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Dietary cholesterol in graded amounts : threshold to ceiling effects upon plasma free cholesterol synthesis,… Li, Zi-Chi 1993

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DIETARY CHOLESTEROL IN GRADED AMOUNTS: THRESHOLD TO CEILING EFFECTS UPON PLASMA FREE CHOLESTEROL SYNTHESIS, EQUILIBRATION AND CIRCULATION LEVELS IN HUMANS BY ZI-CHI LI  M.D., Jinan University, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES SCHOOL OF FAMILY AND NUTRITIONAL SCIENCES DIVISION OF HUMAN NUTRITION We accept this thesis as confirming ^  THE UNIVERSITY OF BRITISH COLUMBIA October 1993 ©ZI-CHL,193  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  ex,e0i-e—a<3  Department of ^ The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  act  (02,  /  ABSTRACT  The purpose of this study was to investigate the effect of dietary cholesterol upon plasma cholesterol concentration and cholesterogenesis; and the aspect of equilibration of newly synthesized cholesterol between plasma and red blood cell (RBC) in humans. Eight healthy subjects (seven men and one woman) at the age of 55.5 ± 4.2 (mean ± SEM) years were recruited for this study. Three experimental diets (55% carbohydrate, 15% protein, 30% fat, P:S = 0.8) containing 50mg (low), 350mg (medium) and 650mg (high) cholesterol per day were randomly consumed by these subjects for four weeks at levels designed to maintain body weight. On day 28 of each diet, subjects were given a priming dose of 0.7 g deuterium oxide (D20)/kg body water followed by maintenance doses over 24 hours. Cholesterol synthesis was determined as the fractional synthetic rate (FSR) of the rapid exchangeable pool calculated by deuterium incorporation from body water into plasma free cholesterol over 24 hours. RBC cholesterol deuterium incorporation was also compared to that of plasma free cholesterol. Plasma cholesterol was significantly elevated by 13% (+26 mg/di, P = 0.002) in the high as compared to low but not medium cholesterol containing diets. Equilibration of deuterium enrichment from the newly synthesized plasma free cholesterol, expressed as parts per thousand  (0 /00)  relative to Standard Mean Ocean Water (SMOW), to RBC cholesterol was significantly delayed over the initial 6 (51.7 ± 15.5 vs 1.8 ± 10.2/ a, oof P = 0.010) and 12 (60.0 ± 12.3 vs 16.1 ± 8.0  ^oof P = 0.005) hours postdose  in the high cholesterol diet. Dietary cholesterol levels of 50, 350, and 650 mg/day did not alter FSR (0.078 ± 0.016, 0.072 ± 0.007 and 0.071 ± 0.014 day-1, respectively, P = 0.913). No relationship between change of  plasma cholesterol and rate of cholesterogenesis was observed among the three experimental diets. These findings suggest that dietary cholesterol levels affect plasma cholesterol concentrations. Deuterium incorporation between plasma free cholesterol and RBC cholesterol is delayed over the initial 12 hours postdose. Use of a deuterium incorporation period of 24 hours, or more, enables more accurate determination of cholesterogenesis when using this methodology. Cholesterogenesis is neither affected by dietary cholesterol nor correlated with alterations of plasma cholesterol concentration.  111  TABLE OF CONTENTS ABSTRACT ^  ii  LIST OF TABLES ^  vi  LIST OF FIGURES ^  vii  ACKNOWLEDGEMENT ^  xi  1. INTRODUCTION ^  1  2. LITERATURE REVIEW ^ 4 2.1 Dietary Cholesterol Metabolism ^ 4 2.2 Effect of Dietary Cholesterol Content on Plasma Cholesterol Levels ^ 6 2.2.1 Animal Studies ^ 6 2.2.2 Human Studies ^ 6 2.3 Cholesterol Compartmentalization ^ 14 2.4 Measurements of Cholesterol Synthesis ^ 18 2.4.1 Sterol Balance Methods ^ 18 2.4.2 HMG-CoA Reductase Activity ^ 18 2.4.3 Kinetic Analysis of the Isotopic Decay of Isotopically Labelled Cholesterol ^ 19 2.4.4 Measurement of Levels of Cholesterol Precursors ^ 20 2.5 Deuterium Incorporation Methodology ^ 22 2.5.1 History ^ 22 2.5.2 Sensitivity ^ 22 2.5.3 Three-Pool Model ^ 23 2.5.4 Assumptions ^ 24 3. EXPERIMENTAL DESIGN AND METHODS ^ 25 3.1 Subject Recruitment ^ 25 3.2 Diet Protocol ^ 26 3.3 Administration of Deuterium Oxide ^ 31 3.4 Blood Sampling ^ 33 3.5 Laboratory Procedures ^ 34 3.5.1 Lipid Extraction ^ 34 3.5.2 Solvent Evaporation ^ 35 3.5.3 Distillation ^ 36 3.5.4 Plasma Water Preparation ^ 36 3.5.5 Mass Spectrometric Determination ^ 37 3.6 Data Calculations ^ 38 3.7 Statistical Analyses ^ 41 4. RESULTS ^ 4.1 Subject Characteristics ^ 4.2 Comparison of the Effect of Dietary Cholesterol on Plasma Total Cholesterol Levels ^ 4.3 Comparison of Deuterium Enrichment and Cholesterol FSR Between Plasma and RBC ^  iv  42 42 44 50  4.4 Comparison of the Effect of Dietary Cholesterol on Cholesterol FSR ^ 4.5 Comparison of the Effect of Plasma Total Cholesterol on Cholesterol FSR ^  57 69  5. DISCUSSION ^ 68 5.1 Subject Charateristics ^ 68 5.2 Effect of Dietary Cholesterol on Plasma Total Cholesterol Levels ^ 70 5.3 Equilibration of Synthesized Cholesterol Between Plasma and RBC ^ 73 5.4 Effect of Dietary Cholesterol on Cholesterolesterogenesis ^ 76 5.5 Relationship Between Plasma Total Cholesterol Levels and Cholesterol FSR ^ 79 5.6 Conclusions ^ 81 BIBLIOGRAPHY ^  83  APPENDICES ^  92  Appendix 1. Lipid Studies Volunteer Information Form ^  92  Appendix 2. Consent and Instruction Form ^ 98 Appendix 3. A Sample of Three Day Food Records ^ 101 Appendix 4. Calculation of Caloric Intake of Selected Subjects Consuming North American Diet ^  103  Appendix 5. Randomization Scheme for Graded Cholesterol Protocol ^  106  Appendix 6. Deuterated Water Test Schedule Sample Form ^  107  Appendix 7. Doses of Deuterated Water ^  108  Appendix 8. Deuterium Enrichment of Plasma and RBC Free Cholesterol From Baseline Plasma Water at Various Time Points Among Three Experimental Diets 109  LIST OF TABLES Table 1.  Table 2.  Food Items Contained in the Three Experimental Diets at the Reference Level of 2800 kcal Per Day ^  28  Order of Randomized Dietary Phase Assignment ^  30  Table 3.  Anthropometric Data and Screening Plasma Lipid Profile of the Selected Subjects ^ 43  Table 4.  Plasma Total Cholesterol Levels in Subjects Before and After Dietary Cholesterol Interventions ^  46  Table 5.  Summary of the P Values on Plasma Cholesterol Concentration and Changes of Plasma Cholesterol Concentration in Subjects Consuming Three Experimental Diets ^ 49  Table 6.  Summary of P values of the Paired Sample t-tests on Deuterium Enrichment of Plasma Versus RBC at Different Time Intervals ^ 54  Table 7.  Plasma and RBC Cholesterol FSR in Subjects Consuming Three Experimental Diets ^ 55  Table 8.  Summary of the P Values on Cholesterol FSR in Subjects Consuming Three Experimental Diets ^  57  Summary of the P Values on the Relationship Between Changes of Cholesterol FSR and Changes of Plasma Total Cholesterol Concentration ^  67  Table 9.  LIST OF FIGURES Figure 1.  Figure 2. Figure 3.  Figure 4.  Figure 5.  Figure 6.  Figure 7.  Figure 8.  Figure 9. Figure 10  The Proposed "S" Shaped Curve of Effects on the Plasma Cholesterol Level of Gradually Increasing the Amount of Dietary Cholesterol in Humans Whose Background Diet is Very Low in Cholesterol Content ^  12  Three-pool Model With Possible Side-Pool Synthesis ^  15  The Influence of Three Graded Amounts of Dietary Cholesterol on Plasma Cholesterol Levels ^  47  The Influence of Dietary Cholesterol on Change of Plasma Cholesterol Concentration From Baseline ^  48  Deuterium Enrichment of Plasma and RBC in Subjects Consuming Low Cholesterol Diet As a Function of Time ^  51  Deuterium Enrichment Between Plasma and RBC in Subjects Consuming Medium Cholesterol Diet As a Function of Time ^  52  Deuterium Enrichment of Plasma and RBC in Subjects Consuming High Cholesterol Diet As a Function of Time ^  53  Equilibrium of Synthesized Cholesterol Between Plasma and RBC on Three Experimental Diets ^  56  Overall Plasma Cholesterol FSR in Subjects Consuming Three Experimental Diets ^  58  Changes of Overall Plasma Cholesterol FSR and Changes of Plasma Cholesterol From Baseline in Subjects Consuming a Low Cholesterol Diet ^  60  Figure 11. Changes of Overall Plasma Cholesterol FSR and Changes of Plasma Cholesterol From Baseline in Subjects Consuming a Medium Cholesterol Diet ^  61  Figure 12. Changes of Overall Plasma Cholesterol FSR and Changes of Plasma Cholesterol From Baseline in Subjects Consuming a High Cholesterol Diet ^  62  vii  Figure 13. Plasma Cholesterol FSR in Subjects Consuming Medium Cholesterol Diet Versus Change of Plasma Cholesterol From Low to Medium Cholesterol Diet ^ 63 Figure 14. Plasma Cholesterol FSR in Subjects Consuming High Cholesterol Diet Versus Change of Plasma Cholesterol From Medium to High Cholesterol Diet ^  64  Figure 15. Plasma Cholesterol FSR in Subjects Consuming High Cholesterol Diet Versus Change of Plasma Cholesterol From Low to High Cholesterol Diet ^  65  Figure 16. Change of Plasma FSR Versus Increase in Plasma Cholesterol From Low to High Cholesterol Diet ^  66  ACKNOWLEDGEMENT The present research was conducted under the supervision of Dr. Peter Jones, Division of Human Nutrition, University of British Columbia. Many thanks to Dr. Peter Jones for his constructive guidance and valuable instruction. Many thanks to Dr. Jiri Frohlich, Dr. David Kitts, Dr. Linda McCargar and Dr. Joseph Leichter for serving in my thesis committee. Their invaluable inputs to my thesis are highly appreciated. I am deeply indebted to the financial support from Heart & Stroke Foundation of British Columbia, Canada. I would like to thank Dr. William Connor, Ms. Lauren Hatcher and other CRC staff as well as the study subjects for their hospitalities and assistance during my visit to the CRC at the Oregon Health Sciences University, Portland, Oregon. U.S.A.. My gratitude is also extended to Dr. Catherine Leitch and Gayle Wickens for the countless hours they contributed to me on taming the mass spectrometer. Many thanks are expressed to Brian Toy for his great statistics assistance. I further wish to acknowledge the generous aid from the fellow graduate students and staff in the School of Family and Nutritional Sciences, University of British Columbia over the period of my study. Finally, very special thanks are directed to my wife, Sandra Jian-Hua Liang, for her constant support and encouragement throughout the completion of my study.  ix  1. INTRODUCTION  Coronary heart disease (CUD) is the leading cause of death in Western industrialized countries. A direct relationship between elevated plasma cholesterol levels and the incidence of CHD has been well established (Connor et a/. 1972, Stamler et a/. 1986). When plasma total cholesterol concentration reaches 240 mg/di, each additional 1% elevation of plasma cholesterol level is predicted to increase the risk of CUD by about 2% (LRCP 1984). The effects of dietary cholesterol on plasma cholesterol levels are still controversial. This is partially due to variability in experimental design of the research in this area and individual response (Slater et al. 1976, Connor et al. 1964, McNamara et a/. 1987). For many years, due to inadequate experimental design, dietary cholesterol was considered of little importance in human lipid metabolism and to have no effect on plasma cholesterol levels. Egg yolk was usually added to the usual diet as a source of dietary cholesterol which would also provide calories in the form of fat and protein (Slater et al. 1976, Porter et a/. 1977). However, in carefully controlled environments such as metabolic wards, most studies demonstrated that dietary cholesterol exerts decisive effects on plasma cholesterol levels, and this effect can be best described by a S-shaped curve (Connor et a/. 1961a, Connor and Connor 1985, Hopkins 1992). Furthermore, cholesterol synthesis significantly contributes to the total body pool of cholesterol in humans and thus plays an important role in determining body cholesterol homeostasis (Dietschy 1984). In response to dietary cholesterol, however, cholesterol synthesis has been reported to be frequently (Lin and Connor 1980, McNamara et a/. 1987) but not consistently (Kern 1991, Everson et a/. 1991) down-regulated. Factors  1  regulating cholesterol synthesis in humans remain poorly understood. The relative inadequacy of knowledge on cholesterol synthesis in humans is mainly due to methodological constraints. Cholesterol synthesis could be determined by several methods which include sterol balance (Nestel et a/. 1973), 3-hydroxy-3-methylgutaryl coenzyme A (HMG CoA) reductase activity (Brown et a/. 1979), kinetic analysis of the isotopic decay of isotopelabelled cholesterol (Dell et a/. 1985) and measurement of cholesterol precursor levels (Parker et a/. 1984). These techniques are accurate but time-consuming, quick but invasive, or they are indirect in determination of cholesterol synthesis. The use of deuterium uptake method for assessment of cholesterol synthesis overcomes the above drawbacks and has been successfully applied in humans (Jones et a/. 1993b). In the deuterium uptake method, cholesterol synthesis is determined by the rate of deuterium incorporation from deuterium oxide (D20) in body water into plasma. Both plasma and red blood cell (RBC) have been selected for cholesterol measurement (Jones et a/. 1993b, Wong et al. 1991) since they are within the central pool comprising synthesized cholesterol from liver and intestine (Dietschy 1984). However, the exchange rate of D20 between plasma free cholesterol and RBC has not been fully investigated. The objectives of the present study are to examine the S-shaped relationship between dietary cholesterol and plasma total cholesterol; the influence of dietary cholesterol on cholesterol synthesis; and the correspondence between plasma free cholesterol deuterium and RBC deuterium uptake. The formal statements of the null hypothesis for the current study are as follows:  Hl: There is no change in plasma free cholesterol level with  2  an increase in dietary cholesterol.  H2: The rate of equilibration of de novo synthesized cholesterol between RBC and plasma free cholesterol is constant throughout the measurement time intervals.  H3: There is no change in plasma free cholesterol synthesis with a gradual increase in dietary cholesterol.  2. LITERATURE REVIEW  2.1 Dietary Cholesterol Metabolism  Dietary cholesterol is absorbed by the gut in amounts proportional to the intake up to a dietary level of perhaps 1,200 to 1,500 mg/day. Only about 30 - 60% of the usual intake of cholesterol is absorbed (Connor and Lin 1974, Grundy et al. 1969); the remaining unabsorbed cholesterol passes out in the stool. In humans, absorbed cholesterol is transported initially in chylomicrons, largely as cholesterol ester, reaching a peak concentration in the plasma some 48 hours after a meal. This cholesterol contributes its mass to the total body pools (Bhattacharyya et al. 1976). Cholesterol-rich chylomicron remnants are metabolized by the liver. Dietary cholesterol entering hepatocytes inhibits synthesis both of cholesterol and of LDL receptors. Overall it contributes to the major cholesterol pools of the body. Feedback inhibition of cholesterol biosynthesis in the body only partially occurs in humans, even when a large amount of dietary cholesterol is ingested (Lin and Connor 1981). Because the clearance of LDL cholesterol from the bloodstream depends on the activity of hepatic LDL receptors, down-regulation of these receptors will tend to elevate plasma cholesterol. The extent of down-regulation of the hepatic LDL receptors by dietary cholesterol depends on the ability of the hepatocyte to excrete intracellular cholesterol into the enterohepatic circulation as bile acids. The input of sterols into the plasma-tissue occurs firstly from dietary cholesterol (0 - 500 mg/day) and secondly from synthesized cholesterol derived from the liver and intestinal tract (700 - 900 mg/day) (Dietschy 1984). The rate of cholesterol biosynthesis in human appears not to be  4  greatly altered, as it is in most experimental animals by the ingestion of dietary cholesterol (Connor and Connor 1985). This means that total amount of sterol entering the body from diet, in addition to the synthesized cholesterol, may be much greater in individuals consuming a highcholesterol diet than in individuals consuming a low-cholesterol diet. The output of sterols from the body is largely by way of feces, including cholesterol and bile acids excreted in the bile by the liver and not reabsorbed by the gut via the enterohepatic circulation. Because the ring structure of the sterol nucleus cannot be broken down by the tissues of the body, it must either be excreted or stored. However, most studies published to date have indicated a failure of bile acid and neutral steroid excretion to increase very much after the ingestion of dietary cholesterol (McMurry et a/. 1985, Grundy et a/. 