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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 CEILINGEFFECTS UPON PLASMA FREE CHOLESTEROL SYNTHESIS,EQUILIBRATION AND CIRCULATION LEVELS IN HUMANSBYZI-CHI LIM.D., Jinan University, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESSCHOOL OF FAMILY AND NUTRITIONAL SCIENCESDIVISION OF HUMAN NUTRITIONWe accept this thesis as confirming^THE UNIVERSITY OF BRITISH COLUMBIAOctober 1993© ZI-CHI LI, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of ^ex,e0i-e—a<3The University of British ColumbiaVancouver, CanadaDate act (02, / DE-6 (2/88)ABSTRACTThe purpose of this study was to investigate the effect of dietarycholesterol upon plasma cholesterol concentration and cholesterogenesis;and the aspect of equilibration of newly synthesized cholesterol betweenplasma and red blood cell (RBC) in humans. Eight healthy subjects (sevenmen and one woman) at the age of 55.5 ± 4.2 (mean ± SEM) years wererecruited for this study. Three experimental diets (55% carbohydrate, 15%protein, 30% fat, P:S = 0.8) containing 50mg (low), 350mg (medium) and650mg (high) cholesterol per day were randomly consumed by these subjectsfor four weeks at levels designed to maintain body weight. On day 28 ofeach 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 frombody water into plasma free cholesterol over 24 hours. RBC cholesteroldeuterium incorporation was also compared to that of plasma freecholesterol. Plasma cholesterol was significantly elevated by 13% (+26mg/di, P = 0.002) in the high as compared to low but not medium cholesterolcontaining diets. Equilibration of deuterium enrichment from the newlysynthesized plasma free cholesterol, expressed as parts per thousand (0/00)relative to Standard Mean Ocean Water (SMOW), to RBC cholesterol wassignificantly 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 postdosein the high cholesterol diet. Dietary cholesterol levels of 50, 350, and650 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 ofplasma cholesterol and rate of cholesterogenesis was observed among thethree experimental diets. These findings suggest that dietary cholesterollevels affect plasma cholesterol concentrations. Deuterium incorporationbetween plasma free cholesterol and RBC cholesterol is delayed over theinitial 12 hours postdose. Use of a deuterium incorporation period of 24hours, or more, enables more accurate determination of cholesterogenesiswhen using this methodology. Cholesterogenesis is neither affected bydietary cholesterol nor correlated with alterations of plasma cholesterolconcentration.111TABLE OF CONTENTSABSTRACT^ iiLIST OF TABLES^ viLIST OF FIGURES viiACKNOWLEDGEMENT^ xi1. INTRODUCTION 12. LITERATURE REVIEW^ 42.1 Dietary Cholesterol Metabolism^ 42.2 Effect of Dietary Cholesterol Contenton Plasma Cholesterol Levels 62.2.1 Animal Studies^ 62.2.2 Human Studies 62.3 Cholesterol Compartmentalization^ 142.4 Measurements of Cholesterol Synthesis ^ 182.4.1 Sterol Balance Methods 182.4.2 HMG-CoA Reductase Activity^ 182.4.3 Kinetic Analysis of the Isotopic Decayof Isotopically Labelled Cholesterol^ 192.4.4 Measurement of Levels of CholesterolPrecursors^ 202.5 Deuterium Incorporation Methodology^ 222.5.1 History 222.5.2 Sensitivity^ 222.5.3 Three-Pool Model 232.5.4 Assumptions 243. EXPERIMENTAL DESIGN AND METHODS^ 253.1 Subject Recruitment^ 253.2 Diet Protocol^ 263.3 Administration of Deuterium Oxide^ 313.4 Blood Sampling 333.5 Laboratory Procedures^ 343.5.1 Lipid Extraction 343.5.2 Solvent Evaporation 353.5.3 Distillation^ 363.5.4 Plasma Water Preparation^ 363.5.5 Mass Spectrometric Determination^ 373.6 Data Calculations^ 383.7 Statistical Analyses 414. RESULTS^ 424.1 Subject Characteristics^ 424.2 Comparison of the Effect of DietaryCholesterol on Plasma Total CholesterolLevels^ 444.3 Comparison of Deuterium Enrichment andCholesterol FSR Between Plasma and RBC^ 50iv4.4 Comparison of the Effect of DietaryCholesterol on Cholesterol FSR^ 574.5 Comparison of the Effect of Plasma TotalCholesterol on Cholesterol FSR 695. DISCUSSION^ 685.1 Subject Charateristics^ 685.2 Effect of Dietary Cholesterol on PlasmaTotal Cholesterol Levels 705.3 Equilibration of Synthesized CholesterolBetween Plasma and RBC^ 735.4 Effect of Dietary Cholesterol onCholesterolesterogenesis 765.5 Relationship Between Plasma TotalCholesterol Levels and Cholesterol FSR^ 795.6 Conclusions^ 81BIBLIOGRAPHY^ 83APPENDICES 92Appendix 1. Lipid Studies VolunteerInformation Form^ 92Appendix 2. Consent and Instruction Form^ 98Appendix 3. A Sample of Three Day Food Records^ 101Appendix 4. Calculation of Caloric Intakeof Selected Subjects ConsumingNorth American Diet^ 103Appendix 5. Randomization Scheme for GradedCholesterol Protocol^ 106Appendix 6. Deuterated Water TestSchedule Sample Form^ 107Appendix 7. Doses of Deuterated Water 108Appendix 8. Deuterium Enrichment of Plasma andRBC Free Cholesterol From BaselinePlasma Water at Various Time PointsAmong Three Experimental Diets 109LIST OF TABLESTable 1. Food Items Contained in the ThreeExperimental Diets at the ReferenceLevel of 2800 kcal Per Day^ 28Table 2. Order of Randomized DietaryPhase Assignment^ 30Table 3. Anthropometric Data and Screening PlasmaLipid Profile of the Selected Subjects^ 43Table 4. Plasma Total Cholesterol Levels inSubjects Before and After DietaryCholesterol Interventions^ 46Table 5. Summary of the P Values on PlasmaCholesterol Concentration and Changes ofPlasma Cholesterol Concentration in SubjectsConsuming Three Experimental Diets^ 49Table 6. Summary of P values of the Paired Samplet-tests on Deuterium Enrichment of PlasmaVersus RBC at Different Time Intervals^ 54Table 7. Plasma and RBC Cholesterol FSR inSubjects Consuming Three Experimental Diets^ 55Table 8. Summary of the P Values on CholesterolFSR in Subjects Consuming ThreeExperimental Diets^ 57Table 9. Summary of the P Values on the RelationshipBetween Changes of Cholesterol FSR andChanges of Plasma Total CholesterolConcentration^ 67Figure 1.LIST OF FIGURESThe Proposed "S" Shaped Curve ofEffects on the Plasma CholesterolLevel of Gradually Increasing theAmount of Dietary Cholesterol inHumans Whose Background Diet isVery Low in Cholesterol Content^ 12Figure 2. Three-pool Model With PossibleSide-Pool Synthesis^ 15Figure 3. The Influence of Three Graded Amountsof Dietary Cholesterol on PlasmaCholesterol Levels^ 47Figure 4. The Influence of Dietary Cholesterol onChange of Plasma Cholesterol ConcentrationFrom Baseline^ 48Figure 5. Deuterium Enrichment of Plasma and RBCin Subjects Consuming Low CholesterolDiet As a Function of Time^ 51Figure 6. Deuterium Enrichment Between Plasmaand RBC in Subjects Consuming MediumCholesterol Diet As a Function of Time^ 52Figure 7. Deuterium Enrichment of Plasmaand RBC in Subjects Consuming HighCholesterol Diet As a Function of Time^ 53Figure 8. Equilibrium of Synthesized CholesterolBetween Plasma and RBC on Three ExperimentalDiets^ 56Figure 9. Overall Plasma Cholesterol FSR in SubjectsConsuming Three Experimental Diets^ 58Figure 10 Changes of Overall Plasma CholesterolFSR and Changes of Plasma CholesterolFrom Baseline in Subjects Consuming aLow Cholesterol Diet^ 60Figure 11. Changes of Overall Plasma CholesterolFSR and Changes of Plasma CholesterolFrom Baseline in Subjects Consuming aMedium Cholesterol Diet^ 61Figure 12. Changes of Overall Plasma CholesterolFSR and Changes of Plasma CholesterolFrom Baseline in Subjects Consuming aHigh Cholesterol Diet^ 62viiFigure 13. Plasma Cholesterol FSR in SubjectsConsuming Medium Cholesterol DietVersus Change of Plasma CholesterolFrom Low to Medium Cholesterol Diet^ 63Figure 14. Plasma Cholesterol FSR in SubjectsConsuming High Cholesterol Diet VersusChange of Plasma Cholesterol FromMedium to High Cholesterol Diet^ 64Figure 15. Plasma Cholesterol FSR in SubjectsConsuming High Cholesterol Diet VersusChange of Plasma Cholesterol From Lowto High Cholesterol Diet^ 65Figure 16. Change of Plasma FSR Versus Increasein Plasma Cholesterol From Low to HighCholesterol Diet^ 66ACKNOWLEDGEMENTThe present research was conducted under the supervision of Dr. PeterJones, Division of Human Nutrition, University of British Columbia. Manythanks to Dr. Peter Jones for his constructive guidance and valuableinstruction. Many thanks to Dr. Jiri Frohlich, Dr. David Kitts, Dr. LindaMcCargar and Dr. Joseph Leichter for serving in my thesis committee. Theirinvaluable inputs to my thesis are highly appreciated. I am deeply indebtedto the financial support from Heart & Stroke Foundation of BritishColumbia, Canada.I would like to thank Dr. William Connor, Ms. Lauren Hatcher and otherCRC staff as well as the study subjects for their hospitalities andassistance during my visit to the CRC at the Oregon Health SciencesUniversity, Portland, Oregon. U.S.A..My gratitude is also extended to Dr. Catherine Leitch and Gayle Wickensfor the countless hours they contributed to me on taming the massspectrometer. Many thanks are expressed to Brian Toy for his greatstatistics assistance.I further wish to acknowledge the generous aid from the fellow graduatestudents 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-HuaLiang, for her constant support and encouragement throughout the completionof my study.ix1. INTRODUCTIONCoronary heart disease (CUD) is the leading cause of death in Westernindustrialized countries. A direct relationship between elevated plasmacholesterol levels and the incidence of CHD has been well established(Connor et a/. 1972, Stamler et a/. 1986). When plasma total cholesterolconcentration reaches 240 mg/di, each additional 1% elevation of plasmacholesterol level is predicted to increase the risk of CUD by about 2%(LRCP 1984). The effects of dietary cholesterol on plasma cholesterollevels are still controversial. This is partially due to variability inexperimental 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, dietarycholesterol was considered of little importance in human lipid metabolismand to have no effect on plasma cholesterol levels. Egg yolk was usuallyadded to the usual diet as a source of dietary cholesterol which would alsoprovide calories in the form of fat and protein (Slater et al. 1976, Porteret a/. 1977). However, in carefully controlled environments such asmetabolic wards, most studies demonstrated that dietary cholesterol exertsdecisive effects on plasma cholesterol levels, and this effect can be bestdescribed by a S-shaped curve (Connor et a/. 1961a, Connor and Connor 1985,Hopkins 1992). Furthermore, cholesterol synthesis significantly contributesto the total body pool of cholesterol in humans and thus plays an importantrole in determining body cholesterol homeostasis (Dietschy 1984). Inresponse to dietary cholesterol, however, cholesterol synthesis has beenreported to be frequently (Lin and Connor 1980, McNamara et a/. 1987) butnot consistently (Kern 1991, Everson et a/. 1991) down-regulated. Factors1regulating cholesterol synthesis in humans remain poorly understood. Therelative inadequacy of knowledge on cholesterol synthesis in humans ismainly due to methodological constraints. Cholesterol synthesis could bedetermined 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 isotope-labelled cholesterol (Dell et a/. 1985) and measurement of cholesterolprecursor levels (Parker et a/. 