+6% 2 3-48% 0.37+0.22 0.12-1.15 418+156 200-783 Correlations Between Dietary and Lipid Parameters Very few significant correlations were observed between dietary intake and plasma lipids (Table 4). Higher cholesterol intakes were associated with lower phospholipid and total lipid concentrations after supplementation. In contrast to previous studies 40 (Connor, Stone and Hodge, 1964; Wilson et aL , 1971), no relationship between dietary cholesterol and plasma cholesterol was found. It was also expected, based on the findings of Okey et al. (I960) and MacDonald (1972), that the amount and quality of dietary fat would affect plasma lipids. However, no significant correlations were observed between plasma lipids and the fat as percent of Calories or the P/S ratio. This is in part due to the wide variation that existed in the self-chosen diets, whereas the investigators cited above used a metabolic ward or more rigidly controlled diets. Other variables, such as activity levels, body weight and stress, were not controlled in the present study and may have influenced the plasma lipid concentrations sufficiently to mask the effect of the dietary parameters. Plasma Lipids and Additional Variables The average activity level and relative weight are given in Table 2. Significant correlations between these variables and plasma lipids are presented in Table 4. More vigorous physical activity ■was associated with lower initial triglyceride concentrations and with higher proportions of cholesterol ester. This confirms the work of Lopez-S (1974) and Hoffman (1967) who have reported lower triglycerides in subjects participating in daily exercise programs. However, these investigators also found that exercise decreased 41 plasma cholesteroL the present study. No such hypocholesteremic effect was noted in The significant relationship observed between cholesterol ester and physical activity is not comparable to previous studies since neither Lopez-S nor Hoffman measured cholesterol ester. Triglyceride concentrations and the final total lipid concentration correlated positively with relative body weight. The initial proportion of cholesterol ester, on the other hand, was lower in those whose relative weight was high. Other investigators have measured only cholesterol when evaluating the effect of relative weight on plasma lipids. Tanner (1951) established a highly signi- ficant relationship between cholesterol and the thickness of supscapular subcutaneous tissue. On the other hand, Thomas (I960) found no difference in the cholesterol concentrations of young men with different relative weights. The present study shed no light on this controversy; no correlation was observed between total cholesterol and body weight. Triglyceride was the only plasma lipid which correlated significantly With smoking habits. The four subjects who smoked all had elevated triglyceride concentrations. The hypertriglyceridemic effect of smoking has been demonstrated by Billimoria et al. (1975). These investigators also found significantly increased concentrations of phospholipids and total cholesterol in chronic smokers. Although 42 cholesterol and phospholipids tended to be higher in the smokers in this study, the relationship was not statistically significant. This study indicates that triglycerides and the proportion of cholesterol ester are more closely associated with activity, body weight and smoking than is the total cholesterol. It has been demon- strated that elevated triglycerides have predictive value for heart disease (Albrink, Meigs and Man, 1961; Hatch et al. , 