1969). Therefore, there are four consequences of ingestion of dietary cholesterol. First, cellular cholesterol in the liver increases resulting from dietary cholesterol absorbed into chylomicrons and removed from plasma by the liver as chylomicron remnants. Second, a decrease in the number of LDL receptors in the liver occurs. Third, there is a rise in the plasma cholesterol concentration. And last, the increased amounts of cholesterol deposit in the tissues, particularly in the coronary arteries to initiate and sustain the atherosclerosis process (Connor and Connor 1985). The present study was in part designed to investigate the mechanism of the effect on plasma cholesterol concentration in response to increasing amounts of dietary cholesterol levels and cholesterol synthesis. It is proposed that the elevation of both dietary cholesterol and cholesterol synthesis might result in the increase in plasma cholesterol concentration leading to the development of CHD.  5  2.2 Effect of Dietary Cholesterol Content on Plasma Cholesterol Levels  2.2.1 Animal Studies  Species vary in their response to dietary cholesterol. Humans are not as sensitive to dietary cholesterol as are certain animal species, such as the rabbit. Experiments done with animal species showed a more consistent relationship between dietary cholesterol and plasma cholesterol levels. Turley et a/. (1983) found a significantly decreased liver cholesterol synthesis as measured by tritium incorporation and increased plasma cholesterol levels in female hamsters fed with cholesterol (0.12% w/w). In addition, Spady and Dietschy (1983) observed a significant decrease in HMGCoA reductase activity in 18 tissues of squirrel, monkey, guinea pig, rabbit, hamster and rat, following cholesterol feeding. Even in animals such as the rat and the dog, whose plasma cholesterol levels are resistant to change as a result of cholesterol feeding because of their great capacity to excrete the ingested dietary cholesterol as bile acids, experimental manipulations to block such excretion can lead to increase in plasma cholesterol levels (Dietschy 1984). It appears from these studies that dietary cholesterol may have an effect on plasma cholesterol levels.  2.2.2 Human Studies  Many epidemiological studies suppport the overall dietary hypothesis about the development of CHD; namely, that high cholesterol diets relate to  6  the occurrence of CUD in entire populations (Connor et a/. 1972, LRCP 1984). There is evidence that plasma cholesterol levels greater than 220 mg/d1 would increase the incidence of CHD (The Pooling Project Research Group 1978, Turley and West 1976), and it has been established that the risk of CHD from hypercholesterolemia tends to increase 1% for every 1 mg/d1 rise in plasma total cholesterol when plasma total cholesterol is above 240 mg/d1 (LRCP 1984, Grundy et a/. 1988). Autopsy studies carried out in people who have died of CHD indicate that, in almost all cases, the reduction of blood flow to the heart muscle was caused by atherosclerosis. A major chemical constituent of the atherosclerosis in coronary arteries in both man and animals is cholesterol (Armstrong and Megan 1972, Bottcher and Woodford 1962). Radioactive cholesterol from the diet has been traced into the very matrix of severe atherosclerotic lesions in both humans and animals (Jagannathan et a/. 1974, Newman and Zilversmit 1962). Despite the very large number of studies undertaken during the last 30 years, the effect of dietary cholesterol upon plasma cholesterol levels in humans remains controversial. This reflects the complexity of the factors that determine the response: the variability among individuals, the significant fluctuation in an individual's plasma cholesterol, the interaction between dietary cholesterol and other food components, the exaggerated expectation and interpretation from experiments in animals, variable experimental design and inexact measurements. For many years, however, dietary cholesterol was thought to have little impact on human lipid metabolism and to have no effect on plasma cholesterol concentrations (Slater et a/. 1976, Porter et al. 1977, Kummerow et a/. 1977). The experimental conclusions of these studies were  7  derived from experiments that were incorrectly designed. On the other hand, many separate metabolic experiments have demonstrated that dietary cholesterol exerts decisive effects on plasma cholesterol levels (Becker et a/. 1983, Connor et a/. 1961a, Connor et a/. 1964, Steiner et al. 1962). Two kinds of dietary cholesterol experiments have been conducted historically to determine their effects on the plasma cholesterol level. In the first group of experiments, dietary cholesterol was added to the usual diet in the form of egg yolk which would also supply calories in the form of fat and protein. Frequently, these experiments were carried out in outpatients who simply added eggs to their diets (Steiner and Domanki 1941, Messinger et al. 1950, Porter et al. 1977, Kummerow et a/. 1977, Slater et al. 1976, Key et al. 1956). Therefore, any effect could not be attributed directly to dietary cholesterol alone but had to include the other components of the egg yolk (e.g., saturated fats, proteins). In some experiments, 2-4 egg yolks per day were added to diets already cholesterolrich (Kummerow et al. 1977, Slater et al. 1976, Key et a/. 1956). Dietary fat, fatty acid composition, protein and calories had not been properly controlled in some, but not all, of these studies. Such experiments lacked precision and strict metabolic control. Another type of experiments involved accurate metabolic studies. Early experiments of this variety were inconclusive or negative about effects of dietary cholesterol upon plasma cholesterol levels, because cholesterol was added in a form (as crystalline cholesterol) not well absorbed by the gut to a cholesterol-free, very low fat diet and no effects were observed (Beveridge et al. 1960). Furthermore, there were a series of experiments in which cholesterol, either as butterfat or egg yolk, was incorporated into the total diet, so that the effects of dietary cholesterol could be  8  directly observed (Connor et a/. 1961a, Connor et a/. 1964, Connor et al. 1961b, Mattson et a/. 1972, Erichson et al. 1964, Grande et al. 1965, Hegsted et a/. 1965, Anderson et al. 1976, Flaim et a/. 1981). The egg yolk experiments are most pertinent because they were carried out for longer periods of time and because all of the nutrients contained in the egg yolk (fat, protein, minerals and vitamins) were compensated for by appropriate subtractions from a baseline cholesterol-free diet. Thus, the number of calories fed to the experimental subjects remained the same as did the amounts of fat, protein and carbohydrate; the only difference was the addition of egg yolk cholesterol. In addition, the P/S (polyunsaturated fatty acids : saturated fatty acids) ratio of the fat and the fatty acid composition was the same in both the control and experimental diets. The results of closely controlled metabolic experiments, ordinarily conducted in a metabolic ward of a Clinical Research Center with the food completely prepared and supplied to the experimental subjects as outpatients, consistently showed that dietary cholesterol caused an elevation of plasma cholesterol concentrations (Connor et a/. 1961a, Connor et a/. 1961b, Connor et a/. 1964, Mattson et a/. 1972, Erichson et a/. 1964, Grande et a/. 1965, Hegsted et a/. 1965, Anderson et a/. 1976, Packard et a/. 1983). In other well controlled experiments, some subjects increased plasma cholesterol concentrations and others did not in response to different dietary cholesterol levels (Mistry et a/. 1981, Langer et a/. 1972, Cole et a/. 1985). Variability in response was stressed. Jacobs et a/.(1983) suggested that the average standard deviation for this variability was around 15 mg/d1. Some of this variability can be explained on the basis of cholesterol regulation and on the effectiveness of metabolic compensation in the face of increased cholesterol absorption. This has important  9  implications in dietary counselling in that it explains unexpected results, such as apparent deterioration in a patient who has complied. Analytical variability of cholesterol assay may also be a factor contirbuting to the lack of effect of dietary cholesterol on plasma cholesterol concentrations. However, most of the studies involved normal subjects consuming the baseline diet, which contained 300 mg of cholesterol, rather than high in cholesterol content. In one study, patients with familial hypercholesterolemia had no increase in plasma cholesterol level after being fed a high cholesterol diet (Connor and Connor 1985). The effects upon plasma cholesterol levels as the amount of dietary cholesterol gradually increased are depicted in Figure 1. These data are supported by both animal and human experiments (Connor et a/. 1961a, Connor et a/. 1961b, Connor et al. 1964, Mattson et a/. 1972, Mahley et a/. 1978, Keys et al. 1965). The rise in plasma cholesterol in response to dietary cholesterol levels between 0 and 500 mg/day has been considered to be linear (Mattson et a/. 1972) or curvilinear (Keys et a/. 1965). With a baseline cholesterol-free diet, the amount of dietary cholesterol necessary to produce a rise in the plasma cholesterol concentration is termed the threshold amount. The ceiling amount is defined as the amount of dietary cholesterol increases until the ceiling point is reached on this curve. Further increases in dietary cholesterol do not lead to higher levels of plasma cholesterol. The concepts of the "threshold" and the "ceiling" may explain many of the apparent conflicting results in the literature. If a certain individual already consuming a high cholesterol diet is given additional dietary cholesterol, it is quite likely that further increase in plasma cholesterol will not occur because the ceiling for the individual  10  may have already been reached (Wells and Bronte 1963). If it has not been reached yet, plasma cholesterol level may continue to increase f-rther.  11  Figure 1. The proposed "S" Shaped Curve of Effects on the Plasma Cholesterol Level of Gradually Increasing the Amount of Dietary Cholesterol in Humans Whose Background Diet is Very Low in Cholesterol Content.  Each animal or human may have its own distinctive threshold and ceiling amounts. Beveridge et a/.(1960) fed increasing amounts of cholesterol as butterfat to experimental subjects and found that dietary cholesterol did not significantly affect plasma cholesterol concentrations until intake reached about 200-300 mg/day. Furthermore, at about 600-900 mg/day the response curve plateaued. Wells and Bronte-Stewart (1963) fed three men diets containing cholesterol from 17 to 3017 mg/day. Serum cholesterol levels increased with dietary cholesterol levels until intake was about 500 mg/day. Thereafter, the response curve flattened out. Two experiments conducted by Connor et a/.(1961b, 1964) showed that dietary cholesterol in amounts from 475 mg to well over 4000 mg per day produced similar increments in plasma cholesterol concentration. In another preliminary unpublished study conducted by Connor et al. with six men (two per group), 110 mg of cholesterol per day did not affect plasma cholesterol levels while higher levels (310 mg and 610 mg/day) gave similar and significant elevations. Based on these previous experimental findings (Connor et a/. 1964, Beveridge et a/. 1960, Connor et a/. 1961b, Mahley et al. 1978), it is estimated that an average threshold amount for human beings is 100 mg/day; an average ceiling of dietary cholesterol is in the range of 300-400 mg/day. Further experiments are necessary to provide more precise information about the cholesterol threshold and ceiling amount.  13  2.3 Cholesterol Compartmentalization  During the past decade a great deal of knowledge has become available on the biochemistry of cholesterol, including its synthesis from acetate, and on the properties of the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Dempsey 1974). Advances have been slower in the area of in vivo cholesterol metabolism in humans. In order to complement the biochemical knowledge, it is important to investigate the in vivo metabolism of cholesterol in humans so that this common health problem can be managed. One valuable approach to the study of in vivo metabolism of cholesterol is kinetic analysis of data obtained following the administration of isotopes (Schwartz 1982). Whole-body cholesterol metabolism has been studied by analyzing the turnover of plasma cholesterol following injection of radiolabeled cholesterol. Such an analysis estimates the amount of cholesterol in the whole body and how rapidly cholesterol is turning over in the intact subject (Schwartz 1982). A pool model or compartmental analysis approach is one of the mathematical approaches used to analyze plasma cholesterol specific activity-time curves. A simple theoretical three-compartment model for whole-body cholesterol kinetics in humans has been developed by Goodman et a/.(1973, 1980, 1983). Although cholesterol in the body must actually exist in many small pools all exchanging with plasma, assuming that these compartments can be grouped into three pools based upon their turnover rates allows one to generate quantitative information about the rate of turnover of cholesterol and the amounts of cholesterol stored.  14  The three pools of the compartmental model are mathematical constructs and do not have precise physical or anatomical meaning. This model suggests that all of the various sites of cholesterol in the body turn over at rates that can be separated into three groups. Pool 1 consists of cholesterol which turns over very rapidly and includes plasma, erythrocyte, liver, and GI tract cholesterol as well as much of the cholesterol in the pancreas, spleen, kidneys, and lungs. The bulk of cholesterol in the adipose tissue and muscle turns over slowly and probably contributes to most of pool 3. Pool 2 represents cholesterol turning over at rates intermediate between plasma cholesterol and cholesterol in pool 3 and includes some cholesterol  in the viscera as well as peripheral tissue.  20  R  10  PR  Figure 2. Three-Pool Model With Possible Side-Pool Synthesis. R's represent mass flow rates in grams cholesterol per day, k's are rate constants in days-1 (M2k12 = M1k21 R201 etc.), M's are mass of cholesterol in grams, and PR (production rate) is mass outflow from the system in g/day. Subscripts for R and k denote movement into one compartment from another--e.g., 12 means into compartment 1 from compartment 2, and 30 means into compartment 3 from outside the system.  15  The three-pool model has eight unknown parameters depicted in Figure 2 (Dell and Ramarkrishnan 1982). These parameters are the degradation rate (R01), which equals production rate (PR) in a steady state; the mass of pool 1 (M1); four exchange rates (k21, k12, k31, and k13); and the synthesis rates in the side pools (R20 and R30). All other parameters are derivable from these basic eight parameters. However, only six of the eight fundamental parameters can be estimated from the data. What cannot be determined uniquely are the two side pool synthesis rates and hence the masses of the side pools (Goodman et a/. 1973, Goodman et a/. 1980, Goodman et al. 1983, Dell and Ramarkarishnan 1982). The assumptions necessary for this model are the following (Lieberman and Samuel 1982): (1) The system is in steady state with respect to body cholesterol, metabolism, and distribution. (2) The system is not appreciably disturbed by introduction of tracer cholesterol. (3)  Both body cholesterol and tracer cholesterol are conserved between inlets and outlets.  (4)  The exponential extrapolation of the plasma specific activity curve to infinite time is valid.  (5)  The only exit of cholesterol from the system is from pool 1. This makes total production rate equal to production rate in pool 1.  (6)  The only entrance of dietary and/or synthetic cholesterol into the system is into pool 1. This gives the minimum value of total exchangeable mass of  16  cholesterol.  However, the three-pool model is not without limits and shortcomings. Little is known about exchange of cholesterol between lipoproteins and specific tissues that comprise the rapidly miscible pool. By subjecting the experimentally observed mass and specific activity data from several types of isotopic preparations to multicompartmental analysis using the SAAM program, a recent more comprehensive multicompartmental model for cholesterol within the rapidly miscible pool has been developed by Schwartz et a/.(1993). This model has enabled us to identify and quantitate  cholesterol transport between all major compartments of blood, several tissues and bile.  17  2.4 Measurements of Cholesterol Synthesis  At present, a variety of techniques have been employed to measure cholesterol synthesis in animals and humans. These approaches include: (1) sterol balance methods, (2) HMG-CoA reductase activity, (3) kinetic analysis of the isotopic decay of stable isotope-labelled or radioactive isotope-labelled cholesterol, (4) measurement of precursors, and (5) deuterium incorporation methodology.  