1984). These techniques are accurate buttime-consuming, quick but invasive, or they are indirect in determinationof cholesterol synthesis. The use of deuterium uptake method for assessmentof cholesterol synthesis overcomes the above drawbacks and has beensuccessfully applied in humans (Jones et a/. 1993b). In the deuteriumuptake method, cholesterol synthesis is determined by the rate of deuteriumincorporation from deuterium oxide (D20) in body water into plasma. Bothplasma and red blood cell (RBC) have been selected for cholesterolmeasurement (Jones et a/. 1993b, Wong et al. 1991) since they are withinthe central pool comprising synthesized cholesterol from liver andintestine (Dietschy 1984). However, the exchange rate of D20 between plasmafree cholesterol and RBC has not been fully investigated.The objectives of the present study are to examine the S-shapedrelationship between dietary cholesterol and plasma total cholesterol; theinfluence of dietary cholesterol on cholesterol synthesis; and thecorrespondence between plasma free cholesterol deuterium and RBC deuteriumuptake. The formal statements of the null hypothesis for the current studyare as follows:Hl: There is no change in plasma free cholesterol level with2an increase in dietary cholesterol.H2: The rate of equilibration of de novo synthesizedcholesterol between RBC and plasma free cholesterol isconstant throughout the measurement time intervals.H3: There is no change in plasma free cholesterol synthesiswith a gradual increase in dietary cholesterol.2. LITERATURE REVIEW2.1 Dietary Cholesterol MetabolismDietary cholesterol is absorbed by the gut in amounts proportional tothe intake up to a dietary level of perhaps 1,200 to 1,500 mg/day. Onlyabout 30 - 60% of the usual intake of cholesterol is absorbed (Connor andLin 1974, Grundy et al. 1969); the remaining unabsorbed cholesterol passesout in the stool. In humans, absorbed cholesterol is transported initiallyin chylomicrons, largely as cholesterol ester, reaching a peakconcentration in the plasma some 48 hours after a meal. This cholesterolcontributes its mass to the total body pools (Bhattacharyya et al. 1976).Cholesterol-rich chylomicron remnants are metabolized by the liver. Dietarycholesterol entering hepatocytes inhibits synthesis both of cholesterol andof LDL receptors. Overall it contributes to the major cholesterol pools ofthe body. Feedback inhibition of cholesterol biosynthesis in the body onlypartially occurs in humans, even when a large amount of dietary cholesterolis ingested (Lin and Connor 1981). Because the clearance of LDL cholesterolfrom 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 dietarycholesterol depends on the ability of the hepatocyte to excreteintracellular cholesterol into the enterohepatic circulation as bile acids.The input of sterols into the plasma-tissue occurs firstly from dietarycholesterol (0 - 500 mg/day) and secondly from synthesized cholesterolderived from the liver and intestinal tract (700 - 900 mg/day) (Dietschy1984). The rate of cholesterol biosynthesis in human appears not to be4greatly altered, as it is in most experimental animals by the ingestion ofdietary cholesterol (Connor and Connor 1985). This means that total amountof sterol entering the body from diet, in addition to the synthesizedcholesterol, may be much greater in individuals consuming a high-cholesterol 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 andnot reabsorbed by the gut via the enterohepatic circulation. Because thering structure of the sterol nucleus cannot be broken down by the tissuesof the body, it must either be excreted or stored. However, most studiespublished to date have indicated a failure of bile acid and neutral steroidexcretion to increase very much after the ingestion of dietary cholesterol(McMurry et a/. 1985, Grundy et a/. 1969). Therefore, there are fourconsequences of ingestion of dietary cholesterol.First, cellular cholesterol in the liver increases resulting fromdietary cholesterol absorbed into chylomicrons and removed from plasma bythe liver as chylomicron remnants. Second, a decrease in the number of LDLreceptors in the liver occurs. Third, there is a rise in the plasmacholesterol concentration. And last, the increased amounts of cholesteroldeposit in the tissues, particularly in the coronary arteries to initiateand sustain the atherosclerosis process (Connor and Connor 1985).The present study was in part designed to investigate the mechanism ofthe effect on plasma cholesterol concentration in response to increasingamounts of dietary cholesterol levels and cholesterol synthesis. It isproposed that the elevation of both dietary cholesterol and cholesterolsynthesis might result in the increase in plasma cholesterol concentrationleading to the development of CHD.52.2 Effect of Dietary Cholesterol Content on PlasmaCholesterol Levels2.2.1 Animal StudiesSpecies vary in their response to dietary cholesterol. Humans are notas sensitive to dietary cholesterol as are certain animal species, such asthe rabbit. Experiments done with animal species showed a more consistentrelationship between dietary cholesterol and plasma cholesterol levels.Turley et a/. (1983) found a significantly decreased liver cholesterolsynthesis as measured by tritium incorporation and increased plasmacholesterol levels in female hamsters fed with cholesterol (0.12% w/w). Inaddition, Spady and Dietschy (1983) observed a significant decrease in HMG-CoA 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 cholesterollevels are resistant to change as a result of cholesterol feeding becauseof their great capacity to excrete the ingested dietary cholesterol as bileacids, experimental manipulations to block such excretion can lead toincrease in plasma cholesterol levels (Dietschy 1984). It appears fromthese studies that dietary cholesterol may have an effect on plasmacholesterol levels.2.2.2 Human StudiesMany epidemiological studies suppport the overall dietary hypothesisabout the development of CHD; namely, that high cholesterol diets relate to6the occurrence of CUD in entire populations (Connor et a/. 1972, LRCP1984). There is evidence that plasma cholesterol levels greater than 220mg/d1 would increase the incidence of CHD (The Pooling Project ResearchGroup 1978, Turley and West 1976), and it has been established that therisk of CHD from hypercholesterolemia tends to increase 1% for every 1mg/d1 rise in plasma total cholesterol when plasma total cholesterol isabove 240 mg/d1 (LRCP 1984, Grundy et a/. 1988).Autopsy studies carried out in people who have died of CHD indicatethat, in almost all cases, the reduction of blood flow to the heart musclewas caused by atherosclerosis. A major chemical constituent of theatherosclerosis in coronary arteries in both man and animals is cholesterol(Armstrong and Megan 1972, Bottcher and Woodford 1962). Radioactivecholesterol from the diet has been traced into the very matrix of severeatherosclerotic 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 30years, the effect of dietary cholesterol upon plasma cholesterol levels inhumans remains controversial. This reflects the complexity of the factorsthat determine the response: the variability among individuals, thesignificant fluctuation in an individual's plasma cholesterol, theinteraction between dietary cholesterol and other food components, theexaggerated expectation and interpretation from experiments in animals,variable experimental design and inexact measurements.For many years, however, dietary cholesterol was thought to have littleimpact on human lipid metabolism and to have no effect on plasmacholesterol concentrations (Slater et a/. 1976, Porter et al. 1977,Kummerow et a/. 1977). The experimental conclusions of these studies were7derived from experiments that were incorrectly designed. On the other hand,many separate metabolic experiments have demonstrated that dietarycholesterol exerts decisive effects on plasma cholesterol levels (Becker eta/. 1983, Connor et a/. 1961a, Connor et a/. 1964, Steiner et al. 1962).Two kinds of dietary cholesterol experiments have been conductedhistorically to determine their effects on the plasma cholesterol level.In the first group of experiments, dietary cholesterol was added to theusual diet in the form of egg yolk which would also supply calories in theform of fat and protein. Frequently, these experiments were carried out inoutpatients who simply added eggs to their diets (Steiner and Domanki 1941,Messinger et al. 1950, Porter et al. 1977, Kummerow et a/. 1977, Slater etal. 1976, Key et al. 1956). Therefore, any effect could not be attributeddirectly to dietary cholesterol alone but had to include the othercomponents of the egg yolk (e.g., saturated fats, proteins). In someexperiments, 2-4 egg yolks per day were added to diets already cholesterol-rich (Kummerow et al. 1977, Slater et al. 1976, Key et a/. 1956). Dietaryfat, fatty acid composition, protein and calories had not been properlycontrolled in some, but not all, of these studies. Such experiments lackedprecision and strict metabolic control.Another type of experiments involved accurate metabolic studies. Earlyexperiments of this variety were inconclusive or negative about effects ofdietary cholesterol upon plasma cholesterol levels, because cholesterol wasadded in a form (as crystalline cholesterol) not well absorbed by the gutto a cholesterol-free, very low fat diet and no effects were observed(Beveridge et al. 1960). Furthermore, there were a series of experiments inwhich cholesterol, either as butterfat or egg yolk, was incorporated intothe total diet, so that the effects of dietary cholesterol could be8directly 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 yolkexperiments are most pertinent because they were carried out for longerperiods of time and because all of the nutrients contained in the egg yolk(fat, protein, minerals and vitamins) were compensated for by appropriatesubtractions from a baseline cholesterol-free diet. Thus, the number ofcalories fed to the experimental subjects remained the same as did theamounts of fat, protein and carbohydrate; the only difference was theaddition of egg yolk cholesterol. In addition, the P/S (polyunsaturatedfatty acids : saturated fatty acids) ratio of the fat and the fatty acidcomposition was the same in both the control and experimental diets. Theresults of closely controlled metabolic experiments, ordinarily conductedin a metabolic ward of a Clinical Research Center with the food completelyprepared and supplied to the experimental subjects as outpatients,consistently showed that dietary cholesterol caused an elevation of plasmacholesterol concentrations (Connor et a/. 1961a, Connor et a/. 1961b,Connor et a/. 1964, Mattson et a/. 1972, Erichson et a/. 1964, Grande eta/. 1965, Hegsted et a/. 1965, Anderson et a/. 1976, Packard et a/. 1983).In other well controlled experiments, some subjects increased plasmacholesterol concentrations and others did not in response to differentdietary cholesterol levels (Mistry et a/. 1981, Langer et a/. 1972, Cole eta/. 1985). Variability in response was stressed. Jacobs et a/.