1966). It has also been suggested that a decreased proportion of cholesterol ester raay be associated with atherosclerosis (Soloff, Rutenberg and Lacko, 1973). For these reasons, it seems important that cholesterol ester and triglyceride analyses be included in studies examining the salutory effects of exercise, weight reduction and decreased smoking. Conclusions In this study, dietary supplementation with soy lecithin was effective in increasing the proportion of cholesterol ester and the LCAT activity in plasma. It is impossible to know if these changes improved latent atherosclerotic conditions since there is no way to examine the arteries of human subjects. There was also no way of knowing if the changes brought about by lecithin supplementation decreased the likelihood of developing heart disease, since this was a short-term study with only healthy subjects. Nevertheless, the results of other studies which have compared healthy and diseased 43 subjects or have been done with animals suggest that an increase in cholesterol ester and LCAT activity may indeed be beneficial. More research needs to be done to clarify the interaction between lecithin and plasma lipids. Since the action of lecithin appears to be a dose-related phenomenon, it might be helpful to use different dosages of lecithin. A larger sample size, stricter controls on dietary intake, physical activity and smoking habits could help to decrease the variability of the data and make it easier to identify which effects can be attributed to lecithin. The results of this study demonstrate that cholesterol ester analysis should be carried out routinely in investigations dealing with interactions of plasma lipids with dietary and supplementary components or with descriptive parameters. 44 SUMMARY The effect of lecithin supplementation on free and esterified cholesterol in plasma was studied. Thirty-one apparently healthy middle-aged men were given 7.2 gm/day "Natural Needs" soy lecithin. Blood samples were drawn both before and after the two- month period of supplementation. The blood was analyzed for total cholesterol, cholesterol ester, total lipids, total phospholipids, individual phospholipids, triglycerides and LCAT enzyme activity. Information was also collected about the subjects' diets, physical activity, smoking habits and relative body weight. Changes in plasma lipids with supplementation were evaluated. Significant increases were observed in the proportion of cholesterol ester (p < 0. 01) and in the LCAT activity (p < 0. 025). It was sug- gested that the increase in plasma enzyme activity might be due to the increase of one of the substrates for the enzyme, lecithin, in the diet. Since LCAT is largely responsible for cholesterol esteri- fication in plasma, the increased proportion of cholesterol ester was explained by the increased LCAT activity. None of the other lipid parameters changed significantly during the experimental period. This may be because lecithin has no effect on these para- meters or may be due to the contradictory influences of uncontrolled variables. 45 Correlation coefficients were calculated on all pertinent combinations of variables. Strong positive correlations were observed among all lipid parameters, excepting cholesterol ester as percent of total. The cholesterol ester fraction varied inversely with most other lipids. 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APPENDIX ss O tn OJ ■U i^ tn *>• 0^ w O O O O O ^ Oi i- *. •vi O O fe O O 00 >0 0^ !-■ O O O w N ^> oo OOOi-O^OOO OOOi^.«3^00 ^^.