2.4.1 Sterol Balance Methods  Sterol balance methods (Nestel et al. 1973, Bennion and Grundy 1975) have been used to determine the whole body net cholesterol synthesis by feeding the subjects with cholesterol diets and collecting feces. This method is based on an assumption of steady state (cholesterol input = output) for cholesterol metabolism, that the elimination of endogenous cholesterol and its metabolites occurs only in the feces. Although this method is accurate, it relies on precise food intake and complete stool collection; it takes up to three weeks for the subjects to reach a steady state (Nestel and Poyser 1976).  2.4.2 HMG-CoA Reductase Activity  HMG-CoA reductase is the rate-limiting enzyme in cholesterol biosynthesis. Measurement of the HMG-CoA reductase activity (Brown et a/. 1979, Dietschy and Spady 1984) has been used as a qualitative method to determine relative cholesterol synthetic rates in a given tissue  18  preparation, such as liver and small intestine, which are the primary sites for cholesterol synthesis in humans (Dietschy and Wilson 1970, Norum et al. 1983). This method should provide the direct real-time index of cholesterol synthetic rate (Bjorkhem et a/. 1987). However, it has not been widely applied to human study because of the need of biopsy specimens (Carulli et a/. 1989).  2.4.3 Kinetic Analysis of the Isotopic Decay of Isotopically Labelled Cholesterol  Cholesterol synthesis and distribution in various tissues throughout the body have been measured by injection of 14C or 3H labelled cholesterol in animals (Dell et a/. 1985) and in humans (Goodman et a/. 1973, 1980, 1983, Schwartz et a/. 1993). Following the injection of labelled cholesterol, the decay of plasma cholesterol specific activity at certain time points over a period of time reflects the changes in body cholesterol synthesis and turnover rate among the theoretical three-pool model (Goodman et al. 1973). Unfortunately, this kind of measurement is lengthy and cannot  detect short-term cholesterol synthetic rate. In a recent study of cholesterol kinetics in subjects with bile fistula, Schwartz et a/.(1993) employed several types of isotopic preparations to simultaneously label separate cholesterol pools and sample all components of blood and bile at frequent intervals, which led to the development of a comprehensively multicompartmental model for cholesterol kinetics. It was found that free cholesterol was extensively exchanged between HDL and liver, RBC, and other tissues. A large portion of total hepatic cholesterol comprised a pool that turned over rapidly by exchanging mainly with plasma HDL and was the major  19  source of bile acids and biliary cholesterol. The analysis also showed that 94% of de novo synthesized cholesterol was partitioned into the large hepatic pool which exchanged rapidly with plasma lipoproteins.  2.4.4 Measurement of Levels of Cholesterol Precursors  Other approaches available for determination of cholesterol synthesis are to measure the cholesterol precursors such as plasma and 24-h urinary mevalonic acid (MVA) (Parker et a/. 1982, Parker et al. 1984), plasma squalene (Nestel et al. 1975) and methyl sterols (Miettinen 1982). Previous studies have shown that, compared to cholesterol synthesis measured by sterol balance methods, concentrations of plasma and 24-h urinary MVA exhibit changes that parallel the rate of whole body cholesterol biosynthesis (Miettinen 1982, Kopito et a/. 1980). Although plasma concentrations of mevalonate display diurnal variations (Kopito et a/. 1982), the mean 24-h plasma concentration can be used, on a comparative basis under different dietary, or pharmaceutical manipulations. The 24-h urinary MVA excretion reflects the integrated plasma concentration and provides a more practical way of assessing the cholesterol synthesis compared with the conventional sterol balance techniques. The presence of these cholesterol precursors in the plasma differs with the HMG-CoA reductase activity during cholesterol synthesis. But these techniques only serve as indirect indicators of cholesterol synthesis. Radiolabelled cholesterol precursors such as 14C- acetate and 14Cmevalonate (Andersen and Dietschy 1979, Liu et al. 1975, Ferezou et a/. 1982) have been used to determine the fractional synthetic rate of cholesterol in animals and humans. However, there are three major  20  limitations to this 14C-labelled substrate technique. Firstly, it requires periods of measurement up to 28 weeks. Secondly, it may underestimate cholesterol synthesis because of the dilution of the acetyl CoA intracellular pool specific activity by mixing of labelled precursor with unlabelled substrates; Thirdly, it is associated with radioactive hazards due to the use of 14C. In addition, measurement of incorporation of tritium from tritiated water (Dietschy and Spady 1984) has been reported to be a more useful method than 14C-labelled substrates since the water pool of the precursors is less diluted by unlabelled substrates which reduce its specific radioactivity. Although tritiated water uptake can be used to determine short-term cholesterol synthesis, the radiation hazards from the large dose of tritium required in this method preclude its use in human subjects.  21  2.5 Deuterium Incorporation Methodology  An accurate, direct, non hazardous, short-term method for assessment of cholesterol synthetic rates in humans has been successfully carried out by Jones et a/. (1988) using deuterium incorporation methodology. The use of a stable isotopically labelled precursor, D20 eliminates several drawbacks encountered by other techniques discussed previously.  2.5.1 History  Deuterium incorporation method was initially employed to measure human fat and cholesterol synthesis by Rittenberg and Schoenheimer (1937). Taylor et a/.(1966) measured human cholesterol synthesis with the deuterium label in 1966. Deuterium enrichment of body water was maintained at 5.0 g deuterium oxide/kg body water (0.5 atom % excess), a 33-fold increase above the baseline level. This high level of deuterium enrichment was necessary to ensure that the incorporation of the deuterium atom into the de novo synthesized cholesterol was measurable by the insensitive mass spectrometric techniques available at that time. Some subjects reported experiencing side-effects such as severe vertigo following the given such large priming dose. Also, it took about 40 days to achieve maximum deuterium oxide enrichment.  2.5.2 Sensitivity  The isotope ratio mass spectrometer analytical sensitivity has been improved so that the required dosage of deuterium enrichment is  22  considerably reduced. The double-comparison method for mass spectrometric determination of hydrogen isotopic abundances developed by Schoeller et a/.(1983) has been contributed to the better precision of deuterium enrichment analysis. Jones et a1.(1988) found that deuterium enrichment of body water maintained at 0.5 g deuterium oxide/kg body water (0.05 atom % excess) was sufficient to detect the enrichment of plasma cholesterol within 12 hours following oral dosage of deuterium. This level of deuterium intake is 10 times less than that in the previous studies. Deuterium incorporation method enables us to examine the short-term perturbations in cholesterol synthesis. Compared with the measurement of plasma mevalonic acid concentrations, Jones et a/. (1992) confirmed that the deuterium uptake method was suitable for relatively uninvasive, shortterm detection of cholesterol synthesis. Some theoretical and procedural considerations of the deuterium uptake method were also addressed (Jones et a/. 1993a).  2.5.3 Three-Pool Model  The accurate use of deuterium incorporation methodology is based on a three-pool model developed by Goodman et a/.(1973) as in the previous discussion. This model theoretically compartmentalizes the body cholesterol within various tissues into three pools, based on the rate of exchangeable tissues cholesterol equilibrates with plasma cholesterol.  2.5.4 Assumptions  The accurate measurement of fluctuations in rates of cholesterol synthesis using three-pool model and deuterium incorporation methodology rely on three major assumptions. First, all of de novo cholesterol only occurs in pool 1 through absorption of exogenous cholesterol or the majority of endogenous newly synthesized cholesterol (Goodman et a/. 1980, Dietschy and Wilson 1970). Under this assumption, the amount of dietary cholesterol entering this pool will likely contribute to the dilution of the labelled cholesterol. Therefore, different dietary cholesterol level may cause variations in cholesterol fractional synthetic rate (FSR). Second, the losses of cholesterol from the body occur solely via pool 1 (Goodman et a/. 1973, 1980). Third, the exchange of cholesterol between pool 2 and pool 3 is conducted only by pool 1 (Goodman et a/. 1973, 1980). And last, the amount of free cholesterol entering pool 1 from pool 2 and pool 3 is minimal because of the slow turnover rates of these pools with plasma cholesterol as discussed above (Goodman et a/. 1973). Cholesterol fractional synthetic rate (FSR) can be calculated through measuring the ratio of deuterium enrichment of plasma free cholesterol to the deuterium enrichment of plasma water contained within pool 1. Additional assumptions are necessary for the valid calculation. The first, stated by Dietschy and Spady (1984), is that almost all cell membranes are permeable to D20 ensuring the equal enrichment between the intracellular enrichment and that of plasma. The second additional assumption is that the same number of hydrogen atoms from water will be taken up in all cholesterol synthetic tissues, independent of metabolic state.  24  3. EXPERIMENTAL DESIGN AND METHODS  This experiment was part of a joint study in collaboration with Dr. William E. Connor and co-workers at the Clinical Research Center of Oregon Health Sciences University in Portland, Oregon, U.S.A. (CRC-OSHU). Subject screening, selecting, and testing were carried out on an outpatient basis at the CRC. All lipid profiles were determined at the CRC-OSHU. Assessment of cholesterol synthesis using deuterium incorporation methodology was carried out by the candidate at Dr. Peter Jones laboratory in Division of Human Nutrition, School of Family and Nutritional Sciences, University of British Columbia. The candidate had visited the CRC for one week to gain experience in the above procedures.  3.1 Subject Recruitment  Subjects were selected using advertisements through local radio stations and newspapers. Volunteers were screened by a form (Appendix 1). A general description of the study was provided to each respondent at this time. In the initial screening, normolipidemic subjects in good health, aged 30-70 years, with plasma cholesterol levels between 190 and 250 mg/d1 and consuming the usual high-cholesterol American diet were recruited for pre-study. Other criteria for subject selection of the study were as follows: (1) normal weight, (2) fasting plasma triglyceride levels below 200 mg/di, (3) no history of lipid disorder, (4) no antihypertensive medication or other medications affecting lipid metabolism, and (5) nonsmokers. Volunteers were requested to fast overnight prior to the screening blood tests. Informed consent was obtained from each subject prior to this  25  study (Appendix 2). The study protocol was approved by the Ethical Review Committee of the Oregon Health Sciences University. Anthropometric Measurements: Height was measured with the subject in stocking feet using an anatomical anthropometric plane as reference. Body weight was measured daily with the subject clothed and shoeless on a beam type balance scale calibrated with standard weights. Body mass index (BMI) was calculated as weight (kg) divided by height (m) 2 .  3.2 Diet Protocol  Three diets were used in this study. Diet 1 contained 50 mg/day cholesterol which was to be below the proposed threshold level of 100 mg/day. There was a level of 350 mg/day cholesterol in diet 2 which was around the proposed ceiling point between 300-400 mg/day. Diet 3 contained 650 mg/day cholesterol which was above the proposed ceiling point. In order to gain information on the routine diet which a selected subject consumed, each individual was required to complete food records over three days. A sample of the three day food records is given in Appendix 3. All diets were designed, calculated and prepared according to the guidelines for the protocol by the CRC Dietary Staff at the CRC kitchen. Nutritionist III, a computer software program, was used to analyze the nutritive components in the meals. The three research meals had the same caloric distribution as shown below and were composed of typical mixed foods. An egg yolk supplement was incorporated as a formula into the baseline low cholesterol diet which provided the increased levels of dietary cholesterol, especially for the high cholesterol diet.  26  Caloric Distribution  Dietary Cholesterol Levels  Carbohydrate = 55 %  (Low)  Diet 1 =  Protein  = 15 %  (Mid)  Diet 2 = 350 mg/day  Fat  = 30 %  (High) Diet 3 = 650 mg/day  mono  = 12 %  poly  =  50 mg/day  8%  saturated = 10 % P/S  = 0.8  Nutrient composition was adjusted, so that dietary cholesterol was the only variable. Dietary cholesterol in the diets generally originated from egg yolk, CRC home-made shortbread cookie, CRC home-made custard and meat (ground beef, turkey breast and chicken breast). Calorie allowance were determined according to the Boothby and Berkson Food Nomogram (Jolliffe and Alpert 1950), and adjusted to each subject's individual need for regularly scheduled physical activities to maintain body weight throughout the whole study period (Appendix 4). The research diets were designed and calculated for all three phases, from 2000 to 3200 kcal, in increments of 200 kcal. The reference diet contained 2800 kcal; other calorie levels were extrapolated from that diet. Table 1 lists three sample diets at the reference calorie level of 2800 kcal from low to high cholesterol, respectively. A seven-day menu cycle was employed for this study. Fruits, vegetables, and bread exchanges were based on the individual's personal preference to some extent without altering cholesterol content in the diet.  27  Table 1. Food Items Contained in the Three Experimental Diets at the Reference Level of 2800 kcal Per Day Diet component  Low (gm)  Mid (gm)  Hiah (gm)  Protein^15% Fat^30% mono^12% poly^8% sat.^10% CHO^55% Cholesterol (mg/day)  105 93 37 25 31 385 50  105 93 37 25 31 385 350  105 93 37 25 31 385 650  1 10 7 7 1 500 167 25 7 50 108 -  1 10 7 7 1 500 34 111 25 6 50 54 49  1 10 7 7 1 500 67 83 24 6 50 98  55 31 31 4 8 -  55 19 2 4 11  55 18 22  Exchanges: meat bread vegetable fruit protein Skim milk Whole egg, beaten Eggbeatersa Saffolab margarine Parkayb margarine SWb German chocolate CRCc no chol. shortbread CRC chol. shortbread Custard milk, evap, skim sugar, white eggbeaters Parkay margarine palm oil WFb froz. egg yolk mix.  a a daily product from Fleischmann's which is 99% real egg white, cholesterol-free. b brand name(s). C Clinical Research Center  Each experiment diet was fed for a four week period at the CRC-OSHU. Each subject was assigned by a randomization 'scheme' using numbers (Appendix 5) so that all experimental diets differing in cholesterol level were applied randomly to each subject (Table 2). Participants had their breakfast at the CRC everyday. Before breakfast, the participants' weight and blood pressure was measured by the nursing staff. Their food and beverage consumption, comments on food preference, as well as exercise over the previous day were recorded by the dietary staff. Lunch and supper were taken home by the participants. They were instructed to consume the research meals at fixed times ( Breakfast 08:10; Lunch 12:00; Dinner 18:00). Frequent contact with subjects both at the time of blood drawing and when food was provided every morning ensured subject compliance. Previous data indicated that a new steady state should be established by two to three weeks after such feeding (Nestel and Poyser 1976). After finishing each four-week period of a certain diet and D20 determination, every subject had a four-week washout period to allow body D20 level back to natural enrichment baseline prior to the next diet phase.  29  Table 2. Order of Randomized Dietary Phase Assignment Subject  *  Scheme*  Randomization diet phase  1  B  low - high - mid  2  B  low - high - mid  3  D  mid - high - low  4  C  mid - low - high  5  A  low - mid - high  6  F  high - mid - low  7  F  high - mid - low  8  E  high - low - mid  see Appendix 5.  