(1983)suggested that the average standard deviation for this variability wasaround 15 mg/d1. Some of this variability can be explained on the basis ofcholesterol regulation and on the effectiveness of metabolic compensationin the face of increased cholesterol absorption. This has important9implications in dietary counselling in that it explains unexpected results,such as apparent deterioration in a patient who has complied. Analyticalvariability of cholesterol assay may also be a factor contirbuting to thelack of effect of dietary cholesterol on plasma cholesterol concentrations.However, most of the studies involved normal subjects consuming thebaseline diet, which contained 300 mg of cholesterol, rather than high incholesterol content. In one study, patients with familialhypercholesterolemia had no increase in plasma cholesterol level afterbeing fed a high cholesterol diet (Connor and Connor 1985).The effects upon plasma cholesterol levels as the amount of dietarycholesterol gradually increased are depicted in Figure 1. These data aresupported by both animal and human experiments (Connor et a/. 1961a, Connoret 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 cholesterollevels between 0 and 500 mg/day has been considered to be linear (Mattsonet a/. 1972) or curvilinear (Keys et a/. 1965). With a baselinecholesterol-free diet, the amount of dietary cholesterol necessary toproduce a rise in the plasma cholesterol concentration is termed thethreshold amount. The ceiling amount is defined as the amount of dietarycholesterol increases until the ceiling point is reached on this curve.Further increases in dietary cholesterol do not lead to higher levels ofplasma cholesterol. The concepts of the "threshold" and the "ceiling" mayexplain many of the apparent conflicting results in the literature. If acertain individual already consuming a high cholesterol diet is givenadditional dietary cholesterol, it is quite likely that further increase inplasma cholesterol will not occur because the ceiling for the individual10may have already been reached (Wells and Bronte 1963). If it has not beenreached yet, plasma cholesterol level may continue to increase f-rther.11Figure 1. The proposed "S" Shaped Curve of Effects on thePlasma Cholesterol Level of Gradually Increasing theAmount of Dietary Cholesterol in Humans WhoseBackground Diet is Very Low in Cholesterol Content.Each animal or human may have its own distinctive threshold and ceilingamounts. Beveridge et a/.(1960) fed increasing amounts of cholesterol asbutterfat to experimental subjects and found that dietary cholesterol didnot significantly affect plasma cholesterol concentrations until intakereached about 200-300 mg/day. Furthermore, at about 600-900 mg/day theresponse curve plateaued. Wells and Bronte-Stewart (1963) fed three mendiets containing cholesterol from 17 to 3017 mg/day. Serum cholesterollevels increased with dietary cholesterol levels until intake was about 500mg/day. Thereafter, the response curve flattened out. Two experimentsconducted by Connor et a/.(1961b, 1964) showed that dietary cholesterol inamounts from 475 mg to well over 4000 mg per day produced similarincrements in plasma cholesterol concentration. In another preliminaryunpublished study conducted by Connor et al. with six men (two per group),110 mg of cholesterol per day did not affect plasma cholesterol levelswhile higher levels (310 mg and 610 mg/day) gave similar and significantelevations.Based on these previous experimental findings (Connor et a/. 1964,Beveridge et a/. 1960, Connor et a/. 1961b, Mahley et al. 1978), it isestimated that an average threshold amount for human beings is 100 mg/day;an average ceiling of dietary cholesterol is in the range of 300-400mg/day. Further experiments are necessary to provide more preciseinformation about the cholesterol threshold and ceiling amount.132.3 Cholesterol CompartmentalizationDuring the past decade a great deal of knowledge has become availableon 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 inhumans. In order to complement the biochemical knowledge, it is importantto investigate the in vivo metabolism of cholesterol in humans so that thiscommon health problem can be managed. One valuable approach to the study ofin vivo metabolism of cholesterol is kinetic analysis of data obtainedfollowing the administration of isotopes (Schwartz 1982).Whole-body cholesterol metabolism has been studied by analyzing theturnover of plasma cholesterol following injection of radiolabeledcholesterol. Such an analysis estimates the amount of cholesterol in thewhole body and how rapidly cholesterol is turning over in the intactsubject (Schwartz 1982). A pool model or compartmental analysis approach isone of the mathematical approaches used to analyze plasma cholesterolspecific activity-time curves.A simple theoretical three-compartment model for whole-body cholesterolkinetics in humans has been developed by Goodman et a/.(1973, 1980, 1983).Although cholesterol in the body must actually exist in many small poolsall exchanging with plasma, assuming that these compartments can be groupedinto three pools based upon their turnover rates allows one to generatequantitative information about the rate of turnover of cholesterol and theamounts of cholesterol stored.14The three pools of the compartmental model are mathematical constructsand do not have precise physical or anatomical meaning. This model suggeststhat all of the various sites of cholesterol in the body turn over at ratesthat can be separated into three groups. Pool 1 consists of cholesterolwhich turns over very rapidly and includes plasma, erythrocyte, liver, andGI tract cholesterol as well as much of the cholesterol in the pancreas,spleen, kidneys, and lungs. The bulk of cholesterol in the adipose tissueand muscle turns over slowly and probably contributes to most of pool 3.Pool 2 represents cholesterol turning over at rates intermediate betweenplasma cholesterol and cholesterol in pool 3 and includes some cholesterolin the viscera as well as peripheral tissue.20 R 1 0PRFigure 2. Three-Pool Model With Possible Side-Pool Synthesis.R's represent mass flow rates in grams cholesterolper day, k's are rate constants in days-1 (M2k12 =M1k21 R201 etc.), M's are mass of cholesterol ingrams, and PR (production rate) is mass outflow fromthe system in g/day. Subscripts for R and k denotemovement into one compartment from another--e.g., 12means into compartment 1 from compartment 2, and 30means into compartment 3 from outside the system.15The 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 ofpool 1 (M1); four exchange rates (k21, k12, k31, and k13); and thesynthesis rates in the side pools (R20 and R30). All other parameters arederivable from these basic eight parameters. However, only six of the eightfundamental parameters can be estimated from the data. What cannot bedetermined uniquely are the two side pool synthesis rates and hence themasses of the side pools (Goodman et a/. 1973, Goodman et a/. 1980, Goodmanet al. 1983, Dell and Ramarkarishnan 1982).The assumptions necessary for this model are the following (Liebermanand Samuel 1982):(1) The system is in steady state with respect to bodycholesterol, metabolism, and distribution.(2) The system is not appreciably disturbed by introductionof tracer cholesterol.(3) Both body cholesterol and tracer cholesterol areconserved between inlets and outlets.(4) The exponential extrapolation of the plasma specificactivity curve to infinite time is valid.(5) The only exit of cholesterol from the system is frompool 1. This makes total production rate equal toproduction rate in pool 1.(6) The only entrance of dietary and/or syntheticcholesterol into the system is into pool 1. This givesthe minimum value of total exchangeable mass of16cholesterol.However, the three-pool model is not without limits and shortcomings.Little is known about exchange of cholesterol between lipoproteins andspecific tissues that comprise the rapidly miscible pool. By subjecting theexperimentally observed mass and specific activity data from several typesof isotopic preparations to multicompartmental analysis using the SAAMprogram, a recent more comprehensive multicompartmental model forcholesterol within the rapidly miscible pool has been developed by Schwartzet a/.(1993). This model has enabled us to identify and quantitatecholesterol transport between all major compartments of blood, severaltissues and bile.172.4 Measurements of Cholesterol SynthesisAt present, a variety of techniques have been employed to measurecholesterol synthesis in animals and humans. These approaches include: (1)sterol balance methods, (2) HMG-CoA reductase activity, (3) kineticanalysis of the isotopic decay of stable isotope-labelled or radioactiveisotope-labelled cholesterol, (4) measurement of precursors, and (5)deuterium incorporation methodology.2.4.1 Sterol Balance MethodsSterol balance methods (Nestel et al. 1973, Bennion and Grundy 1975)have been used to determine the whole body net cholesterol synthesis byfeeding the subjects with cholesterol diets and collecting feces. Thismethod is based on an assumption of steady state (cholesterol input =output) for cholesterol metabolism, that the elimination of endogenouscholesterol and its metabolites occurs only in the feces. Although thismethod is accurate, it relies on precise food intake and complete stoolcollection; it takes up to three weeks for the subjects to reach a steadystate (Nestel and Poyser 1976).2.4.2 HMG-CoA Reductase ActivityHMG-CoA reductase is the rate-limiting enzyme in cholesterolbiosynthesis. Measurement of the HMG-CoA reductase activity (Brown et a/.1979, Dietschy and Spady 1984) has been used as a qualitative method todetermine relative cholesterol synthetic rates in a given tissue18preparation, such as liver and small intestine, which are the primary sitesfor cholesterol synthesis in humans (Dietschy and Wilson 1970, Norum et al.1983). This method should provide the direct real-time index of cholesterolsynthetic rate (Bjorkhem et a/. 1987). However, it has not been widelyapplied to human study because of the need of biopsy specimens (Carulli eta/. 1989).2.4.3 Kinetic Analysis of the Isotopic Decay of IsotopicallyLabelled CholesterolCholesterol synthesis and distribution in various tissues throughoutthe body have been measured by injection of 14C or 3H labelled cholesterolin animals (Dell et a/. 1985) and in humans (Goodman et a/. 1973, 1980,1983, Schwartz et a/. 1993). Following the injection of labelledcholesterol, the decay of plasma cholesterol specific activity at certaintime points over a period of time reflects the changes in body cholesterolsynthesis and turnover rate among the theoretical three-pool model (Goodmanet al. 1973). Unfortunately, this kind of measurement is lengthy and cannotdetect short-term cholesterol synthetic rate. In a recent study ofcholesterol kinetics in subjects with bile fistula, Schwartz et a/.(1993)employed several types of isotopic preparations to simultaneously labelseparate cholesterol pools and sample all components of blood and bile atfrequent intervals, which led to the development of a comprehensivelymulticompartmental model for cholesterol kinetics. It was found that freecholesterol was extensively exchanged between HDL and liver, RBC, and othertissues. A large portion of total hepatic cholesterol comprised a pool thatturned over rapidly by exchanging mainly with plasma HDL and was the major19source of bile acids and biliary cholesterol. The analysis also showed that94% of de novo synthesized cholesterol was partitioned into the largehepatic pool which exchanged rapidly with plasma lipoproteins.2.4.4 Measurement of Levels of Cholesterol PrecursorsOther approaches available for determination of cholesterol synthesisare to measure the cholesterol precursors such as plasma and 24-h urinarymevalonic acid (MVA) (Parker et a/. 