^■I^Ln^^uicnvi cnu^o^^-»^-*CnO^-l M M V0 ^O iU Cft VO m wi CTi m o o o <o o >b ^ n> ni ro ft B B o B fD *" fl> X" X- fl> IT (t> IT go go go go go B I o I B o B I B o B wx'iricirx-x'xtcirx'sc goooooooooooo g g g g 3 3 3 3 3 3 1 B 0 B IV>00OlMO^CTlCTia>OC>it».^tnUli(>.00>Uv]O>*.0\CTivlU1^.00Ci^J ^.to>oroo~JooMi-'Oiooi\)0)Voo\to*.M*-odi-'MO>^o~-]^ tnvjtnuivicntnOi>^.tntntnuic/iUivjtnvjoitno>(/iCTiUiuiv)oi5i ft> 0 0 ro re o B I O B 1 0 K>WM<JJrJIV>N)K>WhJ*.N>N)>-'M^ISJMl-*t\)NIN>^NJWN) i-» ^ g g 1o os g rOl— O tn ^ o vovjo^cnj^wroo^oo-^dcn^twisj*-* u> O m 4s. O O '■O 00 W ►-^ UJ *. 4^ on ui ui en oji-.o,£>ooO*tn>t*.c« n 1 -~ 3 x m o 3 B 00 M I 3 ^4 fT ft > re re " > 3 n>' o n> a. T) ft n o n> B a. 13 "a Appendix Table i (continued). 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Subject Number Total CholeiSterol Before After Chole:sterol Ester (%) Before After Total Lipiids Before After Total P-lipi ds Before After Tri-» glycerides Before After P-lipid/ Chole;sterol Before After LPC/PC Before After LCAT Percent Change 35 36 37 195 255 166 203 263 160 66 66 66 70 72 69 550 702 564 645 796 606 182 192 163 172 218 154 56 62 60 56 65 84 0.93 0.75 0.98 0.85 0.83 0.96 0.227 0.135 0. 148 0. 184 0. 119 0.075 - 4 Mean S.D. 206 36 210 37 66 2 67 3 655 164 645 170 181 32 187 27 91 43 105 59 0.88 0. 12 0.90 0. 11 0. 137 0.05 0. 123 0.03 — 10 21 11 CO Table iii. Correlation coefficients among plasma lipids and between plasma lipids and other factors. Total Cholesterol Before After .889 Total Cholesterol Before After % Cholesterol Ester Before After Total Lipids Before After Phospholipids Before After Triglycerides Before After LPC/PC Before After3 % Cholesterol Ester Before After. -.213 -.129 Total Lipids After Before Phospholipids Before After Triglycerides Before After LCAT3 Phospholipid/ Cholesterol Before After -.133 -.032 .660 .625 .645 .632 .745 .803 .699 .778 .566 .544 .455 .316 -.197 -.072 -.305 -.055 -.534 -.634 .489 -.389 -.380 -.390 -.352 -.211 -.104 -.081 -.259 -.507 -.266 -.290 -.494 .096 .335 -.012 .122 .101 -.265 .915 .706 .638 .694 .726 .755 .723 .637 .692 .093 .007 -. 199 -.153 .522 .621 .526 .340 .628 .396 -.204 -.647 -.049 . 113 -.112 -.229 .220 -.139 -.194 .719 -.148 -.048 -.157 -.028 .569 .475 -.096 .125 N.C. N.C. N.C. N.C. -.284 -.159 .213 .069 N.C. N.C. N.C. N.C. -.334 .337 N.C. N.C. N.C. N.C. -.190 -.249 -.220 -,208 .016 .026 -.024 -.188 . 177 . 175 N.C. .214 .119 -.141 -.107 -.097 .095 -.149 -.175 -.187 -. 136 -.259 -.288 N.C. -.035 -.052 -.181 -.260 .001 .018 -.331 -.384 -.054 -.376 -.205 -. 199 N.C. .220 -.059 -.276 -.272 .396 .362 -. 194 -. 144 -.127 -.197 -.457 -.294 N.C. .078 . 144 Smoking .331 .281 -.298 -.245 .238 .335 .346 .240 .477 .521 N.C. .000 -.119 % Ideal Weight .092 .047 -.440 -.240 .333 .358 . 177 .168 .570 .465 N.C. .148 .070 Fat as % Calories b P/S Ratio Diet Cholesterol Activity Level b Number of subjects evaluated equaled 31 for each correlation except a (n= 12) and b(n == 28). N. C. means Not Correlated. 60 Table iii (continued). LPC/PC LPC/PC Before3 .553 Fat as % Calories P/S Ratio b Diet Cholesterol Activity Level Smoking b b a , b P/S Ratio N.C. -.267 b Diet Cholesterol N.C. Activity Level N.C. Smoking N.C. % Ideal Weight N.C. .361 -.226 .206 .258 -.436 .343 -.001 -.180 -.056 .097 -. 181 -.332 -.313 .312 Number of subjects evaluated equaled 31 for each correlation except a (n = 12) and b (n = 28). N.C. means Not Correlated. 61 Table iv. Correlation coefficients between lipids and other factors, and percent change in plasma lipids. Percent Change Total Cholesterol % Cholesterol Ester Total Lipids Phospholipids Triglycerides LPC/PC3 Before After -.187 .272 .038 .075 -.392 -.368 -.270 -.285 -.294 -.468 .171 . 