3.3 Administration of Deuterium Oxide  D20 incorporation measurements were carried out with study participants as day patients of the CRC-OHSU. On approximately the 28th day of each four-week dietary period, 20 ml of blood was collected for determination of the natural enrichments of deuterium in cholesterol and body water at approximately 08:00. The deuterium dosing procedures were as follows. Determination of total body water: The body water volume was calculated using bioelectrical impedance analysis (BIA) or, if BIA calculation was not available, simply multiplying body weight (kg) by 0.6: Body Weight (kg) x 0.6 = Total Body Water (kg) If BIA was the method used, subject did not eat or drink anything for four hours prior to this assessment and voided immediately prior to the measurement. Calculate dose of D20: Calculation of dose of D20 was as follows: Total Body Water (kg) x 0.6 g/kg body water = ^D20 (g) D20 was weighed into small Nalgene bottle, which was then closed tightly and the lid was wrapped with parafilm. Subject then drank this priming dose of above stated D20 (99.8 atom % excess deuterium) per kg estimated body water followed by a 50 ml rinse of distilled water. The time was at 08:0, defined as zero hour timepoint. This dose was sufficient to establish the body water deuterium enrichment, assuming that body water is 73% of the total fat-free mass (Pace and Rathbun 1945). Water consuming during the next 24 hour period was labeled with D20 at 1.2 g per liter of body water deuterium enrichment at plateau. Subjects were asked to curtail any physical exertion for this 24 hour period even though it might be a part of their typical regimen.  31  Further blood samples (20 ml each) were taken at 6, 12, and 24 hours after dosing with D20 for plasma free cholesterol and red blood cell (RBC) cholesterol deuterium enrichment analysis. Plasma water deuterium enrichment were also determined at these timepoints. A E,mple of the D20 test schedule was detailed in Appendix 6. Doses of D20 of all testing subjects were calculated and listed in Appendix 7.  32  3.4 Blood Sampling  One to two weeks before the dietary intervention, two or three overnight fasting blood samples were drawn from the volunteers. These blood samples were used for the measurement of baseline plasma cholesterol and triglyceride levels. During each of the four-week dietary intervention periods, overnight fasting blood samples were obtained every three days to monitor plasma lipid profile. A minimum of two to three plasma cholesterol values were averaged at the end of each dietary period. This value was taken as the end effect of each diet on plasma cholesterol concentration. After four weeks of each diet period, 20 ml of blood was collected at t = 0 hr (day 28, 08:00) for the determination of natural enrichments of deuterium in cholesterol and body water. Further blood samples (20 ml each) were taken at 6, 12, and 24 hours after the prime dosing with D20 into tubes containing EDTA for the deuterium enrichment analysis of plasma free cholesterol, RBC cholesterol and plasma water.  08:00 (day 28)^0 hr^20 cc EDTA 14:00 (day 28)^6 hr^20 cc EDTA 20:00 (day 28)^12 hr^20 cc EDTA 08:00 (day 29)^24 hr^20 cc EDTA  Plasma was promptly separated off by centrifugation. RBC were saved in the vacutainer tube, recapped with stopper. Both RBC and plasma were refrigerated at -10°C until analysis.  33  3.5 Laboratory Procedures  The plasma cholesterol and triglyceride concentrations were quantitated fluorometically on Auto Analyzer II (Technicon Instruments Corp., Tarrytown, NY) using standard Lipid Research Clinic methods (LRCP 1974). Procedures for plasma cholesterol extraction, combustion and mass spectrometric analysis have been established as previously reported by Jones et a/.(1988, 1993a, 1993b). These analytical procedures were also applied to RBC samples in this study.  3.5.1 Lipid Extraction  Lipid was extracted from 4 ml plasma (or RBC) at each timepoint in triplicate with use of 8 ml methanol and heated in water bath at 55°C under nitrogen (N2) gas for 15 minutes. An amount of 24 ml hexane-chloroform (4:1 vol/vol) was next added and shaken for 15 minutes. Another 2 ml distilled water was added and shaken for another 10 minutes followed by centrifugation at 1500 x g for 15 minutes at 4°C. After centrifugation, the upper phase was removed and the total procedure was repeated twice again with no need of adding methanol and distilled water. The upper phases of all three extracts were then combined into one test tube and dried at 55°C water bath under N2 gas and the yellow/orange residue obtained. This precipitate was redissolved in gradually reduced amounts of 10, 6, 3 drops of chloroform, each then spotted onto thin layer chromatography (TLC) silica plates (Silica TLC, Universal Scientific Incorp. Atlanta, Georgia) which had already been activated at 100°C for 30  34  minutes. Plates were developed in petroleum ether/ethyl ether/acetic acid (135:15:1.5 vol/vol/vol) for 1 hour, allowed to dry. Free cholesterol bands on TLC were visualized in iodine vapor against an internal cholesterol standard. Free cholesterol band was then scraped from the silica plate and eluted with 6 ml of hexane/chloroform/diethyl ether (5:2:1 vol/vol/vol). After being shaken for 15 minutes followed by centrifugation (1500 x g) for 15 minutes at 4°C, the supernatant of the sample was removed and the whole procedure was repeated twice with the same solvents in the reduced amounts of 4 ml and 3 ml. The supernatant of all three elutions were then combined into one tube and dried in water bath at  °  55 C under N2 gas. The free cholesterol extract, a white residue, was finally obtained at this step.  3.5.2 Solvent Evaporation  The free cholesterol extract was redissolved by chloroform and transferred to Pyrex tube (18 cm x 6 mm) added with 500 mg copper oxide and one silver wire (2cm x 1 mm). Chloroform was removed under vacuum by freezing the tube in liquid nitrogen, and then evaporated by gradual heating for 10 minutes until the solvent boiled off and finally the internal pressure returns to baseline (less than 50 mtorr). Tubes were then sealed and put into the oven undergoing combustion at 520°C for 4 hours, and cooled down slowly to room temperature. The hydrogen and oxygen elements in free cholesterol were thus converted to water through combustion.  35  3.5.3 Distillation  All water evolved from the combustion of cholesterol was then vacuum distilled into a 18 cm x 6 mm Pyrex tube containing 60 mg zinc which was placed at an 520°C oven for 30 minutes. Water derived from free cholesterol was finally reduced to hydrogen (H2) (Schoeller et a/. 1980). The amount of 2 pl of each D20 standard for cholesterol, SMOW (Standard Mean Ocean Water), SLAP (Standard Light Antarctic), GISP (Greenland Ice Sheet), was also transferred by this vacuum-distillation system into 18 cm x 6 mm Pyrex tubes of which was containing 60 mg zinc reagent. Standards were reduced into H2 at 520°C for 30 minutes.  3.5.4 Plasma Water Preparation  To measure the deuterium enrichment of body water, additional plasma samples from 0, 12, 24 hr timepoint were needed. With the exception of base sample, 50 pl plasma samples from each timepoint were diluted sixfold by using 300 pl tap water to reduced water deuterium enrichment level to within the normal analytical range of standards used by the mass spectrometer. Triplicate 2 pl respective aliquots were vacuum distilled across 60 mg zinc in 10 cm x 6 mm Pyrex tubes and reduced at 520°C for 30 minutes. The amount of 2 pl of each additional plasma water standards, SMOW, Chicago tap water, Chicago stock and Vancouver tap water, was also prepared following the upper same procedure for the observed ratio correction in real time of H3 + contribution in mass spectrometer (Craig 1961, Peterson 1979, Schoeller et a/. 1983).  36  3.5.5 Mass Spectrometric Determination  The deuterium enrichment of samples were measured on a differential isotope ratio mass spectrometer (VG Isomass, 903D, Cheshire, UK) through the determination of cholesterol deuterium/protium (2H/1H) isotope ratio. This mass spectrometer had a double inlet, and the analyses were performed by comparison of the sample against two gas standards. The sample H3+ contribution was measured daily and appropriate corrections were applied (Schoeller et a/. 1983, Jones et a/. 1993b). The mass spectrometer was calibrated daily using water standards of known isotopic composition. Precision level for overall analysis of this study was determined by averaging the standard deviation (SD) of the replicating readings finally gained from the mass spectrometer. As a result, the average SD of deuterium enrichment for plasma water, plasma free cholesterol and RBC cholesterol samples were calculated as -5.0, -3.5 and -5.6 parts per thousand relative to SHOW, respectively.  37  (°/00)  3.6 Data Calculations  Results from the isotope ratio mass spectrometer were defined as parts per mil or:  -1 x 1000, 0/00 [(Rsamplei Rsmow)]  Eq.1  where R was the ratio of heavy (2H) to light (IH) isotopic species. Cholesterol deuterium enrichment was expressed relative to Standard Mean Ocean Water (SHOW). Fractional synthetic rate (FSR) of cholesterol in plasma and RBC was calculated based on the methods of Dietschy and Spady (1984) as adapted by Jones et a/.(1988, 1993b), which have been mentioned previously. One molecule of cholesterol contains 27 carbon atoms and 46 hydrogen atoms. During the synthesis of cholesterol, the hydrogen atoms may be incorporated into the sterol molecule from three different sources: 7 atoms are incorporated directly from water; 15 atoms are inserted from NADPH; or ultimately, hydrogen atoms from water are incorporated into the acetyl CoA pool, which can be used as a cholesterol precursor. Moreover, FSR of plasma cholesterol was determined as the fractional incorporation of precursor deuterium over time. We assumed that over a 48-hour period there was complete equilibration of D20 with plasma water and with NADPH, but that the acetyl CoA pool has not yet been labelled. Therefore, this represented that 22 deuterium atoms were incorporated into each cholesterol molecule, resulting in a D/C ratio of 0.81 in the synthesis of cholesterol. This yielded the equation:  38  delcholesterol (c)/oo) ^Eq.2  FSR (day-1) delp lasma water (°/00) x 0.81 D/C x 27C/46H  and del-lasma water were differences in deuterium where delcholesterol^p enrichment of each tissue expressed as parts per mil versus SMOW. 27C/46H represents the correction for the absolute ratio of carbon to hydrogen atoms within the cholesterol molecule (Jones et al. 1988, 1993b). Using this formula, the FSR of both plasma free cholesterol and RBC cholesterol were calculated for subjects consuming each of the three graded amounts of cholesterol. However, this calculation of FSR could be criticized since certain assumptions used by the deuterium incorporation method remained controversial: first, the central M1 pool is relatively constant in size; and second, newly synthesized cholesterol is solely released to M1 pool. First of all, Dell et a/.(1985) reported contribution of cholesterol from side pool 3 to cholesterol synthesis in a baboon study, although whether total synthesis is distributed in a similar manner in humans remains to be verified. Moreover, with the newly developed multicompartmental model, Schwartz et a/.(1993) recently revealed that incoming synthesized free plasma cholesterol was transported most rapidly between plasma and the liver, RBC, and other tissues among the rapidly miscible pool. It is possible that the newly synthesized cholesterol within the central M1 pool was removed into other tissues, preventing exchange of the labelled cholesterol with the unlabelled cholesterol carried on lipoproteins. It was also indicated by Schwartz et a/.(1993) that bile acid synthetic rate was correlated directly with the size of the large hepatic pool, thus, labelled  39  cholesterol may have been mobilized for bile acid synthesis during the measurement interval. At last, it is also possible that the increase of dietary cholesterol may result in direct dilution of the deuterium enrichment within the central Ml pool. Consequently, substantial underestimation of cholesterol enrichment or FSR could occur with the current D20 uptake method. Further advanced models taking into account the above perturbations will be required to illustrate the kinetics of cholesterol synthesis using D20 uptake method in vivo.  40  3.7 Statistical Analyses  Means and Standard Errors of the Means (± SEM) were calculated to assess each of the specific activity of deuterium enrichment versus plasma free cholesterol and RBC cholesterol, and anthropometric variables during each of the dietary periods. It was statistically desirable to have a randomized experimental design, therefore subjects were randomly assigned to different dietary groups. One-way analysis of variance (ANOVA) was performed to examine the statistical significance among plasma cholesterol concentrations and dietary cholesterol levels, cholesterol FSR and cholesterol diets, as well as cholesterol FSR and plasma cholesterol concentrations (Zar 1984, Wilkinson 1990). Regression analysis was also employed to examine the correlation between dietary cholesterol and plasma cholesterol. All statistical significances detected by one-way ANOVA were further measured by Tukey test to differentiate the real significant difference between variables. Paired sample t-test was employed to compare the difference in both deuterium enrichment and FSR values between plasma and RBC at each diet. Level of statistical significance was expressed as at least a value of P < 0.05. Interaction graphs were also drawn to facilitate visualization of the relationships between factors.  41  4. RESULTS  4.1 Subject Characteristics  After the initial screening procedures as mentioned in subject recruitment section, eight subjects (one female and seven males) were recruited for this study. The relevant mean + SEM values for anthropometric data and plasma lipid profile at baseline of the study population were summarized in Table 3. Among the eight selected healthy subjects, most of them were 40 to 60 years old (55.5 ± 4.2, mean ± SEM) with the exception of subject 2 who was 34 years old, however, his results did not appear to differ from those of the other seven subjects. The majority of subjects were within the acceptable BMI range of 21 to 25 kg/m2 (23.7 ± 0.5). Subject 5 had a slightly higher BMI (25.8) as compared to the others. The baseline plasma total cholesterol levels of these selected subjects before any dietary treatment ranged from 203 to 248 mg/dl (227.3 ± 5.9) which were within the acceptable normal range according to their ages. The subjects had normal triglyceride levels ranged from 71 to 142 mg/d1 (99.8 ± 9.2). Subjects' body weights were monitored carefully and remained unchanged during the study period. Overall, the criteria set for subject recruitment were generally met by the selected subjects.  42  Table 3. Anthropometric Data and Screening Plasma Lipid Profile of the Selected Subjects  Subject  Plasma Lipids BMI^TChol^TG (kg/m2)^(mg/di)  Sex  Age (yrs)  Height (m)  Weight (kg)  1  F  46  1.63  61.6  23.2  222  123  2  M  34  1.85  84.5  24.7  203  118  3  M  66  1.84  73.4  21.7  246  102  4  M  67  1.67  68.3  24.5  248  142  5  M  57  1.67  71.9  25.8  235  84  6  M  63  1.70  63.6  22.0  238  72  7  M  63  1.69  64.7  22.7  212  71  8  M  48  1.92  91.0  24.7  214  86  55.5 4.2  1.75 0.04  72.4 3.7  23.7 0.5  227.3 5.9  99.8 9.2  Mean SEM  4.2 Comparison of the Effect of Dietary Cholesterol on Plasma Total Cholesterol Levels  Table 4 summarizes the changes in plasma total cholesterol after dietary interventions on three graded dietary intakes of cholesterol. Plasma total cholesterol level in subjects on a high cholesterol diet (233.6 ± 4.3 mg/di) was found significantly higher than that of subjects on a low cholesterol diet (207.4 ± 5.7 mg/di, P = 0.002). This effect of three graded amounts of dietary cholesterol upon plasma cholesterol levels is plotted in Figure 3. The statistical significance was reached only between those subjects consuming a low and a high cholesterol diet (P = 0.002). Compared with the subjects on a low cholesterol diet, plasma cholesterol concentration was increased by 13% (+26 mg/di) in subjects on a high cholesterol diet. Although their plasma total cholesterol concentrations were increased by 8% (+17 mg/dl) and 4% (+10 mg/di) in subjects consuming a low cholesterol diet relative to subjects consuming a medium cholesterol diet, and in subjects consuming a medium cholesterol diet relative to subjects consuming a high cholesterol diet, respectively, statistical analysis indicated that these increases of plasma cholesterol level were not statistically significant at the P values of 0.056 and 0.344, respectively. Similar to the above effects of plasma total cholesterol levels in response to different levels of dietary cholesterol, the change of plasma total cholesterol concentration from baseline plasma cholesterol concentration in subjects consuming a high cholesterol diet (6.4 ± 4.8 mg/di) was significantly higher than that of subjects on a low cholesterol diet (-19.9 ± 5.0 mg/di, P = 0.016) (Table 4). Compared to the average of  44  6.4 mg/d1 increase observed in the whole group, subject 3, 4, 5, and 6 on a high cholesterol diet had insignificantly decreased plasma total cholesterol of -3, -1, -5, and -12 mg/d1. This may result from individual variation in response to dietary cholesterol intervention. Figure 4 shows the change of plasma cholesterol from baseline in response to the increase of dietary cholesterol in subjects consuming three experimental cholesterol diets. Again, the statistical significance was only identified between subjects consuming a low and a high cholesterol diet. Changes of plasma cholesterol from baseline were not significantly different in subjects consuming a diet between low and medium (P = 0.188), as well as between medium and high (P = 0.803) cholesterol diets. To obtain information on whether there is a linear or curvilinear association between dietary cholesterol and plasma cholesterol, regression analysis was performed. It was found that there is a strongly positive association between the dietary cholesterol levels and plasma cholesterol concentration, although this association was not linear (r2 = 0.416, P = 0.001). A similar positive association was found between dietary cholesterol and change of plasma cholesterol from baseline (r2 = 0.312, P = 0.005). Table 5 summarizes the P values of statistical analysis for the comparisons.  