1982, Parker et al. 1984), plasmasqualene (Nestel et al. 1975) and methyl sterols (Miettinen 1982). Previousstudies have shown that, compared to cholesterol synthesis measured bysterol balance methods, concentrations of plasma and 24-h urinary MVAexhibit changes that parallel the rate of whole body cholesterolbiosynthesis (Miettinen 1982, Kopito et a/. 1980). Although plasmaconcentrations of mevalonate display diurnal variations (Kopito et a/.1982), the mean 24-h plasma concentration can be used, on a comparativebasis under different dietary, or pharmaceutical manipulations. The 24-hurinary MVA excretion reflects the integrated plasma concentration andprovides a more practical way of assessing the cholesterol synthesiscompared with the conventional sterol balance techniques. The presence ofthese cholesterol precursors in the plasma differs with the HMG-CoAreductase activity during cholesterol synthesis. But these techniques onlyserve as indirect indicators of cholesterol synthesis.Radiolabelled cholesterol precursors such as 14C- acetate and 14C-mevalonate (Andersen and Dietschy 1979, Liu et al. 1975, Ferezou et a/.1982) have been used to determine the fractional synthetic rate ofcholesterol in animals and humans. However, there are three major20limitations to this 14C-labelled substrate technique. Firstly, it requiresperiods of measurement up to 28 weeks. Secondly, it may underestimatecholesterol synthesis because of the dilution of the acetyl CoAintracellular pool specific activity by mixing of labelled precursor withunlabelled substrates; Thirdly, it is associated with radioactive hazardsdue to the use of 14C.In addition, measurement of incorporation of tritium from tritiatedwater (Dietschy and Spady 1984) has been reported to be a more usefulmethod than 14C-labelled substrates since the water pool of the precursorsis less diluted by unlabelled substrates which reduce its specificradioactivity. Although tritiated water uptake can be used to determineshort-term cholesterol synthesis, the radiation hazards from the large doseof tritium required in this method preclude its use in human subjects.212.5 Deuterium Incorporation MethodologyAn accurate, direct, non hazardous, short-term method for assessment ofcholesterol synthetic rates in humans has been successfully carried out byJones et a/. (1988) using deuterium incorporation methodology. The use of astable isotopically labelled precursor, D20 eliminates several drawbacksencountered by other techniques discussed previously.2.5.1 HistoryDeuterium incorporation method was initially employed to measure humanfat and cholesterol synthesis by Rittenberg and Schoenheimer (1937). Tayloret a/.(1966) measured human cholesterol synthesis with the deuterium labelin 1966. Deuterium enrichment of body water was maintained at 5.0 gdeuterium oxide/kg body water (0.5 atom % excess), a 33-fold increase abovethe baseline level. This high level of deuterium enrichment was necessaryto ensure that the incorporation of the deuterium atom into the de novosynthesized cholesterol was measurable by the insensitive massspectrometric techniques available at that time. Some subjects reportedexperiencing side-effects such as severe vertigo following the given suchlarge priming dose. Also, it took about 40 days to achieve maximumdeuterium oxide enrichment.2.5.2 SensitivityThe isotope ratio mass spectrometer analytical sensitivity has beenimproved so that the required dosage of deuterium enrichment is22considerably reduced. The double-comparison method for mass spectrometricdetermination of hydrogen isotopic abundances developed by Schoeller eta/.(1983) has been contributed to the better precision of deuteriumenrichment analysis. Jones et a1.(1988) found that deuterium enrichment ofbody water maintained at 0.5 g deuterium oxide/kg body water (0.05 atom %excess) was sufficient to detect the enrichment of plasma cholesterolwithin 12 hours following oral dosage of deuterium. This level of deuteriumintake is 10 times less than that in the previous studies.Deuterium incorporation method enables us to examine the short-termperturbations in cholesterol synthesis. Compared with the measurement ofplasma mevalonic acid concentrations, Jones et a/. (1992) confirmed thatthe deuterium uptake method was suitable for relatively uninvasive, short-term detection of cholesterol synthesis. Some theoretical and proceduralconsiderations of the deuterium uptake method were also addressed (Jones eta/. 1993a).2.5.3 Three-Pool ModelThe accurate use of deuterium incorporation methodology is based on athree-pool model developed by Goodman et a/.(1973) as in the previousdiscussion. This model theoretically compartmentalizes the body cholesterolwithin various tissues into three pools, based on the rate of exchangeabletissues cholesterol equilibrates with plasma cholesterol.2.5.4 AssumptionsThe accurate measurement of fluctuations in rates of cholesterolsynthesis using three-pool model and deuterium incorporation methodologyrely on three major assumptions.First, all of de novo cholesterol only occurs in pool 1 throughabsorption of exogenous cholesterol or the majority of endogenous newlysynthesized cholesterol (Goodman et a/. 1980, Dietschy and Wilson 1970).Under this assumption, the amount of dietary cholesterol entering this poolwill likely contribute to the dilution of the labelled cholesterol.Therefore, different dietary cholesterol level may cause variations incholesterol fractional synthetic rate (FSR). Second, the losses ofcholesterol from the body occur solely via pool 1 (Goodman et a/. 1973,1980). Third, the exchange of cholesterol between pool 2 and pool 3 isconducted only by pool 1 (Goodman et a/. 1973, 1980). And last, the amountof free cholesterol entering pool 1 from pool 2 and pool 3 is minimalbecause of the slow turnover rates of these pools with plasma cholesterolas discussed above (Goodman et a/. 1973).Cholesterol fractional synthetic rate (FSR) can be calculated throughmeasuring the ratio of deuterium enrichment of plasma free cholesterol tothe 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 arepermeable to D20 ensuring the equal enrichment between the intracellularenrichment and that of plasma. The second additional assumption is that thesame number of hydrogen atoms from water will be taken up in allcholesterol synthetic tissues, independent of metabolic state.243. EXPERIMENTAL DESIGN AND METHODSThis experiment was part of a joint study in collaboration with Dr.William E. Connor and co-workers at the Clinical Research Center of OregonHealth Sciences University in Portland, Oregon, U.S.A. (CRC-OSHU). Subjectscreening, selecting, and testing were carried out on an outpatient basisat the CRC. All lipid profiles were determined at the CRC-OSHU. Assessmentof cholesterol synthesis using deuterium incorporation methodology wascarried out by the candidate at Dr. Peter Jones laboratory in Division ofHuman Nutrition, School of Family and Nutritional Sciences, University ofBritish Columbia. The candidate had visited the CRC for one week to gainexperience in the above procedures.3.1 Subject RecruitmentSubjects were selected using advertisements through local radiostations and newspapers. Volunteers were screened by a form (Appendix 1). Ageneral description of the study was provided to each respondent at thistime. In the initial screening, normolipidemic subjects in good health,aged 30-70 years, with plasma cholesterol levels between 190 and 250 mg/d1and consuming the usual high-cholesterol American diet were recruited forpre-study. Other criteria for subject selection of the study were asfollows: (1) normal weight, (2) fasting plasma triglyceride levels below200 mg/di, (3) no history of lipid disorder, (4) no antihypertensivemedication or other medications affecting lipid metabolism, and (5) non-smokers. Volunteers were requested to fast overnight prior to the screeningblood tests. Informed consent was obtained from each subject prior to this25study (Appendix 2). The study protocol was approved by the Ethical ReviewCommittee of the Oregon Health Sciences University.Anthropometric Measurements: Height was measured with the subject instocking feet using an anatomical anthropometric plane as reference. Bodyweight was measured daily with the subject clothed and shoeless on a beamtype balance scale calibrated with standard weights. Body mass index (BMI)was calculated as weight (kg) divided by height (m) 2 .3.2 Diet ProtocolThree diets were used in this study. Diet 1 contained 50 mg/daycholesterol which was to be below the proposed threshold level of 100mg/day. There was a level of 350 mg/day cholesterol in diet 2 which wasaround the proposed ceiling point between 300-400 mg/day. Diet 3 contained650 mg/day cholesterol which was above the proposed ceiling point. In orderto gain information on the routine diet which a selected subject consumed,each individual was required to complete food records over three days. Asample of the three day food records is given in Appendix 3. All diets weredesigned, calculated and prepared according to the guidelines for theprotocol by the CRC Dietary Staff at the CRC kitchen. Nutritionist III, acomputer software program, was used to analyze the nutritive components inthe meals. The three research meals had the same caloric distribution asshown below and were composed of typical mixed foods. An egg yolksupplement was incorporated as a formula into the baseline low cholesteroldiet which provided the increased levels of dietary cholesterol, especiallyfor the high cholesterol diet.26Caloric Distribution Dietary Cholesterol LevelsCarbohydrate = 55 % (Low) Diet 1 = 50 mg/dayProtein = 15 % (Mid) Diet 2 = 350 mg/dayFat = 30 % (High) Diet 3 = 650 mg/daymono = 12 %poly = 8%saturated = 10 %P/S = 0.8Nutrient composition was adjusted, so that dietary cholesterol was theonly variable. Dietary cholesterol in the diets generally originated fromegg yolk, CRC home-made shortbread cookie, CRC home-made custard and meat(ground beef, turkey breast and chicken breast). Calorie allowance weredetermined according to the Boothby and Berkson Food Nomogram (Jolliffe andAlpert 1950), and adjusted to each subject's individual need for regularlyscheduled physical activities to maintain body weight throughout the wholestudy period (Appendix 4). The research diets were designed and calculatedfor all three phases, from 2000 to 3200 kcal, in increments of 200 kcal.The reference diet contained 2800 kcal; other calorie levels wereextrapolated from that diet. Table 1 lists three sample diets at thereference 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 personalpreference to some extent without altering cholesterol content in the diet.27Table 1. Food Items Contained in the Three ExperimentalDiets at the Reference Level of 2800 kcal Per DayDiet component Low Mid Hiah(gm) (gm) (gm)Protein^15% 105 105 105Fat^30% 93 93 93mono^12% 37 37 37poly 8% 25 25 25sat.