118 % Cholesterol Before Ester After .117 .239 -.320 .670 . 154 . 118 .166 -. 146 .203 -.275 -.384 .134 Total Cholesterol Total Lipids Before After -.015 .004 -.087 -.057 -.577 -.225 -.246 -. 144 -.252 -.132 N.C. N.C. Phospholipids Before After .184 . 178 .066 -.217 -.516 -.266 -.693 .216 -.492 -.038 .231 -. 181 Before After a .011 -.209 . 146 -.290 -.416 -. 146 -.252 . 130 -.276 .337 N.C. N.C. .326 .242 N.C. -.083 N.C. .673 Before After .576 -.300 .138 -.376 -.245 .371 -.633 .765 -.372 .803 -.061 -.603 .010 .087 -.357 -. 114 N.C. N.C. .434 .200 N.C. N.C. -.679 . 179 -. 101 -.032 .039 -. 127 .052 N.C. .083 .018 -.039 .090 .017 N.C. -. 123 .025 .017 -.271 -.088 N.C. .019 .051 . 184 .036 .125 N.C. Smoking -.090 -.005 .035 -. 140 .110 N.C. % Ideal Weight -.036 .121 -.132 -.099 -.057 N.C. .154 -.055 -. 122 -.485 -.082 -.007 -.301 -.473 .307 .418 .504 N.C. .615 -.362 Triglycerides LCAT Activity P-lipid/ Cholesterol Befor|a After* b Fat as % Calories b P/S Ratio b Diet Cholesterol LPC/PC Activity Level Total Cholesterol % Cholesterol Ester Total Lipids Phospholipids Triglycerides N.C. Number of subjects evaluated equaled 31 for each correlation except a (n = 12) and b (n - 28). N.C. means Not Correlated. 62 Table v. Paired_t-statistics to evaluate changes with supplementation. No. _t-statistic Significance Total Cholesterol 31 -1. 185 N.S. Percent Cholesterol Ester 31 -2.963 p<0„01 Total Lipids 31 1. 148 N.S. Phospholipids 31 -0. 773 N.S. Triglycerides 31 -1.828 N.S. Phospholipid/Cholesterol 31 -0.512 N.S. LPC/PC 12 1.244 N, S, 12 2.540 p< 0.025 LCAT Activity * Unpaired _t-test, one-tailed. Table vi. Dietary intake of subjects compared to National Research Council recommended dietary allowances, 1974 (values are the average of two three-day dietaries unless otherwise indicated). Subject Number 1 2 3' 4 5 6 8 9 10 12 13 14 15 a 16 17 20a 23 24 25a 26 28a 29a 30 31 33 35 36 37 Calories % RDA Protein % RDA 72 94 145 149 127 170 160 190 128 175 164 169 192 152 168 169 162 111 154 168 187 244 184 226 184 156 172 236 138 150 63 102 83 122 80 77 88 84 92 76 106 84 86 74 80 128 121 128 89 130 116 101 115 139 93 74 Fat as % Calories Calcium % RDA Iron % RDA 36 40 44 38 29 41 48 99 127 140 114 118 160 142 113 133 136 135 147 157 171 207 134 122 144 157 181 248 140 189 373b 138 152 250 185 99 37 27 37 41 38 42 38 32 37 23 42 46 41 35 43 40 45 34 48 32 32 42 82 172 126 146 100 42 104 96 191 105 80 139 159 121 90 104 137 234 167 149 104 66 69 142 140 158 values from these individuals are taken from only one dietary. Vitamin A % RDA 74 80 131 175 64 116 114 289 144 95 130 100 146 180 182 92 216 117 174 335 82 S3 230b 82 60 116 19 7b 112 Thaimin % RDA Riboflavin % RDA 54 100 96 66 96 136 85 77 84 1325b 64 94 86 122 105 177 68 144 114 1383b 79 270 316b 106 108 216 344b 50 60 97 96 152 119 159 105 138 112 1116b 141 91 110 131 153 164 90 102 128 876b 161 232 317b 106 105 176 330b 128 Niacin % RDA 103 139 92 109 141 130 112 142 122 754b 123 103 114 168 92 103 122 128 106 489b 104 176 304b 154 190 194 264b 120 Ascorbic Acid % RDA 111 205 247 168 354 398 302 154 260 1536b 136 278 230 406 815b 522 251 434 279 2144b 172 446 802b 112 294 242 99 8b 170 Figures include dietary intake plus vitamin-sapplementation. P/S 0.24 0.32 0.30 0.26 0.66 0.20 0.51 0.34 0.26 0.24 0.20 0.30 0.32 0.29 1. 15 0.85 0.28 0.37 0.27 0.24 0. 12 0.40 0.66 0.24 0.45 0.32 0.33 0.26 Cholesterol mg'/day 310 410 200 320 208 640 342 309 548 591 361 332 478 244 264 296 466 652 615 724 783 356 397 331 360 466 328 371 00