45  Table 4. Plasma Total Cholesterol Levels in Subjects Before and After Dietary Cholesterol Interventions  Subject  Baseline TChol (mg/dl)  TChol After treatment Low^Mid^High (mg/di)  Change of TChol from baseline Low^Mid^High (mg/di)  1  222  217  229  245  -5  7  23  2  203  179  214  210  -24  11  7  3  246  229  228  243  -23  -18  -3  4  248  218  228  247  -30  -20  -1  5  235  222  207  230  -13  -28  -5  6  238  192  217  226  -46  -21  -12  7  212  210  246  233  -2  34  21  8  214  198  223  235  -16  9  21  227.5 5.9  207.4 5.7  224.0 4.2  Mean SEM  233.6a 4.3  -19.9 5.0  -3.3 7.6  6.4b 4.8  a significantly different from low cholesterol diet group (P = 0.002). b significantly different from low cholesterol diet group (P = 0.016).  Figure 3. The Influence of Dietary Cholesterol on Plasma Cholesterol Level (a significantly different from low cholesterol (50 mg/day) diet group (P = 0.002)]  240  200  50^350^650 Dietary Cholesterol Levels (mg/day)  Figure 4. The Influence of Dietary Cholesterol on Change of Plasma Cholesterol Concentration From Baseline (a significantly different from low cholesterol (50 mg/day) diet group (P = 0.016)]  50^350^850 Dietary Cholesterol Levels (mg/day)  Table 5. Summary of the P Values on Plasma Cholesterol Cholesterol and Changes of Plasma Cholesterol Concentration in Subjects Consuming Three Experimental Diets  P Value Plasma Cholesterol  Change of Plasma Cholesterol  All Diets  0.003*  0.017 *  Low vs High  0.002 *  0.016 *  Low vs Mid  0.056  0.188  Mid vs High  0.344  0.803  * Significantly different (P < 0.05).  4.3 Comparison of Deuterium Enrichment and Cholesterol FSR Between Plasma and RBC  Deuterium enrichment between plasma and RBC was compared over different time intervals at 6, 12, and 24 hours postdose in subjects consuming each of the three cholesterol test diets. The results of deuterium enrichment, expressed as parts per thousand (°/oo) relative to SMOW were given in appendix 8. For better visualization, all comparisons are shown in Figure 5, Figure 6 and Figure 7, in which testing subjects were consuming a low, a medium and a high cholesterol diet, respectively. Deuterium enrichment of RBC cholesterol lagged behind that of plasma free cholesterol at all diets. However, this lagging equilibration of de novo synthesized cholesterol from plasma to RBC was found significantly different only at the 6 (P = 0.010) and 12 (P= 0.005) hour time interval in subjects consuming high cholesterol diet (Table 6). The least significance of the lagging equilibration was observed in subjects consuming a medium cholesterol diet (Figure 6). Table 6 summarizes the results of paired sample t-test on plasma versus RBC deuterium enrichment in different time intervals.  50  Figure 5. Deuterium Enrichment of Plasma and RBC in Subjects Consuming Low Cholesterol Diet As a Funtion of Time  Figure 6. Deuterium Enrichment of Plasma and RBC in Subjects Consuming Medium Cholesterol Diet As a Funtion of Time  160  _  RBC v Plasma A  -  Mean MEM -4--  c  0 20  a^12^18 Time Interval (hrs)  24  30  Figure 7. Deuterium Enrichment of Plasma and RBC in Subjects Consuming High Cholesterol Diet As a Function of Time [a,^significantly different from RBC at 6 and 12 hour interval (P = 0.010, P = 0.005), respectively]  Table 6. Summary of P Values of the Paired Sample T-Tests on Deuterium Enrichment of Plasma Versus RBC at Different Time Intervals Low^Medium^High Time^ Interval^All Diets^Chol Diet^Chol Diet^Chol Diet  0.006*  0.137  0.363  0.010*  12  0.021  0.157  0.635  0.005*  24  0.290  0.166  0.655  0.219  6  * Significantly different (P < 0.05).  Based on deuterium enrichment of both cholesterol and plasma water, Table 7 lists all the calculated FSR values of plasma and RBC from subjects consuming three different cholesterol diets. Being similar with cholesterol deuterium enrichment, FSR values in plasma were found significantly different from those of RBC at 0-6 (P < 0.02) and 0-12 (P < 0.02) hour time period. For better comparison and visualization, all FSR values from all three experimental diets were plotted against each time period in Figure 8, diversed FSR values between plasma and RBC in the early time periods became constantly intimate towards the increasing time period. At 0-24 time period, none of these plasma and RBC FSR values were significantly different from each other. Similarly, plasma FSR value was constantly higher than that of RBC in each time period of each of the experimental diets.  54  Table 7. Plasma and RBC Cholesterol FSR in Subjects Consuming Three Experimental Diets FSR (Day-1) Subject 0-6  Plasma 0-12  0-24  0-6  RBC 0-12  0-24  Low diet 1^-.0524 2^.1418 3^.0711 4^.1395 5^.0913 6^.0906 7^.2181 8^.0221  -.0206 .1145 .0076 .0925 .1064 .0609 .1484 .0384  .1058 .0949 -.0239 .0712 .1226 .0616 .0990 .0963  .0592 .0208 .2098 .0349 .0273 .0469 .0385 -.1930  .0143 .0663 .1486 .0324 .0702 .0304 .0427 -.0247  .0557 .0700 .0887 .0540 .0922 .0642 .0648 .0756  Mean^.0900 SEM^.0290  .0690 .0200  .0780 .0160  .0310 .0390  .0480 .0180  .0710 .0050  Medium diet 1^.0728 2^.1172 3^.1107 4^.0335 5^.0342 6^.0998 7^.0503 8^.0433  .0869 .1046 .0488 .0768 .0539 .0651 .0527 .0899  .0350 .0428 .1652 .0204 .0720 .0098 -.0559 .1301  .0556  .1299 .0572 .0307 .0703 .0686 .0339 .0853  .0648 .0040 .0635 .0417 .0590 .0947  .0813 .0921 .0569 .0200 .0919 .0326 .0677 .1123  Mean^.0700 SEM^.0120  .0680 .0130  .0720 .0070  .0520 .0250  .0550 .0100  .0690 .0110  High diet 1^.0545 2^.1088 3^.1881 4^.0027 5^.5103 6^.2104 7^.1067 8^.0692  .0560 .1432 .1278 .0194 .2113 .0717 .0501 .0337  .0764 .1364 .0964 .0518 .1065 .0286 .0475 .0251  .0102 .0414 -.1214 .0116 .2104 -.0653 .0178 -.0343  .0300 .0672 -.0298 .0100 .0767 .0035 .0316 -.0081  .0603 .1037 .0253 .0367 .1076 .0540 .0378 .0346  Mean^.1560a SEM^.0560  .0890b .0230  .0710 .0140  .0090 .0340  .0230 .0130  .0580 .0110  a Significantly different (P < 0.02) from RBC 0-6 timepoint in the high diet. b Significantly different (P < 0.02) from RBC 0-12 timepoint in the high diet.  Figure 8. Equilibrium of Synthesized Cholesterol Between Plasma and RBC on Three Experimental Diets [a, b Significantly different from RBC at 0-6 and 0-12 hour time period in high cholesterol diet (P < 0.02)]  0.18  0.12 0.09 cc 0.08 0.03 0.00 0-8^0-12^0-24 Time Interval (hre)  4.4 Comparison of the Effect of Dietary Cholesterol on Cholesterol FSR  The effect of dietary cholesterol on cholesterogenesis was examined by analyzing 0-24 hour plasma FSR values in Figure 9. Cholesterogenesis was not altered in response to increasing dietary cholesterol levels (P = 0.913). A wide range of subject variation of FSR value was observed in both low and high cholesterol diet groups. Table 8 summarizes the results of ANOVA on cholesterol FSR in subjects consuming three different cholesterol diets. No statistical significance was found in effect of dietary cholesterol upon FSR at each time interval of each diet.  Table 8. Summary of the P Values on Cholesterol FSR in Subjects Consuming Three Experimental Diets  Plasma Time Interval (hr)  RBC Time Interval (hr)  6  6  12  12  24  24  All Diets  0.251  0.690  0.913  0.653  0.276  0.574  Low vs Mid  0.534  0.983  0.735  0.640  0.742  0.912  Low vs High  0.312  0.515  0.736  0.680  0.278  0.302  Mid vs High  0.155  0.983  0.735  0.640  0.742  0.912  57  Figure 9. Overall Plasma Cholesterol FSR in Subjects Consuming Three Experimental Diets  4.5 Comparison of the Effect of Plasma Total Cholesterol on Cholesterol FSR  In order to investigate the effect of plasma cholesterol upon cholesterogenesis, plasma 0-24 hour FSR of subjects consuming low, medium and high cholesterol diets was firstly plotted against change of plasma cholesterol from baseline in Figure 10, Figure 11 and Figure 12, respectively. On low cholesterol diet (Figure 10), decreasing plasma cholesterol level from baseline did not alter significantly FSR values (P = 0.298). Also, FSR in either the medium (Figure 11) or the high (Figure 12) cholesterol diet was not affected by change of plasma cholesterol concentration (P = 0.458, P = 0.707, respectively). Secondly, change of plasma cholesterol concentration from low to medium cholesterol level did not appreciably alter cholesterol FSR in subjects on a medium cholesterol diet (Figure 13, P = 0.868), and in subjects on a high cholesterol diet (Figure 14, P = 0.987). Thirdly, even when the change of plasma cholesterol from a low to a high cholesterol diet was plotted against FSR in Figure 15, there was no correlation between plasma cholesterol level and cholesterol FSR (r2 = 0.253, P = 0.204). Finally, no relationship of increase in plasma cholesterol from a low to a high cholesterol diet and decrease in plasma FSR from a low to a high cholesterol diet was observed among the testing subjects (Figure 16, P = 0.454).  59  Figure 10. Changes of Overall Plasma Cholesterol FSR and Changes of Plasma Cholesterol From Baseline in Subjects Consuming a Low Cholesterol Diet  0.2  —  0 0  0  -0.1 -50^-40^-30^-20^-10^0 Change of Plasma Cholesterol From Baseline (mg/d1)  60  Figure 11. Changes of Overall Plasma Cholesterol FSR and Changes of Plasma Cholesterol From Baseline in Subjects Consuming a Medium Cholesterol Diet  0.11 S.: co -o 0.1 0 Cr) a 0.09 cr co u_ 1,--6 0.08 0 zi (L1-5 0.07 0 a)  i^i^i^i^1^i^1  0  _  -  0  0  -  -5 _c  0  0 0.06 E  '40 0.05 -  0^  0  0  -  0.04 -40 -30 -20 -10 0 10 20 30 40 Change of Plasma Cholesterol From Baseline (mg/d!)  61  Figure 12. Changes of Overall Plasma Cholesterol FSR and Changes of Plasma Cholesterol From Baseline in Subjects Consuming a High Cholesterol Diet  62  Figure 13. Plasma Cholesterol FSR in Subjects Consuming Medium Cholesterol Diet Versus Change of Plasma Cholesterol From Low to Medium Cholesterol Diet  Figure 14. Plasma Cholesterol FSR in Subjects Consuming Medium Cholesterol Diet Versus Change of Plasma Cholesterol From Medium to High Cholesterol Diet  Figure 15. Plasma Cholesterol FSR in Subjects Consuming High Cholesterol Diet Versus change of Plasma Cholesterol From Low to High Cholesterol Diet  Figure 16. Change of Plasma FSR Versus Increase in Plasma Cholesterol From Low to High Cholesterol Diet  Table 9. Summary of the P Values Obtained From ANOVA Comparison on Relationship Between Changes of Cholesterol FSR and Changes of Plasma Total Cholesterol Concentration FSR (day-1) Low diet  P value on Change of Plasma Cholesterol baseline Low-medium Medium-high Low-high ^  Medium diet High diet  ^  ^  Low-high diet  0.298  0.458  0.707  ^  0.858  ^  ^  0.987^0.204 0.454  DISCUSSION  5.1 Subject Characteristics  This is a unique study in which plasma cholesterol levels, cholesterol FSR and the equilibration of newly synthesized cholesterol between plasma and RBC have been studied simultaneously in healthy humans consuming three diets with cholesterol content of 50, 350 and 650 mg/day. Based on the three pool model, overall human cholesterol turnover is influenced by certain anthropometric parameters such as age and body weight. In this investigation, subjects were randomly assigned to all of the three dietary treatment groups. Confounding factors such as interindividual variation, difference in physical characteristics and lipid profiles have been eliminated in this study. Therefore, in addition to the with-in person variations, dietary composition was the only variable contributing to any alteration of cholesterol metabolism, since body weight virtually remained unchanged during the investigation period. There were seven males and one female recruited for this study. Results of the female subject (subject 1) in this study did not differ considerably from other male subjects. Despite the fact that subject 2's age was relatively lower than the average age of the study population (34 vs. 55.5 4.2 yrs), and subject 5 had slightly higher BMI (25.8 vs. 23.7 ± 0.5 kg/m2), their experimental data did not markedly vary from those of others. All subjects remained healthy throughout the whole study period. These data were supported by the following two controlled human studies. In a study by Katan and Beynen (1987), 21 men and 11 women aged from 19 to 62 years were fed a low followed by a high cholesterol diet for three to four weeks. They  68  found no relation of responsiveness with age, sex, and intestinal transit time on plasma total cholesterol levels in low-cholesterol and highcholesterol diets. Also in a recent study by Clifton and Nestel (1992), 26 men and 25 women aged from 25 to 65 were matched for age, LDL cholesterol, TG, and BMI. These subjects were given a low-fat, low-cholesterol (<250 mg/day) diet followed by a high-fat, high-cholesterol (650 mg/day) diet each for three weeks. With respect to the plasma total cholesterol levels, no gender difference was found in these subjects in response to the dietary interventions. The overall criteria for subject recruitment as discussed previously in the experimental design section was generally fulfilled in the present study.  69  5.2 Effect of Dietary Cholesterol on Plasma Total Cholesterol Levels  The effect of dietary cholesterol on plasma cholesterol concentration has been studied extensively and the results are inconclusive and controversial. However, in a series of precise, well-controlled metabolic studies, high dietary cholesterol generally increases the plasma levels of total cholesterol (Connor et a/. 1961a, Connor et a/. 1961b, Connor et a/. 1964, Mattson et al. 1972, Erichson et a/. 1964, Grande et al. 1965, Hegsted et a/. 1965, Anderson et al. 1976, Packard et al. 1983, Grundy et a/. 1988). In the present study, an overall 13% increase of plasma cholesterol was observed in subjects consuming a high cholesterol diet (650 mg/day) compared to those consuming a low cholesterol diet (50 mg/day). The relation between the increase of dietary cholesterol from 50 to 650 mg/day and plasma cholesterol was not linear overall, but positive (Figure 3). A similar result was obtained when the increase of dietary cholesterol was plotted against the change of plasma cholesterol from baseline (Figure 4). The present finding is generally in accordance with findings of several other studies (Mattson et al. 1972, Gyling and Miettinen 1992, Hopkins 1992). Mattson et a/.