^10% 31 31 31CHO^55% 385 385 385Cholesterol (mg/day) 50 350 650Exchanges:meat 1 1 1bread 10 10 10vegetable 7 7 7fruit 7 7 7protein 1 1 1Skim milk 500 500 500Whole egg, beaten - 34 67Eggbeatersa 167 111 83Saffolab margarine 25 25 24Parkayb margarine 7 6 6SWb German chocolate 50 50 50CRCc no chol. shortbread 108 54 -CRC chol. shortbread - 49 98Custardmilk, evap, skim 55 55 55sugar, white 31 19 18eggbeaters 31 - -Parkay margarine 4 2 -palm oil 8 4 -WFb froz. egg yolk mix. - 11 22a a daily product from Fleischmann's which is 99% real eggwhite, cholesterol-free.b brand name(s).C Clinical Research CenterEach 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 levelwere applied randomly to each subject (Table 2). Participants had theirbreakfast at the CRC everyday. Before breakfast, the participants' weightand blood pressure was measured by the nursing staff. Their food andbeverage consumption, comments on food preference, as well as exercise overthe previous day were recorded by the dietary staff. Lunch and supper weretaken home by the participants. They were instructed to consume theresearch meals at fixed times ( Breakfast 08:10; Lunch 12:00; Dinner18:00). Frequent contact with subjects both at the time of blood drawingand when food was provided every morning ensured subject compliance.Previous data indicated that a new steady state should be establishedby two to three weeks after such feeding (Nestel and Poyser 1976). Afterfinishing each four-week period of a certain diet and D20 determination,every subject had a four-week washout period to allow body D20 level backto natural enrichment baseline prior to the next diet phase.29Table 2. Order of Randomized Dietary Phase AssignmentSubject Scheme* Randomization diet phase1 B low - high - mid2 B low - high - mid3 D mid - high - low4 C mid - low - high5 A low - mid - high6 F high - mid - low7 F high - mid - low8 E high - low - mid* see Appendix 5.3.3 Administration of Deuterium OxideD20 incorporation measurements were carried out with study participantsas day patients of the CRC-OHSU. On approximately the 28th day of eachfour-week dietary period, 20 ml of blood was collected for determination ofthe natural enrichments of deuterium in cholesterol and body water atapproximately 08:00. The deuterium dosing procedures were as follows.Determination of total body water: The body water volume was calculatedusing bioelectrical impedance analysis (BIA) or, if BIA calculation was notavailable, 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 forfour hours prior to this assessment and voided immediately prior to themeasurement.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 closedtightly and the lid was wrapped with parafilm. Subject then drank thispriming dose of above stated D20 (99.8 atom % excess deuterium) per kgestimated body water followed by a 50 ml rinse of distilled water. The timewas at 08:0, defined as zero hour timepoint. This dose was sufficient toestablish the body water deuterium enrichment, assuming that body water is73% of the total fat-free mass (Pace and Rathbun 1945). Water consumingduring the next 24 hour period was labeled with D20 at 1.2 g per liter ofbody water deuterium enrichment at plateau. Subjects were asked to curtailany physical exertion for this 24 hour period even though it might be apart of their typical regimen.31Further blood samples (20 ml each) were taken at 6, 12, and 24 hoursafter dosing with D20 for plasma free cholesterol and red blood cell (RBC)cholesterol deuterium enrichment analysis. Plasma water deuteriumenrichment were also determined at these timepoints. A E,mple of the D20test schedule was detailed in Appendix 6. Doses of D20 of all testingsubjects were calculated and listed in Appendix 7.323.4 Blood SamplingOne to two weeks before the dietary intervention, two or threeovernight fasting blood samples were drawn from the volunteers. These bloodsamples were used for the measurement of baseline plasma cholesterol andtriglyceride levels.During each of the four-week dietary intervention periods, overnightfasting blood samples were obtained every three days to monitor plasmalipid profile. A minimum of two to three plasma cholesterol values wereaveraged at the end of each dietary period. This value was taken as the endeffect of each diet on plasma cholesterol concentration. After four weeksof 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 incholesterol and body water. Further blood samples (20 ml each) were takenat 6, 12, and 24 hours after the prime dosing with D20 into tubescontaining EDTA for the deuterium enrichment analysis of plasma freecholesterol, RBC cholesterol and plasma water.08:00 (day 28)^0 hr^20 cc EDTA14:00 (day 28)^6 hr^20 cc EDTA20:00 (day 28)^12 hr^20 cc EDTA08:00 (day 29)^24 hr^20 cc EDTAPlasma was promptly separated off by centrifugation. RBC were saved inthe vacutainer tube, recapped with stopper. Both RBC and plasma wererefrigerated at -10°C until analysis.333.5 Laboratory ProceduresThe plasma cholesterol and triglyceride concentrations were quantitatedfluorometically on Auto Analyzer II (Technicon Instruments Corp.,Tarrytown, NY) using standard Lipid Research Clinic methods (LRCP 1974).Procedures for plasma cholesterol extraction, combustion and massspectrometric analysis have been established as previously reported byJones et a/.(1988, 1993a, 1993b). These analytical procedures were alsoapplied to RBC samples in this study.3.5.1 Lipid ExtractionLipid was extracted from 4 ml plasma (or RBC) at each timepoint intriplicate with use of 8 ml methanol and heated in water bath at 55°C undernitrogen (N2) gas for 15 minutes. An amount of 24 ml hexane-chloroform (4:1vol/vol) was next added and shaken for 15 minutes. Another 2 ml distilledwater was added and shaken for another 10 minutes followed bycentrifugation at 1500 x g for 15 minutes at 4°C. After centrifugation, theupper phase was removed and the total procedure was repeated twice againwith no need of adding methanol and distilled water.The upper phases of all three extracts were then combined into one testtube and dried at 55°C water bath under N2 gas and the yellow/orangeresidue obtained. This precipitate was redissolved in gradually reducedamounts of 10, 6, 3 drops of chloroform, each then spotted onto thin layerchromatography (TLC) silica plates (Silica TLC, Universal ScientificIncorp. Atlanta, Georgia) which had already been activated at 100°C for 3034minutes. 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 againstan internal cholesterol standard. Free cholesterol band was then scrapedfrom the silica plate and eluted with 6 ml of hexane/chloroform/diethylether (5:2:1 vol/vol/vol). After being shaken for 15 minutes followed bycentrifugation (1500 x g) for 15 minutes at 4°C, the supernatant of thesample was removed and the whole procedure was repeated twice with the samesolvents in the reduced amounts of 4 ml and 3 ml. The supernatant of allthree elutions were then combined into one tube and dried in water bath at55 °C under N2 gas. The free cholesterol extract, a white residue, wasfinally obtained at this step.3.5.2 Solvent EvaporationThe free cholesterol extract was redissolved by chloroform andtransferred to Pyrex tube (18 cm x 6 mm) added with 500 mg copper oxide andone silver wire (2cm x 1 mm). Chloroform was removed under vacuum byfreezing the tube in liquid nitrogen, and then evaporated by gradualheating for 10 minutes until the solvent boiled off and finally theinternal pressure returns to baseline (less than 50 mtorr). Tubes were thensealed and put into the oven undergoing combustion at 520°C for 4 hours,and cooled down slowly to room temperature. The hydrogen and oxygenelements in free cholesterol were thus converted to water throughcombustion.353.5.3 DistillationAll water evolved from the combustion of cholesterol was then vacuumdistilled into a 18 cm x 6 mm Pyrex tube containing 60 mg zinc which wasplaced at an 520°C oven for 30 minutes. Water derived from free cholesterolwas finally reduced to hydrogen (H2) (Schoeller et a/. 1980).The amount of 2 pl of each D20 standard for cholesterol, SMOW (StandardMean Ocean Water), SLAP (Standard Light Antarctic), GISP (Greenland IceSheet), was also transferred by this vacuum-distillation system into 18 cmx 6 mm Pyrex tubes of which was containing 60 mg zinc reagent. Standardswere reduced into H2 at 520°C for 30 minutes.3.5.4 Plasma Water PreparationTo measure the deuterium enrichment of body water, additional plasmasamples from 0, 12, 24 hr timepoint were needed. With the exception of basesample, 50 pl plasma samples from each timepoint were diluted sixfold byusing 300 pl tap water to reduced water deuterium enrichment level towithin the normal analytical range of standards used by the massspectrometer. Triplicate 2 pl respective aliquots were vacuum distilledacross 60 mg zinc in 10 cm x 6 mm Pyrex tubes and reduced at 520°C for 30minutes.The amount of 2 pl of each additional plasma water standards, SMOW,Chicago tap water, Chicago stock and Vancouver tap water, was also preparedfollowing the upper same procedure for the observed ratio correction inreal time of H3 + contribution in mass spectrometer (Craig 1961, Peterson1979, Schoeller et a/. 1983).363.5.5 Mass Spectrometric DeterminationThe deuterium enrichment of samples were measured on a differentialisotope ratio mass spectrometer (VG Isomass, 903D, Cheshire, UK) throughthe determination of cholesterol deuterium/protium (2H/1H) isotope ratio.This mass spectrometer had a double inlet, and the analyses were performedby 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 wascalibrated daily using water standards of known isotopic composition.Precision level for overall analysis of this study was determined byaveraging the standard deviation (SD) of the replicating readings finallygained from the mass spectrometer. As a result, the average SD of deuteriumenrichment for plasma water, plasma free cholesterol and RBC cholesterolsamples were calculated as -5.0, -3.5 and -5.6 parts per thousand (°/00)relative to SHOW, respectively.373.6 Data CalculationsResults from the isotope ratio mass spectrometer were defined as partsper mil or:-1[(Rsamplei Rsmow)] x 1000, 0/00 Eq.1where R was the ratio of heavy (2H) to light (IH) isotopic species.Cholesterol deuterium enrichment was expressed relative to Standard MeanOcean Water (SHOW). Fractional synthetic rate (FSR) of cholesterol inplasma and RBC was calculated based on the methods of Dietschy and Spady(1984) as adapted by Jones et a/.(1988, 1993b), which have been mentionedpreviously.One molecule of cholesterol contains 27 carbon atoms and 46 hydrogenatoms. During the synthesis of cholesterol, the hydrogen atoms may beincorporated into the sterol molecule from three different sources: 7 atomsare incorporated directly from water; 15 atoms are inserted from NADPH; orultimately, hydrogen atoms from water are incorporated into the acetyl CoApool, which can be used as a cholesterol precursor. Moreover, FSR of plasmacholesterol was determined as the fractional incorporation of precursordeuterium over time. We assumed that over a 48-hour period there wascomplete equilibration of D20 with plasma water and with NADPH, but thatthe acetyl CoA pool has not yet been labelled. Therefore, this representedthat 22 deuterium atoms were incorporated into each cholesterol molecule,resulting in a D/C ratio of 0.81 in the synthesis of cholesterol. Thisyielded the equation:38delcholesterol (c)/oo)FSR (day-1) ^Eq.2delp lasma water (°/00) x 0.81 D/C x 27C/46Hwhere delcholesterol^pand del-lasma water were differences in deuteriumenrichment of each tissue expressed as parts per mil versus SMOW. 27C/46Hrepresents the correction for the absolute ratio of carbon to hydrogenatoms within the cholesterol molecule (Jones et al. 1988, 1993b). Usingthis formula, the FSR of both plasma free cholesterol and RBC cholesterolwere calculated for subjects consuming each of the three graded amounts ofcholesterol.However, this calculation of FSR could be criticized since certainassumptions used by the deuterium incorporation method remainedcontroversial: 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 fromside pool 3 to cholesterol synthesis in a baboon study, although whethertotal synthesis is distributed in a similar manner in humans remains to beverified. Moreover, with the newly developed multicompartmental model,Schwartz et a/.(1993) recently revealed that incoming synthesized freeplasma cholesterol was transported most rapidly between plasma and theliver, RBC, and other tissues among the rapidly miscible pool. It ispossible that the newly synthesized cholesterol within the central M1 poolwas removed into other tissues, preventing exchange of the labelledcholesterol with the unlabelled cholesterol carried on lipoproteins. It wasalso indicated by Schwartz et a/.(1993) that bile acid synthetic rate wascorrelated directly with the size of the large hepatic pool, thus, labelled39cholesterol may have been mobilized for bile acid synthesis during themeasurement interval. At last, it is also possible that the increase ofdietary cholesterol may result in direct dilution of the deuteriumenrichment within the central Ml pool. Consequently, substantialunderestimation of cholesterol enrichment or FSR could occur with thecurrent D20 uptake method. Further advanced models taking into account theabove perturbations will be required to illustrate the kinetics ofcholesterol synthesis using D20 uptake method in vivo.403.7 Statistical AnalysesMeans and Standard Errors of the Means (± SEM) were calculated toassess each of the specific activity of deuterium enrichment versus plasmafree cholesterol and RBC cholesterol, and anthropometric variables duringeach of the dietary periods. It was statistically desirable to have arandomized experimental design, therefore subjects were randomly assignedto different dietary groups. One-way analysis of variance (ANOVA) wasperformed to examine the statistical significance among plasma cholesterolconcentrations and dietary cholesterol levels, cholesterol FSR andcholesterol diets, as well as cholesterol FSR and plasma cholesterolconcentrations (Zar 1984, Wilkinson 1990). Regression analysis was alsoemployed to examine the correlation between dietary cholesterol and plasmacholesterol. All statistical significances detected by one-way ANOVA werefurther measured by Tukey test to differentiate the real significantdifference between variables. Paired sample t-test was employed to comparethe difference in both deuterium enrichment and FSR values between plasmaand RBC at each diet. Level of statistical significance was expressed as atleast a value of P < 0.05. Interaction graphs were also drawn to facilitatevisualization of the relationships between factors.414. RESULTS4.1 Subject CharacteristicsAfter the initial screening procedures as mentioned in subjectrecruitment section, eight subjects (one female and seven males) wererecruited for this study. The relevant mean + SEM values for anthropometricdata and plasma lipid profile at baseline of the study population weresummarized in Table 3.Among the eight selected healthy subjects, most of them were 40 to 60years old (55.5 ± 4.2, mean ± SEM) with the exception of subject 2 who was34 years old, however, his results did not appear to differ from those ofthe other seven subjects. The majority of subjects were within theacceptable BMI range of 21 to 25 kg/m2 (23.7 ± 0.5). Subject 5 had aslightly higher BMI (25.8) as compared to the others. The baseline plasmatotal cholesterol levels of these selected subjects before any dietarytreatment ranged from 203 to 248 mg/dl (227.3 ± 5.9) which were within theacceptable normal range according to their ages. The subjects had normaltriglyceride levels ranged from 71 to 142 mg/d1 (99.8 ± 9.2). Subjects'body weights were monitored carefully and remained unchanged during thestudy period. Overall, the criteria set for subject recruitment weregenerally met by the selected subjects.42Table 3. Anthropometric Data and Screening Plasma LipidProfile of the Selected SubjectsSubject Sex Age(yrs)Height(m)Weight(kg)Plasma LipidsBMI^TChol^TG(kg/m2) (mg/di)1 F 46 1.63 61.6 23.2 222 1232 M 34 1.85 84.5 24.7 203 1183 M 66 1.84 73.4 21.7 246 1024 M 67 1.67 68.3 24.5 248 1425 M 57 1.67 71.9 25.8 235 846 M 63 1.70 63.6 22.0 238 727 M 63 1.69 64.7 22.7 212 718 M 48 1.92 91.0 24.7 214 86Mean 55.5 1.75 72.4 23.7 227.3 99.8SEM 4.2 0.04 3.7 0.5 5.9 9.24.2 Comparison of the Effect of Dietary Cholesterol onPlasma Total Cholesterol LevelsTable 4 summarizes the changes in plasma total cholesterol afterdietary 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 ona low cholesterol diet (207.4 ± 5.7 mg/di, P = 0.002). This effect of threegraded amounts of dietary cholesterol upon plasma cholesterol levels isplotted in Figure 3. The statistical significance was reached only betweenthose subjects consuming a low and a high cholesterol diet (P = 0.002).Compared with the subjects on a low cholesterol diet, plasma cholesterolconcentration was increased by 13% (+26 mg/di) in subjects on a highcholesterol diet. Although their plasma total cholesterol concentrationswere increased by 8% (+17 mg/dl) and 4% (+10 mg/di) in subjects consuming alow cholesterol diet relative to subjects consuming a medium cholesteroldiet, and in subjects consuming a medium cholesterol diet relative tosubjects consuming a high cholesterol diet, respectively, statisticalanalysis indicated that these increases of plasma cholesterol level werenot statistically significant at the P values of 0.056 and 0.344,respectively.Similar to the above effects of plasma total cholesterol levels inresponse to different levels of dietary cholesterol, the change of plasmatotal cholesterol concentration from baseline plasma cholesterolconcentration in subjects consuming a high cholesterol diet (6.4 ± 4.8mg/di) was significantly higher than that of subjects on a low cholesteroldiet (-19.9 ± 5.0 mg/di, P = 0.016) (Table 4). Compared to the average of446.4 mg/d1 increase observed in the whole group, subject 3, 4, 5, and 6 on ahigh cholesterol diet had insignificantly decreased plasma totalcholesterol of -3, -1, -5, and -12 mg/d1. This may result from individualvariation in response to dietary cholesterol intervention. Figure 4 showsthe change of plasma cholesterol from baseline in response to the increaseof dietary cholesterol in subjects consuming three experimental cholesteroldiets. Again, the statistical significance was only identified betweensubjects consuming a low and a high cholesterol diet. Changes of plasmacholesterol from baseline were not significantly different in subjectsconsuming a diet between low and medium (P = 0.188), as well as betweenmedium and high (P = 0.803) cholesterol diets.To obtain information on whether there is a linear or curvilinearassociation between dietary cholesterol and plasma cholesterol, regressionanalysis was performed. It was found that there is a strongly positiveassociation between the dietary cholesterol levels and plasma cholesterolconcentration, although this association was not linear (r2 = 0.416, P =0.001). A similar positive association was found between dietarycholesterol and change of plasma cholesterol from baseline (r2 = 0.312, P =0.005). Table 5 summarizes the P values of statistical analysis for thecomparisons.45Table 4. Plasma Total Cholesterol Levels in Subjects Beforeand After Dietary Cholesterol InterventionsSubject BaselineTChol(mg/dl)TCholAfter treatmentLow^Mid^High(mg/di)Change of TCholfrom baselineLow^Mid^High(mg/di)1 222 217 229 245 -5 7 232 203 179 214 210 -24 11 73 246 229 228 243 -23 -18 -34 248 218 228 247 -30 -20 -15 235 222 207 230 -13 -28 -56 238 192 217 226 -46 -21 -127 212 210 246 233 -2 34 218 214 198 223 235 -16 9 21Mean 227.5 207.4 224.0 233.6a -19.9 -3.3 6.4bSEM 5.9 5.7 4.2 4.3 5.0 7.6 4.8a significantly different from low cholesterol diet group (P =0.002).b significantly different from low cholesterol diet group (P =0.016).24020050^350^650Figure 3. The Influence of Dietary Cholesterol on PlasmaCholesterol Level (a significantly different fromlow cholesterol (50 mg/day) diet group (P = 0.002)]Dietary Cholesterol Levels (mg/day)Figure 4. The Influence of Dietary Cholesterol on Change ofPlasma Cholesterol Concentration From Baseline (asignificantly different from low cholesterol (50mg/day) diet group (P = 0.016)]50^350^850Dietary Cholesterol Levels (mg/day)Table 5. Summary of the P Values on Plasma CholesterolCholesterol and Changes of Plasma CholesterolConcentration in Subjects Consuming ThreeExperimental DietsP ValuePlasma Cholesterol Change of PlasmaCholesterolAll Diets 0.003* 0.017 *Low vs High 0.002 * 0.016 *Low vs Mid 0.056 0.188Mid vs High 0.344 0.803* Significantly different (P < 0.05).4.3 Comparison of Deuterium Enrichment and Cholesterol FSRBetween Plasma and RBCDeuterium enrichment between plasma and RBC was compared over differenttime intervals at 6, 12, and 24 hours postdose in subjects consuming eachof the three cholesterol test diets. The results of deuterium enrichment,expressed as parts per thousand (°/oo) relative to SMOW were given inappendix 8. For better visualization, all comparisons are shown in Figure5, Figure 6 and Figure 7, in which testing subjects were consuming a low, amedium and a high cholesterol diet, respectively. Deuterium enrichment ofRBC cholesterol lagged behind that of plasma free cholesterol at all diets.However, this lagging equilibration of de novo synthesized cholesterol fromplasma 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 cholesteroldiet (Table 6). The least significance of the lagging equilibration wasobserved in subjects consuming a medium cholesterol diet (Figure 6). Table6 summarizes the results of paired sample t-test on plasma versus RBCdeuterium enrichment in different time intervals.50Figure 5. Deuterium Enrichment of Plasma and RBC inSubjects Consuming Low Cholesterol Diet Asa Funtion of Time160A RBC_ v Plasma -Mean MEM-4--c0 20a^12^18 3024Figure 6. Deuterium Enrichment of Plasma and RBC inSubjects Consuming Medium Cholesterol Diet Asa Funtion of TimeTime Interval (hrs)Figure 7. Deuterium Enrichment of Plasma and RBC inSubjects Consuming High Cholesterol Diet Asa Function of Time [a,^significantlydifferent 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-Testson Deuterium Enrichment of Plasma Versus RBC atDifferent Time IntervalsTime^ Low^Medium^HighInterval^All Diets^Chol Diet^Chol Diet^Chol Diet6 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* 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 subjectsconsuming three different cholesterol diets. Being similar with cholesteroldeuterium enrichment, FSR values in plasma were found significantlydifferent from those of RBC at 0-6 (P < 0.02) and 0-12 (P < 