(1972) observed that plasma cholesterol concentration was linearly increased by cholesterol intake of up to 400 mg/day. In a study by Gylling and Miettinen (1992), 29 home-living men with an average age of 55 were placed on a low-fat, low cholesterol diet (208 mg/day) for 6 weeks followed by a low-fat high cholesterol diet (878 mg/day) for 5 weeks. The high dietary cholesterol was achieved by adding three egg yolks per day to the previous low-fat low cholesterol diet. It was found that plasma  70  cholesterol was increased significantly by 10% in subjects on the low-fat high cholesterol diet. In a meta-analysis and review of the effect of dietary cholesterol on plasma cholesterol concentration, Hopkins (1992) combined 27 studies in which controlled diets were supplied by metabolic kitchens. It was found that an approximate 12-15% increase in plasma cholesterol level could be predicted when 500 mg/day cholesterol was added to a cholesterol-free diet in normal subjects. This finding was also confirmed by the present study. However, results of the present study are not fully in agreement with the proposed S-shaped threshold and ceiling amounts of dietary cholesterol in Figure 1. Based on the proposed "S" shaped curve, the threshold point on plasma cholesterol is 100 mg/day of dietary cholesterol, and the ceiling point on plasma cholesterol is reached when dietary cholesterol is raised to 350 mg/day. As a result, the present finding (Figure 3, 4) is partially in agreement with regard to the ceiling point, since a further increase of dietary cholesterol to 650 mg/day only elevated plasma cholesterol by 4% which was not significant. When the threshold point is considered, the increase of plasma cholesterol should be significant at 350 mg/day as compared to 50 mg/day of dietary cholesterol. However, the present study reveals that although there is an 8% increase in plasma cholesterol, it is of borderline statistical significance (P = 0.056). The lack of significance of the present data might be mainly due to the experimental design itself. The three cholesterol diets used in this experiment may not have generated enough data points for the appropriate comparison. At least three additional diets are required to obtain enough points to plot and compare the results with those summarized and proposed in Figure 1. In addition to the 50 mg/day cholesterol diet, a dietary  71  cholesterol of approximately around 100 - 150 mg/day is also needed for accurate determination of the threshold point. Furthermore, two other additional cholesterol diets in the range of 450 - 500 and up to 800 - 900 mg cholesterol per day are required to well define the proposed ceiling point. By combining data from 27 studies, Hopkins (1992) recently did observe the S-shaped relationship between change in plasma cholesterol and added dietary cholesterol. Alternatively, the relatively large variability of subjects plasma cholesterol level in response to dietary cholesterol seen in this study may also be partially responsible for difficulties in data interpretation. There were some individuals who were much more responsive than others (Figure 2). Nevertheless, the present results suggest that plasma cholesterol concentration is clearly increased due to an increase in dietary cholesterol from 50 mg/day to 650 mg/day. The magnitude of change in plasma cholesterol in response to dietary cholesterol tended to plateau when the level of dietary cholesterol reached 650 mg/day. Additional diets containing different levels of cholesterol are required to define the threshold and ceiling effect of dietary cholesterol upon plasma cholesterol concentration.  72  5.3 Equilibration of Synthesized Cholesterol Between Plasma and RBC  For cholesterol deuterium uptake measurement in normolipidemic subjects, a relatively large amount of initial plasma volume (2m1) per replicate is required to yield 1 mg free cholesterol, the amount needed to produce 1 pl combustion water necessary for isotopic mass spectrometric analysis. This blood volume may not be easily obtained in certain subjects such as infants. Alternatively, the use of smaller amount of blood in the analysis can be achieved by employing RBC samples. This is due to the fact that plasma comprises both free and esterified cholesterol. However, RBC do not synthesize cholesterol and contain solely free cholesterol exchanged from plasma (London and Schwartz 1953). Thus, compared to plasma samples, smaller amount of blood is needed to get the necessary amount of free cholesterol for analysis when RBC samples are used. Both plasma and RBC were considered to be within the central pool comprising cholesterol synthesized from liver and intestine (Goodman et a/. 1973). So far, either plasma (Jones et a/. 1993b) or RBC (Wong et a/. 1991) have been sampled for determination of cholesterol deuterium uptakes. However, an intraindividual cross-comparison of the correspondence between plasma and RBC deuterium enrichment in measuring human cholesterol synthesis has not been fully investigated. The rate of equilibration between plasma and RBC remains unclear. An early study conducted by Hagerman and Gould (1951) incorporating C14-acetate carbon into cholesterol in vitro between plasma and RBC showed that equilibration of free cholesterol between plasma and RBC was closely approached in four hours in a dog previously fed a cholesterol-free diet.  73  In a recent human study using 3H and "C radiolabelled cholesterol in vivo, Schwartz et ai.(1993) found that free cholesterol was transported rapidly between plasma and RBC in an exchange rate of 11.6 mg/min. Equilibration of plasma and RBC free cholesterol specific activities was reached by 6.7 - 10 hours corresponding to the time when RBC free cholesterol specific activity reached a peak. By employing the deuterium uptake method, in vivo cholesterol synthesis has been estimated using sample derived from either plasma in humans consuming low and high cholesterol diets (Jones et a/. 1993b) or RBC in piglets fed a cholesterol-free diet and a diet containing 0.5% cholesterol (Wong et a/. 1991). Results of the present study indicate that D20 (Figure 5-7) was initially incorporating more slowly into RBC from plasma over the initial 6-12 hour time interval in all three cholesterol diets. This lagging deuterium enrichment from plasma to RBC was found to be significant in the high cholesterol diet only at 6 and 12 hour time intervals (Figure 7). Likewise, FSR values between plasma and RBC, interpreted from deuterium enrichment data, were not correlated with each other in all three diets until the 24 hour time interval was reached. The difference was significant on high cholesterol diet at 0-6 and 0-12 time period (Figure 8). The present results were mostly confirmed by data from Schwartz et a/.(1993),  in which 6.7 to 10 hours were required for the thorough equilibration of radiolabelled free cholesterol between plasma and RBC. These findings generally indicate the substantial interpool exchange of free cholesterol between plasma and RBC over the initial postdose time intervals up to 12 hours. The complete equilibration of deuterium enrichment between plasma and RBC was reached in 24 hours postdose on all subjects consuming three experimental diets.  74  However, the reason for the much slower deuterium incorporation from plasma to RBC in subjects consuming high cholesterol diet remains unknown. We postulated that the actual central pool size was enlarged and/or deuterium enrichment in the pool is diluted out by the incremental incoming dietary cholesterol during the initial 12 hours when cholesterol intake was high. As a result, deuterium enrichment in plasma was decreased, which might contribute to the slower exchange of deuterium into RBC. Nevertheless, a more advanced mathematical model is required in which all variables or parameters would be factored in for cholesterol kinetic analysis using the deuterium uptake method. Current results imply that over the initial 6-12 hour post-dose interval, measurement of RBC deuterium uptake may result in underestimation of cholesterogenesis when cholesterol intake is high. The use of 24 hour interval from either plasma or RBC sample best reflects cholesterol synthesis.  75  5.4 Effect of Dietary Cholesterol on Cholesterogenesis  The metabolic response to increased dietary cholesterol in humans might include a decreased absorption of dietary cholesterol, a reduction in de novo cholesterol synthesis and an increase in the excretion of biliary cholesterol. Several feedback responses have been documented during high cholesterol intake in humans. However, cholesterol synthesis is found frequently (Nestel and Poyser 1976, Lin and Connor 1981, McMurry et al. 1985, McNamara et a/. 1987, Miettinen and Kesaniemi 1989) but not consistently to be down-regulated (Kern 1991, Everson et al. 1991, Jones et a/. 1993b). In the study conducted by Nestel and Poyser (1976), two normolipdemic and seven hyperlipidemic subjects consumed a low cholesterol diet (250 mg/day) and a high cholesterol diet (750 mg/day) each for a period of four to six weeks. Cholesterol synthesis measured by sterol balance method was suppressed in five, including the two normolipidemic, subjects on the high cholesterol diet. However, the amounts of fat and the high range of cholesterol used in this experiment were higher than ours (40% vs. 30%, and 750 vs. 650 mg/day, respectively). Furthermore, in a long-term sterol balance study, cholesterol synthesis was inhibited by a high cholesterol intake of 1000 mg/day compared to a very low cholesterol diet in one normocholesterolemic and one hypercholesterolemic subjects (Lin and Connor 1981). Finally, eight human subjects were fed sequentially a cholesterolfree diet for three weeks followed by a diet containing 900 mg cholesterol for another three weeks under controlled conditions (McMurry et al. 1985). Cholesterol biosynthesis measured by sterol balance method decreased significantly by 49%. Therefore, we speculated that the high cholesterol  76  intake of 650 mg/day used in the present study may not be high enough to induce the feedback inhibition of cholesterol synthesis. However, in another study conducted by Kern (1991), cholesterol synthesis was quantified by measuring the 14C-acetate incorporated into cholesterol in mononuclear cells. It was found that cholesterogenesis in a subject consuming 25 eggs per day remained equal to the mean synthetic rate in normal subjects. Individual variability in the response to a given change in the dietary cholesterol level vary widely. It was found that this individual had a great reduction in cholesterol absorption and a marked increase in the hepatic conversion of cholesterol into bile acids. Using deuterium uptake methodology, our data show that cholesterol FSR is not significantly changed (P = 0.913) by increasing cholesterol intake from low, medium to high (Figure 9). This finding suggests that cholesterol synthesis is independent to changes in dietary cholesterol. It is suggested that cholesterogenesis is primarily mediated by the differences in cholesterol absorption efficiency, neutral sterol excretion and conversion of hepatic cholesterol to bile acids. We speculate that with incremental cholesterol intake, cholesterol synthesis can remain constant due to many homeostatic and regulatory mechanisms such as reduction in the efficiency of cholesterol absorption, down-regulating of LDL-receptor activity, increased bile acid synthesis and increased excretion and re-excretion of absorbed cholesterol. Unfortunately, we did not examine these parameters in the present project. There is no doubt, however, that the absorption of cholesterol influences the rate of synthesis, at least in the liver by a feedback control. Grundy et a/.(1969) reported that the total body cholesterol synthesis measured by sterol balance method did not reduce when a large amount of dietary cholesterol was given to a normocholesterolemic  77  subject. However, the synthesis of cholesterol rose strikingly when cholesterol absorption was suppressed with plant sterol. They concluded that feedback control of cholesterol synthesis by dietary cholesterol was relatively unimportant for the regulation of cholesterol metabolism under the normal metabolic condition. This was because that cholesterol absorption mechanism was primarily saturated by the large amount of endogenous cholesterol available for reabsorption. Again, marked individual variations in cholesterogenesis in response to dietary cholesterol level, especially to low and high cholesterol diets (Figure 9), may somewhat explain the small effect of dietary cholesterol upon cholesterol synthesis in this study.  78  5.5 Relationship between Plasma Total Cholesterol Levels and Cholesterol FSR  Results of previous studies examining the effects of plasma cholesterol upon cholesterol synthesis have not been consistent. Katan and Beynen (1987) reported that whole body cholesterol synthesis was inversely correlated with responsiveness of plasma cholesterol concentration to a dietary cholesterol challenge in humans consuming low cholesterol diet. They concluded that higher cholesterol synthetic rate in subjects whose plasma cholesterol levels respond little to dietary cholesterol (hyporesponders) would enable stronger feedback inhibition of cholesterol synthesis during the period of dietary cholesterol challenge. However in a recent study, Jones et al. (Jones et a/. 1993b) reported that such a negative association with low cholesterol diet was not observed, yet there was a significant inverse association between FSR and the increase in plasma cholesterol in subjects on high compared to low cholesterol diet. Data of the present study did not show any significant influence of plasma cholesterol concentration upon cholesterol synthesis (Figures 1016). The mechanisms involved in the regulation of cholesterol synthesis in response to changes in plasma cholesterol levels are still unclear. It is proposed that regulation of cholesterol synthesis may be a relatively passive component of overall regulatory process, and its suppression occurs only when plasma cholesterol levels are not normally controlled by other mechanisms such as decreased cholesterol absorption from the gut, increased bile acid synthesis and excretion (Jones et a/. 1993b). As a result, we postulate that plasma cholesterol level in response to dietary cholesterol is primarily governed by other regulatory mechanisms including alterations  79  of the intestinal absorption efficiency, LDL-receptor activity, secretion of cholesterol into bile, and hepatic conversion of cholesterol into bile acid. The response of plasma cholesterol concentration to cholesterol synthesis is only secondary to the failure of these compensating mechanisms.  80  5.6 Conclusions  Mechanism of cholesterol homeostasis was investigated by identifying differences in dietary cholesterol level and endogenous cholesterol production. These factors may contribute to variations in plasma cholesterol concentration which are closely associated with the development of CHD. Plasma cholesterol concentration was clearly increased in response to an increase of dietary cholesterol. In the subjects on a high cholesterol diet, plasma cholesterol was increased by 13% (P = 0.002) as compared to the low cholesterol diet. The threshold and ceiling amount of dietary cholesterol, however, could not be determined due to an inadequate number of levels of cholesterol in the experimental diets. Although there was an observable increase of plasma cholesterol in response to change of dietary cholesterol from low to medium, the magnitude of increase in plasma cholesterol tended to flatten out when dietary cholesterol was changed from medium to high. Prospectively, graded cholesterol diets utilizing an increased number of at least six cholesterol levels are needed to well define the threshold and ceiling amount of dietary cholesterol. During the initial 12 hour postdose interval, deuterium incorporation from plsama to RBC cholesterol was delayed in all three cholesterol diets. This lag in deuterium enrichment in RBC was significantly different in high compared with low and medium cholesterol diets. Therefore, use of RBC deuterium uptake results may lead to an underestimation of cholesterol synthesis over the initial 12 hour interval when cholesterol intake is high. 