0.02) hour timeperiod. For better comparison and visualization, all FSR values from allthree experimental diets were plotted against each time period in Figure 8,diversed FSR values between plasma and RBC in the early time periods becameconstantly intimate towards the increasing time period. At 0-24 timeperiod, none of these plasma and RBC FSR values were significantlydifferent from each other. Similarly, plasma FSR value was constantlyhigher than that of RBC in each time period of each of the experimentaldiets.54Table 7. Plasma and RBC Cholesterol FSR in SubjectsConsuming Three Experimental DietsSubject0-6Plasma0-12 0-24FSR (Day-1)0-6RBC0-12 0-24Low diet1^-.0524 -.0206 .1058 .0592 .0143 .05572 .1418 .1145 .0949 .0208 .0663 .07003^.0711 .0076 -.0239 .2098 .1486 .08874 .1395 .0925 .0712 .0349 .0324 .05405^.0913 .1064 .1226 .0273 .0702 .09226 .0906 .0609 .0616 .0469 .0304 .06427^.2181 .1484 .0990 .0385 .0427 .06488 .0221 .0384 .0963 -.1930 -.0247 .0756Mean^.0900 .0690 .0780 .0310 .0480 .0710SEM .0290 .0200 .0160 .0390 .0180 .0050Medium diet1^.0728 .0869 .0350 .0556 .08132 .1172 .1299 .1046 .0428 .09213^.1107 .0572 .0488 .1652 .0648 .05694 .0335 .0307 .0768 .0204 .0040 .02005^.0342 .0703 .0539 .0720 .0635 .09196 .0998 .0686 .0651 .0098 .0417 .03267^.0503 .0339 .0527 -.0559 .0590 .06778 .0433 .0853 .0899 .1301 .0947 .1123Mean^.0700 .0680 .0720 .0520 .0550 .0690SEM .0120 .0130 .0070 .0250 .0100 .0110High diet1^.0545 .0560 .0764 .0102 .0300 .06032 .1088 .1432 .1364 .0414 .0672 .10373^.1881 .1278 .0964 -.1214 -.0298 .02534 .0027 .0194 .0518 .0116 .0100 .03675^.5103 .2113 .1065 .2104 .0767 .10766 .2104 .0717 .0286 -.0653 .0035 .05407^.1067 .0501 .0475 .0178 .0316 .03788 .0692 .0337 .0251 -.0343 -.0081 .0346Mean^.1560a .0890b .0710 .0090 .0230 .0580SEM .0560 .0230 .0140 .0340 .0130 .0110a Significantly different (P < 0.02) from RBC 0-6timepoint in the high diet.b Significantly different (P < 0.02) from RBC 0-12timepoint in the high diet.Figure 8. Equilibrium of Synthesized Cholesterol BetweenPlasma and RBC on Three Experimental Diets [a, bSignificantly different from RBC at 0-6 and 0-12hour time period in high cholesterol diet (P <0.02)]0.180.120.09cc0.080.030.000-8^0-12^0-24Time Interval (hre)4.4 Comparison of the Effect of Dietary Cholesterol onCholesterol FSRThe effect of dietary cholesterol on cholesterogenesis was examined byanalyzing 0-24 hour plasma FSR values in Figure 9. Cholesterogenesis wasnot altered in response to increasing dietary cholesterol levels (P =0.913). A wide range of subject variation of FSR value was observed in bothlow and high cholesterol diet groups. Table 8 summarizes the results ofANOVA on cholesterol FSR in subjects consuming three different cholesteroldiets. No statistical significance was found in effect of dietarycholesterol upon FSR at each time interval of each diet.Table 8. Summary of the P Values on Cholesterol FSR inSubjects Consuming Three Experimental DietsPlasmaTime Interval (hr)RBCTime Interval (hr)6 12 24 6 12 24All Diets 0.251 0.690 0.913 0.653 0.276 0.574Low vs Mid 0.534 0.983 0.735 0.640 0.742 0.912Low vs High 0.312 0.515 0.736 0.680 0.278 0.302Mid vs High 0.155 0.983 0.735 0.640 0.742 0.91257Figure 9. Overall Plasma Cholesterol FSR in SubjectsConsuming Three Experimental Diets4.5 Comparison of the Effect of Plasma Total Cholesterol onCholesterol FSRIn order to investigate the effect of plasma cholesterol uponcholesterogenesis, plasma 0-24 hour FSR of subjects consuming low, mediumand high cholesterol diets was firstly plotted against change of plasmacholesterol from baseline in Figure 10, Figure 11 and Figure 12,respectively. On low cholesterol diet (Figure 10), decreasing plasmacholesterol 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 cholesterolconcentration (P = 0.458, P = 0.707, respectively).Secondly, change of plasma cholesterol concentration from low to mediumcholesterol level did not appreciably alter cholesterol FSR in subjects ona medium cholesterol diet (Figure 13, P = 0.868), and in subjects on a highcholesterol diet (Figure 14, P = 0.987).Thirdly, even when the change of plasma cholesterol from a low to ahigh cholesterol diet was plotted against FSR in Figure 15, there was nocorrelation between plasma cholesterol level and cholesterol FSR (r2 =0.253, P = 0.204).Finally, no relationship of increase in plasma cholesterol from a lowto a high cholesterol diet and decrease in plasma FSR from a low to a highcholesterol diet was observed among the testing subjects (Figure 16, P =0.454).59Figure 10. Changes of Overall Plasma Cholesterol FSR andChanges of Plasma Cholesterol From Baseline inSubjects Consuming a Low Cholesterol Diet0.2 000—-0.1-50^-40^-30^-20^-10^0Change of Plasma Cholesterol From Baseline (mg/d1)60i^i^i^i^1^i^10_ -- 0--000^0- -0Figure 11. Changes of Overall Plasma Cholesterol FSR andChanges of Plasma Cholesterol From Baseline inSubjects Consuming a Medium Cholesterol Diet0.11coS.: 0.1 0-oCr)a0.09crcou_1,--6 0.080ziL(1-5 0.070a)-5_c0 0.06E'40 0.050.04-40 -30 -20 -10 0 10 20 30 40Change of Plasma Cholesterol From Baseline (mg/d!)61Figure 12. Changes of Overall Plasma Cholesterol FSR andChanges of Plasma Cholesterol From Baseline inSubjects Consuming a High Cholesterol Diet62Figure 13. Plasma Cholesterol FSR in Subjects ConsumingMedium Cholesterol Diet Versus Change of PlasmaCholesterol From Low to Medium Cholesterol DietFigure 14. Plasma Cholesterol FSR in Subjects ConsumingMedium Cholesterol Diet Versus Change of PlasmaCholesterol From Medium to High Cholesterol DietFigure 15. Plasma Cholesterol FSR in Subjects ConsumingHigh Cholesterol Diet Versus change of PlasmaCholesterol From Low to High Cholesterol DietFigure 16. Change of Plasma FSR Versus Increase in PlasmaCholesterol From Low to High Cholesterol DietTable 9. Summary of the P Values Obtained From ANOVAComparison on Relationship Between Changes ofCholesterol FSR and Changes of Plasma TotalCholesterol ConcentrationFSR(day-1)P value on Change of Plasma Cholesterol baseline Low-medium Medium-high Low-high Low diet^0.298Medium diet^0.458^0.858High diet^0.707 0.987^0.204Low-high diet 0.454DISCUSSION5.1 Subject CharacteristicsThis is a unique study in which plasma cholesterol levels, cholesterolFSR and the equilibration of newly synthesized cholesterol between plasmaand RBC have been studied simultaneously in healthy humans consuming threediets with cholesterol content of 50, 350 and 650 mg/day. Based on thethree pool model, overall human cholesterol turnover is influenced bycertain anthropometric parameters such as age and body weight. In thisinvestigation, subjects were randomly assigned to all of the three dietarytreatment groups. Confounding factors such as interindividual variation,difference in physical characteristics and lipid profiles have beeneliminated in this study. Therefore, in addition to the with-in personvariations, dietary composition was the only variable contributing to anyalteration of cholesterol metabolism, since body weight virtually remainedunchanged during the investigation period.There were seven males and one female recruited for this study. Resultsof the female subject (subject 1) in this study did not differ considerablyfrom other male subjects. Despite the fact that subject 2's age wasrelatively lower than the average age of the study population (34 vs. 55.54.2 yrs), and subject 5 had slightly higher BMI (25.8 vs. 23.7 ± 0.5kg/m2), their experimental data did not markedly vary from those of others.All subjects remained healthy throughout the whole study period. These datawere supported by the following two controlled human studies. In a study byKatan and Beynen (1987), 21 men and 11 women aged from 19 to 62 years werefed a low followed by a high cholesterol diet for three to four weeks. They68found no relation of responsiveness with age, sex, and intestinal transittime on plasma total cholesterol levels in low-cholesterol and high-cholesterol diets. Also in a recent study by Clifton and Nestel (1992), 26men 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 (<250mg/day) diet followed by a high-fat, high-cholesterol (650 mg/day) dieteach for three weeks. With respect to the plasma total cholesterol levels,no gender difference was found in these subjects in response to the dietaryinterventions. The overall criteria for subject recruitment as discussedpreviously in the experimental design section was generally fulfilled inthe present study.695.2 Effect of Dietary Cholesterol on Plasma TotalCholesterol LevelsThe effect of dietary cholesterol on plasma cholesterol concentrationhas been studied extensively and the results are inconclusive andcontroversial. However, in a series of precise, well-controlled metabolicstudies, high dietary cholesterol generally increases the plasma levels oftotal 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 eta/. 1988).In the present study, an overall 13% increase of plasma cholesterol wasobserved in subjects consuming a high cholesterol diet (650 mg/day)compared to those consuming a low cholesterol diet (50 mg/day). Therelation between the increase of dietary cholesterol from 50 to 650 mg/dayand plasma cholesterol was not linear overall, but positive (Figure 3). Asimilar result was obtained when the increase of dietary cholesterol wasplotted against the change of plasma cholesterol from baseline (Figure 4).The present finding is generally in accordance with findings of severalother studies (Mattson et al. 1972, Gyling and Miettinen 1992, Hopkins1992). Mattson et a/.(1972) observed that plasma cholesterol concentrationwas linearly increased by cholesterol intake of up to 400 mg/day. In astudy by Gylling and Miettinen (1992), 29 home-living men with an averageage of 55 were placed on a low-fat, low cholesterol diet (208 mg/day) for 6weeks 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 dayto the previous low-fat low cholesterol diet. It was found that plasma70cholesterol was increased significantly by 10% in subjects on the low-fathigh cholesterol diet. In a meta-analysis and review of the effect ofdietary cholesterol on plasma cholesterol concentration, Hopkins (1992)combined 27 studies in which controlled diets were supplied by metabolickitchens. It was found that an approximate 12-15% increase in plasmacholesterol level could be predicted when 500 mg/day cholesterol was addedto a cholesterol-free diet in normal subjects. This finding was alsoconfirmed by the present study.However, results of the present study are not fully in agreement withthe proposed S-shaped threshold and ceiling amounts of dietary cholesterolin Figure 1. Based on the proposed "S" shaped curve, the threshold point onplasma cholesterol is 100 mg/day of dietary cholesterol, and the ceilingpoint on plasma cholesterol is reached when dietary cholesterol is raisedto 350 mg/day. As a result, the present finding (Figure 3, 4) is partiallyin agreement with regard to the ceiling point, since a further increase ofdietary cholesterol to 650 mg/day only elevated plasma cholesterol by 4%which was not significant. When the threshold point is considered, theincrease of plasma cholesterol should be significant at 350 mg/day ascompared to 50 mg/day of dietary cholesterol. However, the present studyreveals that although there is an 8% increase in plasma cholesterol, it isof borderline statistical significance (P = 0.056).The