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Am J din Nutr 29:11841189, 1976 Armstrong ML and Megan MB: Lipid depletion in atheromatous coronary arteries in rhesus monkeys after regression diets. Circ Res 30:675-680, 1972 Becker N, Illingworth DR, Alaupovic P et a/.: Effects of saturated, monounsaturated, and omega-6 polyunsaturated fatty acids on plasma lipids, lipoproteins and apolipoproteins in humans. Am J din Nutr 37:355-360, 1983 Bennion LJ and Grundy SM: Effect of obesity and caloric intake on biliary lipid metabolism in man. J din Invest 56:9961011, 1975 Beveridge JMR, Connell WF, Mayer GA et al.: The response of man to dietary cholesterol. J Nutr 71:61-65, 1960 Bhattacharyya AK, Connor WE, Mausolf FA and Flatt A: Turnover of xanthoma cholesterol in hyperlipoproteinemic patients. J Lab din Med 87:503-518, 1976 Bjorkhem I, Miettinen T, Reihner E et a/.: Correlation between serum levels of some cholesterol precursors and activity of HMG-CoA reductase in human liver. J Lipid Res 28:11371143, 1987 Bottcher CJF and Woodford FP: Chemical changes in arterial wall associated with atherosclerosis. Fed Proc 21 (Suppl. II):15-19, 1962 Brown MS, Goldstein JL and Dietschy JM: Active and inactive forms of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver of rat. J Biol Chem 254:5144-5149, 1979 Carulli N, Tripodi A and Carubbi F: Assay of HMG-CoA reductase activity in the evaluation of cholesterol cholesterol. Clinica Chimica Acta 83:77-82, 1989 Clifton PM and Nestel PJ: Influence of gender, BMI, and age on response of plasma lipids to dietary fat plus cholesterol. Arteriosclerosis and Thrombosis 12:995-962, 1992  Cole T, Pfleger B, Hitchins 0 and Schonfeld G: Effects of high fat diet on plasma lipoproteins in familial hypercholesterolemia. Metabolism 34:486-493, 1985 Connor WE, Hodges RE and Bleiler RE: The serum lipids in men receiving high cholesterol and cholesterol-free diets. J din Invest 40:894-900, 1961a Connor WE, Hodges RE and Bleiler RE: Effect of dietary cholesterol upon serum lipids in man. J Lab din Med 57:331-342, 1961b Connor WE, Stone DB and Hodges RE: The interrelated effects of dietary cholesterol and fat upon the human serum lipid levels. J din Invest 43:1691-1696, 1964 Connor WE and Connor SL: The key role of nutritional factors in the prevention of coronary heart disease. Prey Med 1:49-83, 1972 Connor WE and Lin DS: The intestinal absorption of dietary cholesterol by hypercholesterolemia (type II) and normocholesterolemic humans. J Clin Invest 53:1062-1070, 1974 Connor WE: In Garry PJ, ed: Human nutrition, Clinical and biochemical aspects. Washington, DC: American Association for Clinical Chemistry, pp. 44, 1980 Connor WE and Connor SL: The dietary prevention and treatment of coronary heart disease. In: Coronary Heart Disease: Prevention, complications, and treatment, edited by Connor WE and Bristow JD. Lippincott JB Comp., Philadelphia. pp. 43-64, 1985 Connor WE and Connor SL: Dietary cholesterol and fat and the prevention of coronary heart disease: Risks and benefits of nutritional change. In: Diet and prevention of coronary disease and cancer, edited by Hallgren B, Levin 0, Rossner S and Vessby B. Raven Press, New York. pp. 113-147, 1986 Craig H: Standard for reporting concentrations of deuterium and oxygen-18 in natural waters. Sciences 131:1833-1834, 1961 Dell RB and Ramakrishnan R: A mathematical model for cholesterol kinetics. In: Lipoprotein Kinetics and Modeling, edited by Berman M, Grundy SM and Howard G. Academic Press Inc., New York. pp. 313-330, 1982 Dell RB, Mott GE, Jackson EM, Ramakrishnan R, Carney KD,HC McGill Jr. et al.: Whole body and tissue cholesterol turnover in the baboon. J Lipid Res 26:327-337, 1985  Dempsey ME: Regulation of steroid biosynthesis. Annu Rev Biochem 43:967-990, 1974 Dietschy JM and Wilson JD: Regulation of cholesterol metabolism. N Eng J Med 282:1128-1138, 1179-1183, 12411249, 1970 Dietschy JM: Regulation of cholesterol metabolism in man and in other species. Klin Wochesnschr 62:338-345, 1984 Dietschy JM and Spady DK: Measurement of rates of cholesterol synthesis using tritiated water. J lipid Res 25:1469-1476, 1984 Erichson BA, Coots RH, Mattson FH et al.: The effect of partial hydrogenation of dietary fats, and of the ratio of polyunsaturated to saturated fatty acids, and of dietary cholesterol upon plasma lipids in man. J Clin Invest 43:2017-2025, 1964 Everson GT, McKinley C and Kern FJ: Mechanisms of gallstone formation in women. Effects of exogenous estrogen (Premarin) and dietary cholesterol on hepatic lipid metabolism. J din Invest 87:237-246, 1991 Ferezou J, Rautreau J, Coste T, Gouffrier E and Chevallier F: Cholesterol turnover in human plasma lipoproteins: Studies with stable and radioactive isotopes. Am J Clin Nutr 36:235-244, 1982 Flaim E, Ferreri L, Thye F, Hill J and Ritchey S: Plasma lipid and lipoprotein cholesterol concentrations in adult males consuming normal and high cholesterol diets under controlled conditions. Am J din Nutr 34:1103-1108, 1981 Goodman DS, Noble RP and Dell RB: Three pool model of the long-term turnover of plasma cholesterol in man. J Lipid Res 14:178-188, 1973 Goodman DS, Smith FR, Seplowitz AH, Ramakrishnan R and Dell RB: Prediction of the parameters of whole body cholesterol metabolism in humans. J Lipid Res 21:699-713, 1980 Goodman DS, Deckelbaum RJ, Palmer RH, Dell RB, Ramakrishnan R, Delpre G et a/.: Cholesterol turnover and metabolism in two patients with abetalipoproteinemia. J Lipid Res 24:1605-1611, 1983 Grande F, Anderson JT, Chlouverakis C et al.: Effect of dietary cholesterol on man's serum lipids. J Nutr 87:5262, 1965 Grundy SM, Ahrens EHJ and Davignon J: The interaction of cholesterol absorption and cholesterol synthesis in man. J  85  Lipid Res 10:304-315, 1969 Grundy S, Barrett-Connor E, Rudel L, Miettinen T and Spector A: Workshop on the impact of dietary cholesterol on plasma lipoproteins and atherogenesis. Arteriosclerosis 8:95-101, 1988 Gylling H and Miettinen TA: Cholesterol absorption and synthesis related to low density lipoprotein metabolism during varying cholesterol intake in men with different apoE phenotypes. J Lipid Res 33:1361-1371, 1992 Hagerman JS and Gould RG: The in vitro interchange of cholesterol synthesis between plasma and red cells. Proc Soc Exp Biol Med 78:329-332, 1951 Hegsted DM, McGandy RB, Meyers ML et al.: Quantitative effects of dietary fat on serum cholesterol in man. Am J din Nutr 17:281-295, 1965 Hopkins PN: Effect of dietary cholesterol on serum cholesterol: a meta-analysis and review. Am J Clin Nutr 55:1060-1070, 1992 Jacobs DR, Anderson JT, Hannan P, Keys A and Blackburn H: Variability in individual serum cholesterol response to change in diet. Arteriosclerosis 3:349-356, 1983 Jagannathan SN, Connor WE, Baker WH and Bhattacharyya AK: The turnover of cholesterol in human atherosclerotic arteries. J din Invest 54:366-377, 1974 Jolliffe N and Alpert E: The "performance index" as a method for estimating effectiveness of reducing regimens. Postgraduate Medicine 9:106-115, 1950 Jones PJH, Scanu AM and Schoeller DA: Plasma cholesterol synthesis using deuterated water in humans: Effect of short-term food restriction. J Lab din Med 111:627-633, 1988 Jones PJH, Illingworth DR and Leitch CA: Correspondence between plasma mevalonic acid levels and deuterium uptake in measuring human cholesterol synthesis. Europ J din Invest 22:609-613, 1992 Jones PJH, Leitch CA, Li ZC and Connor WE: Human cholesterol synthesis measurement using deuterated water: theoretical and procedural considerations. Arteriosclerosis & Thrombosis 13:247-253, 1993a Jones PJH, Main BF and Frohlich JJ: Response of cholesterol synthesis to cholesterol feeding in men with different apolipoprotein E genotypes. Metabolism 42:1-9, 1993b  86  Katan MB and Beynen AC: Hypo- and hyperresponders: Individual differences in the response of serum cholesterol concentration to changes in diet, in Paoloetti R, Kritchevsky D (eds): Advances in Lipid Research, vol 22, San Diego, CA, Academic Press, pp115-171, 1987 Kern FJ: Normal plasma cholesterol in an 88-year-old man who eats 25 eggs a day. Mechanisms of adaptation. N Engl J Med 324:896-899, 1991 Keys A, Anderson JT, Mickelson 0 et al.: Diet and serum cholesterol in man: lack of effect of dietary cholesterol. J Nutr 59:39-56, 1956 Keys A, Anderson JT and Grande F: Serum cholesterol response to changes in the diet. II The effect of cholesterol in the diet. Metabolism 14:759-765, 1965 Kopito RR and Brunengraber H: (R)-mevalonate excretion in human and rat urines. Proc Natl Acad Sci USA 77:5738-5740, 1980 Kopito RR, Weinstock SB et al.: Metabolism of plasma mevalonate in rats and humans. J Lipid Res 23:577-583, 1982 Kummerow FA, Kim Y, Pollard J et al.: The influence of egg consumption on the serum cholesterol level in human subjects. Am J Clin Nutr 30:664-673, 1977 Langer T, Strober W and Levy RI: The metabolism of low density lipoprotein in familial type II hyperlipo-proteinemia. J din Invest 51:1528-1536, 1972 Lieberman S and Samuel P: Determination of total body cholesterol: Input-output analysis versus compartmental analysis. In: Lipoprotein Kinetics and Modeling, edited by Berman M, Grundy SM and Howard G. Academic Press Inc., New York. pp. 331-336, 1982 Lin DS and Connor WE: The long-term effects of dietary cholesterol upon the plasma lipids, lipoproteins,cholesterol absorption, and the sterol balance in man: The demonstration of feedback inhibition of cholesterol biosynthesis and increased bile acid excretion. J Lipid Res 21:1042-1052, 1981 Lipid Research Clinic Program manual of laboratory operations. Vol. 1. Bethesda, MD: Clinics Program. National Institutes of Health, 1974. [DHEW publications (NIH) 75-628]. Lipid Research Clinics Program. The Lipid Research Clinics Coronary Primary Prevention Trial results. II. The  87  relationship of reduction in incidence of coronary heart disease to cholesterol lowing. JAMA 251:365-374, 1984 Liu GCK, Ahrens EH Jr, Schreibman PH, Samuel P, McNamara DJ and Crouse R: Measurement of cholesterol synthesis in man by isotope kinetics of squalene. Proc Natl Acad Sci USA 72:4612-4616, 1975 London IM and Schwartz H: Erythrocyte cholesterol : the metabolic behavior of the cholesterol of human erythrocytes. J din Invest 32:1248-1252, 1953 Mahley RW, Innerarity TL, Bersot TP et al.: Alteration in human high-density lipoproteins with or without increased plasma cholesterol, induced by diets high in cholesterol. Lancet 11:807-809, 1978 Mattson FH, Erickson BA and Klingman AM: Effect of dietary cholesterol on serum cholesterol in man. Am J din Nutr 25:589-594, 1972 McMurry MP, Connor WE, Lin DS, Cerqueira MT and Connor SL: The absorption of cholesterol and the sterol balance in the Taraumara Indians of Mexico fed cholesterol-free and high cholesterol diets. Am J din Nutr 41:1289-1298, 1985 McNamara DJ, Kolb R, Parker TS et a/.: Heterogeneity of cholesterol homeostasis in man, Response to changes in dietary fat quality and cholesterol quantity. J din Invest 79:1729-1739, 1987 Messinger WJ, Porosowska Y and Steele JM: Effect of feeding egg yolk and cholesterol on serum cholesterol levels. Arch Intern Med 86:189-195, 1950 Miettinen TA: Diurnal variation of cholesterol precursors squalene and methyl sterols in human plasma lipoproteins. J Lipid Res 23:466-473, 1982 Miettinen TA and Kesaniemi YA: Cholesterol absorption: regulation of cholesterol synthesis and elimination and within-population variations of serum levels. Am J Clin Nutr 49:629-635, 1989 Mistry P, Miller NE, Laker M, Hazzard WR and Lewis B: Individual variation in the effects of dietary cholesterol on plasma lipoproteins and cellular cholesterol homeostasis in man. Studies of low density lipoprotein receptor activity and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in blood mononuclearcells. J Clin Invest 67:493-502, 1981 Nestel PJ, Schreibman PH and Ahrens EH: Cholesterol metabolism in human obesity. J din Invest 52:2389-2397, 1973 88  Nestel PJ and Kudchodkar B: Plasma squalene as an index of cholesterol synthesis. Clin Sci Mol Med 49:621-624, 1975 Nestel PJ and Poyser A: Changes in cholesterol synthesis and excretion when cholesterol intake is increased. Metabolism 25:1591-1599, 1976 Newman HAI and Zilversmit DB: Quantitative aspects of cholesterol flux in rabbit atherosclerosis lesions. J Biol Chem 237:2078-2084, 1962 Norum KR, Berg T, Helgerud P and Drevon CA: Transport of cholesterol. Phys Rev 63:1343-1419, 1983 Packard CJ, McKinney L, Carr K et a/.: Cholesterol feeding increases low density lipoprotein synthesis. J din Invest 72:45-51, 1983 Parker TS, McNamara DJ, Brown CD, Garrigan 0, Kolb R, Batwin H et al.: Mevalonic acid in human plasma: relationship of concentration and circadian rhythm to cholesterol synthesis rates in man. Proc Natl Acad Sci USA 79:30373041, 1982 Parker TS, McNamara DJ, Brown CD, Kolb R, EH Ahrens Jr, Albert AW et al.: Plasma mevalonate as a measure of cholesterol synthesis in man. J din Invest 74:795-804, 1984 Pace N and Rathbun EN: Studies on body composition. III. The body water and chemically combined nitrogen content in relation to fat content. J Biol Chem 158:685-691, 1945 Peterson DW: High precision mass spectrometric hydrogen isotope ratio measurements. Ph.D. Thesis, Indiana University, 1979 Porter MW, Yamanaka W, Carlson SD et a/.: Effect of dietary egg on serum cholesterol and triglyceride of human males. Am J din Nutr 30:490-495, 1977 Rittenberg D and Schoenheimer R: Deuterium as an indicator in the study of intermediary metabolism. J Biol Chem 121:235251, 1937 Schoeller DA, Van Santen E, Peterson DW, Dietz W, Jaspan J and Klein PD: Total body water measurement in humans with 180 and 2H water. Am J din Nutr 33:2686-2693, 1980 Schoeller DA, Peterson DW and Hayes JM: Double-comparison method for mass spectrometric determination hydrogen isotopic abundances. Anal Chem 55:827-832, 1983 Schwartz CC: Cholesterol kinetics and modeling: introduction.  89  In: Lipoprotein Kinetics and Modelina, edited by Berman M, Grundy SM and Howard G. Academic Press Inc., New York. pp. 309-312, 1982 Schwartz CC, Zech LA, VandenBroek JM and Cooper PS: Cholesterol kinetics in subjects with bile fistula: Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate. J din Invest 91:923-938, 1993 Slater G, Mead J, Dhopeshwarkar G et a/.: Plasma cholesterol and triglyceride in men with added eggs in the diet. Nutr Dept Intl 14:249-259, 1976 Spady D and Dietschy J: Sterol synthesis in vivo in 18 tissues of the squirrel, monkey, guinea pig, rabbit, hamster and rat. J Lipid Res 24:363-475, 1983 Stamler J, Wentworth D and Neaton J: Is the relationship between serum cholesterol and risk of death from coronary heart disease continuous and graded? JANA 256:2823-2828, 1986 Steiner A and Domanki B: Dietary hypercholesterolemia. Am J Med Sci 201:820-824, 1941 Steiner A, Howard EJ and Akgun S: Importance of dietary cholesterol in man. JAMA 181:186-190, 1962 Taylor CB, Mikkelson B, Anderson JA and Forman DT: Human serum cholesterol synthesis measured with the deuterium label. Arch Path 81:213-231, 1966 The Pooling Project Research Group: Relationship of blood pressure, serum cholesterol, smoking habit, relative weight and ECG abnormalities to incidence of major coronary events: Final report of the pooling project research group. J Chron Dis 31:201-306, 1978 Turley S and West C: Effect of cholesterol and cholestyramine feeding and of fasting on sterol synthesis in liver, ileum, and lung of the guinea pig. Lipids 11:571-577, 1976 Turley S, Spady D and Dietschy J: Alteration of the degree of manipulation of the pools of preformed and newly synthesized cholesterol. Gastroen 84:253-264, 1983 Wells VM and Bronte-Stewart B: Egg yolk and serum cholesterol levels. Brit Med J 1:577-581, 1963 Wilkinson L.. SYSTAT: The system for statistics. Evanston, IL: SYSTAT, Inc., 1990 Wong WW, Hachey DL, Feste A et al.: Measurement of in vivo  90  cholesterol synthesis from 2820: a rapid procedure for the isolation, combustion, and isotopic assay of erythrocyte cholesterol. J Lipid Res 32:1049-1056, 1991 Zar JH: Multiple hypotheses. In: Biostatistical analysis. 2nd edition. pp. 162-184, 1984  Appendix 1. Lipid Studies Volunteer Information Form Date Name Last^First^MI^(Maiden Name) Birthday ^ Age ^ Sex (Circle) M F Phone (home) ^ (work) ^ message ^ Address^ . Street^City^State^Zip OHSU Clinic Card # ^ Social Security # State/Country of Birth ^ Mather's Maiden Name ^ . Typical Work Schedule ^ Days^Hours^Occupation Do you smoke? yes^no ^ If yes, how much? ^ Number of years?^If you quit smoking, how long ago?^. Recent weight changes? yes no Weight gain Weight loss_. If yes, please indicate how much^Over what time period_. How long have you been at your current weight? ^ What was your weight at high school? ^ Alcohol consumption (indicate type and amount) ^ Do you drink milk? yes no ^If yes, number of cups per day_. Do you have problems digesting milk or do you have a milk allergy? yes^no^. Please explain ^ Typical Meal Times: Breakfast ^ (Indicate Meals Skipped) Lunch^ Dinner Do you typically snack? yes ^no^When? ^ Do you have a microwave at home? yes^no^ Do you have access to a microwave at work? ^ Do you have any known food allergies? ^ Do you have any difficult chewing foods? yes^ no^ If yes, please list foods ^  92  Exercise Regimen:^Type^Minutes/Session^Sessions/Week  Marital Status: Do you have children? yes^no^How many?^Ages^ Is your mother living? yes ^no^. If yes, present age ^ If no, age at death and cause of death ^ Is your father living? yes^no^. If yes, present age ^. If no, age at death and cause of death ^ What is your most recent cholesterol level? ^ Date:^. If cholesterol level is elevated, when did you first know your cholesterol was high? Approximate date or number of years: ^ Do any of your relatives have an elevated cholesterol level? . How did you first hear about our research studies? [] Newspaper article asking for volunteers [] Radio announcement [] Campusgram announcement [] Other; Please specify ^ Your physician's name ^ May we contact him for information if you participate in a study? [] Yes^[] No Please list any other health problems: (Include major illnesses and/or surgeries along with approximate dates)  Are you taking any medicines? [] Yes^[] No If yes, please indicate below: Name of medication Dose For how long Diuretic (water pill) Diabetes medication Medication to lower cholesterol and/or triglycerides Thyroid medication Hormones Birth control pills Blood pressure medication^  93  Aspirin or Anti-flammatory^ Anticoagulates Others (Identify)  Vitamins (Identify)  Antacids (Identify) Are you allergic to any medications?^yes^no^. If yes, please list: ^ Are you following any kind of special diet recommended by a physician or other health professional? ^  For Females Only: Are you currently having regular menstrual periods? ^ Date of last period? ^ Have you reached menopause (change of life)? ^ Have you had your uterus (womb) removed? ^ If you are eligible for a dietary research study, are there any times in the next six months that you would not want to be involved in a study (vacations, holidays, out of town business?) If so, please indicate the approximate dates below:  Please check below any of health problems experienced by you or a family member such as grandfather, sister, brother, aunt, or uncle:  YOU^MOTHER^FATHER^OTHER FAMILY MEMBER (SPECIFY) HIGH CHOLESTEROL HIGH TRIGLYCERIDE STROKE HIGH BLOOD PRESSURE HEART ATTACK ANGINA DIABETES OTHER HEART PROBLEMS (SPECIFY) GALL BLADDER PROBLEMS ARTERIOSCLEROSIS OF LEGS (HARDENING OF ARTERIES) LIVER DISEASE KIDNEY DISEASE HYPOTHYROIDISM ALCOHOL ABUSE MILDLY OVERWEIGHT EXTREMELY OVERWEIGHT  Please check how you feel about the following foods:  FRUITS^  VEGETABLES  YUM OK YUK^  Cooked beans: frozen/canned green kidney broccoli carrots Fresh (seasonal):^cauliflower cantaloupe^ cabbage grapes^ mushrooms honeydew melon^ onions nectarines^ peas peaches^ tomatoes pineapples plums^ Raw: strawberries^ broccoli Fresh:^ apples^ bananas^ grapefruit^ oranges^  tangerine^ watermelon^  cauliflower cabbage 95  YUM OK YUK  ^  carrots celery Frozen:^ cucumbers peaches^ green peppers ^ lettuce, iceberg blackberries ^ mushrooms blueberries strawberries^ onions spinach Canned: tomatoes prune vegetable juice applesauce ^ ^ cranberry sauce tomato ^ V-8 (vegetable) peaches pears ^ PROTEIN FOODS pineapples kiwi  YUM OK YUK Others: raisins honey jelly Juices: pineapple apple cranberry grape grapefruit orange  turkey chicken ham beef yogurt, plain fish pork eggbeaters egg white 1% cottage cheese countdown cheese  BREADS AND GRAIN  ENTREES (fat-free, low salt) I'LL TRY IT^YUK  YUM OK YUK Cereal, hot cream of wheat oatmeal Cereal, cold all bran corn flakes puffed wheat rice krispies raisin bran wheat ies Toast, Roll, Bread french bread whole wheat bread white bread branola bread english muffin wheatberry bread bagel banana muffin  Meatless chili w/beans Macaroni & cheese (fat free) Tomato soup vegetable soup Meatless Spaghetti Sauce Stir fry veggies in oriental Baked potato w/mock sour crm Tetrazzini: noodles in white sauce w/ optional peas and mushrooms SALADS & DRESSINGS  96  blueberry bran muf^ YUM OK YUK pocket bread mixed green salad Other Carbohydrate Choices macaroni salad bean salad bread sticks potato salad corn tortillas low calories taco chips french dressing potatoes no oil italian rice cakes dressing brown rice white rice spaghetti macaroni corn angel cake sherbet  Appendix 2. Consent and Instruction Form "Dietary Cholesterol in Graded Amounts: Threshold to Ceiling Effects upon Plasma Lipoproteins, Apoproteins and LDL Turnover" ^ , herewith agree to serve as a subject in the investigation entitled "Dietary Cholesterol in Graded Amounts: Threshold to Ceiling Effects upon Plasma Lipoproteins, Apoproteins and LDL Turnover" under the supervision of William E. Connor, M.D., D. Roger Illingworth, M.D., Ph.D., Dan Ullmann, DSc., M.P.H., Lauren Hatcher, M.S., R.D., Don Lin, M.S.. I. Purpose The aim is to study the effects of different and precise amounts of dietary cholesterol upon blood fat levels. II. Procedures The procedures in which I will participate are: a. Eating Research Diets. There will be three separate dietary periods each lasting four weeks. Between each dietary period, I will be required to return to my typical home diet for at least four weeks. The study diets will consist of whole mixed foods (meats, grains, vegetables, dietary products, fruits) and will be provided as three meals with snacks. In addition, a formula or foods as custard or cookies containing varying amounts of cholesterol will be provided. I agree to eat all of my meals provided by the study and to eat nothing else during the study. I understand that I may drink coffee, tea and sugar-free beverages and chew sugar-free gum, but not consume alcohol because of its excess calories and other effects upon blood fats. The fat content of the diets will be modest (30% of calories) and will be the same in each of the three dietary periods. b. Prestudy Evaluations. Prior to beginning of the study, my blood will be drawn on three separate occasions to establish my baseline cholesterol level. I agree to an initial medical history and examination. Initial blood pressure, height, and weight measurements will be taken. c. Daily Maintenance. During the dietary periods, body weight will be measured daily and blood pressure will be measured twice a week. I will be asked to report my daily physical activity. d. LDL Turnover Studies. To measure how quickly cholesterol is metabolized, I agree to participate in a test involving radio active material, called an LDL turnover, three times during this study. A radioactive isotope (25 microcuries of 1251) will be given each time. The radiation exposure lies within the limit of experimental radiation exposure considered acceptable by the Radiological Health Section of the Oregon State Board of Health. Although no radiation dose has been proven to be entirely safe, the dose to which you will be exposed has not shown to cause cancer or other problems.  98  e. Assessment of cholesterol Synthesis by Administration of "Tagged" Water (Deuterium Labeled Water). Deuterium is a heavy isotope of hydrogen and thus pose no radiation hazard or toxicity with the small amount given, When consumed, it is incorporated in the body's water and can be used to trace an individual's cholesterol metabolism. On the last day of each of the dietary periods, I will be asked to spend the day at the Clinical Research Center for this test. At about 8 AM, I will drink approximately 1 ounce of the "tagged" water to begin the test and then for the reminder of the 24 hour period I will use the "tagged" water provided to me for making any beverages or for any water that I wish to drink. Blood samples will be drawn initially and at 6, 12, and 24 hours after the first dose of tagged water. I will consume the :esearch diet normally provided to me on this test day; however, the meal times will be fixed. In addition, I will be asked to limit my exercise during this 24 hour period. f. Venipuncture or Blood Sampling. Blood will be drawn three times before I begin consuming the study diet to evaluate my baseline cholesterol. During each of the dietary periods blood will be drawn 27 times including the blood sampling for the LDL and deuterated water procedures. The total amount of blood drawn for the three dietary periods will be approximately 1 1/3 cups for each dietary period. I understand that my blood count will be monitored so that this amount of blood sampling will not cause anemia over the lengthy time period of the study. g. 24 Hour Urine Collection. I agree to collect all urine for 24 hours: two times before the first diet period and two times during each of the three dietary periods. I will be instructed on the methods to collect the 24 hour urine samples and these samples will be used to assess the effects of the different diets on cholesterol synthesis as well as determine the urinary excretion of radioactivity following the injection of radioiodinated lipoproteins. III. Cost I understand that during the course of the study, the food and the charges incurred by the blood tests necessary to evaluate the study will be free of charge. I will receive a small payment of $2.50 per day at successful completion of the entire study. This study will involve approximately 14 weeks (22 weeks including break period) of my time during which I will be inconvenienced as little as possible. IV. Risk Venipuncture causes modest discomfort and may be associated with bruising and rarely infection at the venipuncture site and the possibility of clots in the vein or the occurrence of small scars. Collection of a 24 hour urine sample involves my inconvenience but is without risk. Although I may not benefit directly from the study, the information contained may improve the understanding and treatment of patients with disorders of cholesterol metabolism.  99  V. Confidentiality I understand that neither my name nor my identity will be used for publication or publicity purposes. I understand that I am free not to participate, and I may withdraw from participation in this study at any time, and it will in no way affect my relationship with the Oregon Health Sciences University. I understand that Drs. Connor, Illingworth, Ullmann and Lauren Hatcher will answer at any time during the study, all questions I might have about the study or procedures. If I have any questions or if there is an emergency, I may call Dr. Connor at 494-8005 (work) or 226-0529 (home). VI. Liability I understand it is not the policy of the Department of Health , Education, and Welfare, or any other agency funding the research project in which I am participating, to compensate or provide medical treatment for human subjects in the event the research results in physical injury. The Oregon Health Sciences University, as an agency of the state, is covered by the State Liability Fund. If I suffer any injury from the research project, compensation would be available to me only if I establish that the injury occurred through the fault of the University, its officers or employees. If I have further questions, I may call Michael D. Baird, M.D. at (503)4948014. I will receive a copy of this consent form if I desire it. I have read the foregoing and agree to participate.  (Signature of Subject)  (Signature of Witness)  ^  ^  (Signature of Investigator)  ^  (Date)^(Time)  (Date)^(Time)  (Date)^(Time)  100  Appendix 3. A Sample of Three Day Food Records You have been asked to keep food records for the research study you are participating in. The information these records will supply is very important to the study, as it tells us about the composition of the foods you typically eat. What you typically eat can affect the way you may respond to the study diet or supplement. A dietitian will tell which days to write down all the food and beverages that you eat. The following is a list of points to be kept in mind as you keep your food record: 1. Record BRAND NAMES where applicable. This is especially important for foods containing fat. Bring in the label if possible. 2. Specify if foods are COOKED or RAW. 3. For COOKED foods specify METHOD of PREPARATION. Example whether meat is fried, broiled, baked, etc.. Identify fats used to cook foods. 4. Record NAMES of RESTAURANTS. 5. Record INGREDIENTS in RECIPES or ANY "COMBINATION" FOOD. Example - turkey sandwich - list type of bread, amount of meat, amount of mayonnaise, mustard, lettuce, cheese, etc.. 6. Record AMOUNT of foods. Use measuring cups and measuring spoons or copy the weight from the food package if possible. Record dimensions (in inches) if weights or measures are not available. 7. Note AMOUNT of foods NOT consumed. Example - bone and fats in meat. Example: ^ Food^ Time/ Location  Amount  07:30 scrambled eggs, home scrambled with Parkay stick marg. toast - Branola bread Parkay marg. on toast orange juice - from frozen conc.  2 2 2 2 8  10:00 coffee with cream caf.^real cream donut - glazed yeast donut 5"  8 oz 1 tablespoon 1 whole  12:00 sandwich: whitebread home turkey wafer thin slices  2 slices 1/2 of 3 oz pkg 101  whole large eggs teaspoons slices teaspoons oz  mayonnaise "Lite" Best Foods Coke - no ice apple large 4" diameters  1 tablespoon 1 - 12 oz can 1 whole  18:00 baked chicken breast (no skin) rest. mashed potatoes green beans seasoned with marg. Beer 12 oz Lite Budweiser  1 medium 1/2 cup 1/2 cup 1 whole  22:00 milk - 2% home cake - chocolate from Pillsbury, made with whole eggs and Wesson oil  12 oz  102  1 - 4x4x2" square  Appendix 4. Calculation of Caloric Intake of Selected Subjects Consuming North American Diet Estimated by: Checked by: Date:  ^ Name: ^  Height:  ^ Weight: ^  Age:  ^ Birthday:  Occupation: ^ Type of activity in occupation: ^ Exercise (or hobby activities): Type^Frequency^Duration (or distance)^Est. Cals.  Nomogram BMR:^kcal  Appendix 4 (cont'd) Estimation of food factor  % above BMR^Classification^Groups of individuals 20%^limited activity^bed-rest anorexia nervosa 30%^minimum activity^up and about inactive women > 50 years old 40%^limited activity^most in-patient women men > 50 years old 50%^limited to normal most in-patient men activity^most out-patient women 60%^normal lifestyle^most out-patient men 70%^heavy work^physical laborers those who schedule daily rigorous physical exercise e.g. 1 hr/day  Food Factor Food Factor Food Factor  Selected eucaloric level: Explanation:  kcals kcals kcals  Appendix 4 (cont'd) Selected daily caloric allowance of the subjects Subject Body surface* BMR* ^Food factor Food allowance ^ area (m2)^(kcal/day) (kcal/day) 1^1.59^1420^1.4^2000 2^2.00^1950^1.8^3600 3^1.85^1720^1.7^3000 4^1.72^1600^1.6^2500 5^1.76^1620^1.7^2800 6^1.65^1520^1.6^2400 7^1.65^1540^1.4^2200 8^2.12^2100^1.8^3800  * determined from reference Jolliffe and Alpert 1950.  Appendix 5. Randomization Scheme for Graded Cholesterol Protocol Scheme#^  Phase order  First^Second^Third (Dietary cholesterol level of phase, mg/day)  A  50  350  650  B  50  650  350  C  350  50  650  D  350  50  650  E  650  50  350  F  650  350  50  Appendix 6. Deuterated Water Test Schedule Sample Form Volunteer Name: Diet Phase: Unit #: Schedule for consumption of deuterated water on ^day, 199_, at AM. ^grams deuterated water to be consumed. Container to be rinsed with 50 g of distilled water which subject should also drink. to administer the deuterated water. Pt./subject to remain at CRC throughout day due to slight chance of vertigo with the deuterated water. Actual time deuterated water was consume ^ AM. 0 6 12 24  hr hr hr hr  20 20 20 20  cc cc cc cc  EDTA EDTA EDTA EDTA  Send 0 hr and 6 hr blood samples to lipid lab. For 12 hr blood sample, nursing staff to spin, separate off plasma, save red cells in vacutainer, recap with stopper, and refrigerate the red cells and freeze the plasma. This sample to be sent to lipid lab with the 24 hr sample when it is drawn the next morning. (Tubes to be labeled 0, 6, 12, or 24 hr). In addition to beverages which are a part of the research diet, volunteer should consume, throughout the day, 2000 g of distill water containing 2.4 grams deuterated water. This water can be used for making decaffeinated coffee and/or tea and Crystal Lite beverage. Total amount of coffee or tea consumed should be 3 cups or less. No other beverages, including carbonated beverages, are allowed. Dieticians to keep a record of dietary intake -- menu used and time of meals and snack(s). Meal/snack times and menu used to be the same on each diet phase.  107  Appendix 7. Doses of Deuterated Water  Subject Total body water^Estimation method^D20 (kg)^ (g) 1  32.9  BIA  19.7  2  51.0  BIA  30.6  3  40.6  BIA  24.4  4  41.0  body weight x 0.6  24.6  5  43.4  body weight x 0.6  26.1  6  40.0  7  38.8  8  54.0  BIA body weight x 0.6 BIA  24.0 23.3 32.4  ^  Appendix 8. Deuterium Enrichment of Plasma and RBC Free Cholesterol From Baseline Plasma Water at Various Timepoints Among Three Experimental Diets Subject Code  Free cholesterol 2H/1H relative to SMOW (°/oo) Plasma^ RBC 6hr^12hr^24hr^6hr^12hr 24hr  Low diet 1 2 3 4 5 6 7 8  21.6 49.0 10.3 59.3 34.6 38.9 81.6 5.2  -17.0 79.1 2.2 78.7 80.6 52.3 111.0 18.0  174.4 131.1 -14.0 121.1 185.7 105.8 148.1 90.2  24.4 7.2 30.5 14.8 10.3 20.1 14.4 -45.2  11.8 45.8 43.2 27.6 53.2 26.1 31.9 -11.6  91.8 96.7 51.6 91.9 139.6 110.2 97.0 70.8  Mean SEM  32.2 11.7  50.6 15.9  117.8 22.0  9.6 8.3  28.5 7.4  93.7 9.2  Medium diet 33.2 1 2 43.8 3 42.2 4 15.2 5 13.3 6 36.6 7 18.8 8 10.2  97.1 43.7 27.8 54.6 50.4 25.4 40.1  158.4 156.4 74.5 139.2 83.7 95.6 78.8 84.5  15.9 16.0 63.0 9.2 27.9 3.6 -20.9 30.6  50.7 49.5 3.6 49.3 30.6 44.1 44.5  148.2 137.7 85.9 36.3 142.6 47.9 101.3 105.3  Mean SEM  26.7 4.9  48.4 9.1  108.9 12.8  18.2 8.5  38.9 6.4  100.7 15.0  High diet 1 27.2 2 34.9 3 57.7 4 1.1 5 141.9 6 83.3 7 44.4 8 23.5  55.8 91.9 78.4 15.1 117.5 56.8 41.7 22.9  152.3 175.2 118.3 80.8 118.5 45.3 79.0 34.0  5.1 13.3 -37.2 4.5 58.5 -25.9 7.4 -11.6  29.9 43.1 -18.3 7.8 42.7 2.8 26.3 -5.5  120.2 133.2 31.0 57.2 119.7 85.5 62.9 46.9  100.4 17.5  1.8 10.2  16.1 8.0  82.1 13.6  Mean SEM  51.7a 15.5  60.0b 12.3  a Significantly different from that of RBC in the high diet (P = 0.010). b Significantly different from that of RBC in the high diet (P = 0.005).  109  

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