lack of significance of the present data might be mainly due to theexperimental design itself. The three cholesterol diets used in thisexperiment may not have generated enough data points for the appropriatecomparison. At least three additional diets are required to obtain enoughpoints to plot and compare the results with those summarized and proposedin Figure 1. In addition to the 50 mg/day cholesterol diet, a dietary71cholesterol of approximately around 100 - 150 mg/day is also needed foraccurate determination of the threshold point. Furthermore, two otheradditional cholesterol diets in the range of 450 - 500 and up to 800 - 900mg cholesterol per day are required to well define the proposed ceilingpoint. By combining data from 27 studies, Hopkins (1992) recently didobserve the S-shaped relationship between change in plasma cholesterol andadded dietary cholesterol.Alternatively, the relatively large variability of subjects plasmacholesterol level in response to dietary cholesterol seen in this study mayalso 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 cholesterolconcentration is clearly increased due to an increase in dietarycholesterol from 50 mg/day to 650 mg/day. The magnitude of change in plasmacholesterol in response to dietary cholesterol tended to plateau when thelevel of dietary cholesterol reached 650 mg/day. Additional dietscontaining different levels of cholesterol are required to define thethreshold and ceiling effect of dietary cholesterol upon plasma cholesterolconcentration.725.3 Equilibration of Synthesized Cholesterol Between Plasmaand RBCFor cholesterol deuterium uptake measurement in normolipidemicsubjects, a relatively large amount of initial plasma volume (2m1) perreplicate is required to yield 1 mg free cholesterol, the amount needed toproduce 1 pl combustion water necessary for isotopic mass spectrometricanalysis. This blood volume may not be easily obtained in certain subjectssuch as infants. Alternatively, the use of smaller amount of blood in theanalysis can be achieved by employing RBC samples. This is due to the factthat plasma comprises both free and esterified cholesterol. However, RBC donot synthesize cholesterol and contain solely free cholesterol exchangedfrom plasma (London and Schwartz 1953). Thus, compared to plasma samples,smaller amount of blood is needed to get the necessary amount of freecholesterol for analysis when RBC samples are used. Both plasma and RBCwere considered to be within the central pool comprising cholesterolsynthesized from liver and intestine (Goodman et a/. 1973). So far, eitherplasma (Jones et a/. 1993b) or RBC (Wong et a/. 1991) have been sampled fordetermination of cholesterol deuterium uptakes. However, an intraindividualcross-comparison of the correspondence between plasma and RBC deuteriumenrichment in measuring human cholesterol synthesis has not been fullyinvestigated. The rate of equilibration between plasma and RBC remainsunclear.An early study conducted by Hagerman and Gould (1951) incorporatingC14-acetate carbon into cholesterol in vitro between plasma and RBC showedthat equilibration of free cholesterol between plasma and RBC was closelyapproached in four hours in a dog previously fed a cholesterol-free diet.73In a recent human study using 3H and "C radiolabelled cholesterol in vivo,Schwartz et ai.(1993) found that free cholesterol was transported rapidlybetween plasma and RBC in an exchange rate of 11.6 mg/min. Equilibration ofplasma and RBC free cholesterol specific activities was reached by 6.7 - 10hours corresponding to the time when RBC free cholesterol specific activityreached a peak. By employing the deuterium uptake method, in vivocholesterol synthesis has been estimated using sample derived from eitherplasma in humans consuming low and high cholesterol diets (Jones et a/.1993b) or RBC in piglets fed a cholesterol-free diet and a diet containing0.5% cholesterol (Wong et a/. 1991).Results of the present study indicate that D20 (Figure 5-7) wasinitially incorporating more slowly into RBC from plasma over the initial6-12 hour time interval in all three cholesterol diets. This laggingdeuterium enrichment from plasma to RBC was found to be significant in thehigh cholesterol diet only at 6 and 12 hour time intervals (Figure 7).Likewise, FSR values between plasma and RBC, interpreted from deuteriumenrichment data, were not correlated with each other in all three dietsuntil the 24 hour time interval was reached. The difference was significanton high cholesterol diet at 0-6 and 0-12 time period (Figure 8). Thepresent results were mostly confirmed by data from Schwartz et a/.(1993),in which 6.7 to 10 hours were required for the thorough equilibration ofradiolabelled free cholesterol between plasma and RBC. These findingsgenerally indicate the substantial interpool exchange of free cholesterolbetween plasma and RBC over the initial postdose time intervals up to 12hours. The complete equilibration of deuterium enrichment between plasmaand RBC was reached in 24 hours postdose on all subjects consuming threeexperimental diets.74However, the reason for the much slower deuterium incorporation fromplasma to RBC in subjects consuming high cholesterol diet remains unknown.We postulated that the actual central pool size was enlarged and/ordeuterium enrichment in the pool is diluted out by the incremental incomingdietary cholesterol during the initial 12 hours when cholesterol intake washigh. As a result, deuterium enrichment in plasma was decreased, whichmight contribute to the slower exchange of deuterium into RBC.Nevertheless, a more advanced mathematical model is required in which allvariables or parameters would be factored in for cholesterol kineticanalysis using the deuterium uptake method.Current results imply that over the initial 6-12 hour post-doseinterval, measurement of RBC deuterium uptake may result in underestimationof cholesterogenesis when cholesterol intake is high. The use of 24 hourinterval from either plasma or RBC sample best reflects cholesterolsynthesis.755.4 Effect of Dietary Cholesterol on CholesterogenesisThe metabolic response to increased dietary cholesterol in humans mightinclude a decreased absorption of dietary cholesterol, a reduction in denovo cholesterol synthesis and an increase in the excretion of biliarycholesterol. Several feedback responses have been documented during highcholesterol intake in humans. However, cholesterol synthesis is foundfrequently (Nestel and Poyser 1976, Lin and Connor 1981, McMurry et al.1985, McNamara et a/. 1987, Miettinen and Kesaniemi 1989) but notconsistently to be down-regulated (Kern 1991, Everson et al. 1991, Jones eta/. 1993b).In the study conducted by Nestel and Poyser (1976), two normolipdemicand seven hyperlipidemic subjects consumed a low cholesterol diet (250mg/day) and a high cholesterol diet (750 mg/day) each for a period of fourto six weeks. Cholesterol synthesis measured by sterol balance method wassuppressed in five, including the two normolipidemic, subjects on the highcholesterol diet. However, the amounts of fat and the high range ofcholesterol used in this experiment were higher than ours (40% vs. 30%, and750 vs. 650 mg/day, respectively). Furthermore, in a long-term sterolbalance study, cholesterol synthesis was inhibited by a high cholesterolintake of 1000 mg/day compared to a very low cholesterol diet in onenormocholesterolemic and one hypercholesterolemic subjects (Lin and Connor1981). Finally, eight human subjects were fed sequentially a cholesterol-free diet for three weeks followed by a diet containing 900 mg cholesterolfor another three weeks under controlled conditions (McMurry et al. 1985).Cholesterol biosynthesis measured by sterol balance method decreasedsignificantly by 49%. Therefore, we speculated that the high cholesterol76intake of 650 mg/day used in the present study may not be high enough toinduce the feedback inhibition of cholesterol synthesis.However, in another study conducted by Kern (1991), cholesterolsynthesis was quantified by measuring the 14C-acetate incorporated intocholesterol in mononuclear cells. It was found that cholesterogenesis in asubject consuming 25 eggs per day remained equal to the mean synthetic ratein normal subjects. Individual variability in the response to a givenchange in the dietary cholesterol level vary widely. It was found that thisindividual had a great reduction in cholesterol absorption and a markedincrease in the hepatic conversion of cholesterol into bile acids.Using deuterium uptake methodology, our data show that cholesterol FSRis not significantly changed (P = 0.913) by increasing cholesterol intakefrom low, medium to high (Figure 9). This finding suggests that cholesterolsynthesis is independent to changes in dietary cholesterol. It is suggestedthat cholesterogenesis is primarily mediated by the differences incholesterol absorption efficiency, neutral sterol excretion and conversionof hepatic cholesterol to bile acids. We speculate that with incrementalcholesterol intake, cholesterol synthesis can remain constant due to manyhomeostatic and regulatory mechanisms such as reduction in the efficiencyof cholesterol absorption, down-regulating of LDL-receptor activity,increased bile acid synthesis and increased excretion and re-excretion ofabsorbed cholesterol. Unfortunately, we did not examine these parameters inthe present project. There is no doubt, however, that the absorption ofcholesterol influences the rate of synthesis, at least in the liver by afeedback control. Grundy et a/.(1969) reported that the total bodycholesterol synthesis measured by sterol balance method did not reduce whena large amount of dietary cholesterol was given to a normocholesterolemic77subject. However, the synthesis of cholesterol rose strikingly whencholesterol absorption was suppressed with plant sterol. They concludedthat feedback control of cholesterol synthesis by dietary cholesterol wasrelatively unimportant for the regulation of cholesterol metabolism underthe normal metabolic condition. This was because that cholesterolabsorption mechanism was primarily saturated by the large amount ofendogenous cholesterol available for reabsorption.Again, marked individual variations in cholesterogenesis in response todietary cholesterol level, especially to low and high cholesterol diets(Figure 9), may somewhat explain the small effect of dietary cholesterolupon cholesterol synthesis in this study.785.5 Relationship between Plasma Total Cholesterol Levels andCholesterol FSRResults of previous studies examining the effects of plasma cholesterolupon cholesterol synthesis have not been consistent. Katan and Beynen(1987) reported that whole body cholesterol synthesis was inverselycorrelated with responsiveness of plasma cholesterol concentration to adietary cholesterol challenge in humans consuming low cholesterol diet.They concluded that higher cholesterol synthetic rate in subjects whoseplasma cholesterol levels respond little to dietary cholesterol(hyporesponders) would enable stronger feedback inhibition of cholesterolsynthesis during the period of dietary cholesterol challenge. However in arecent study, Jones et al. (Jones et a/. 1993b) reported that such anegative association with low cholesterol diet was not observed, yet therewas a significant inverse association between FSR and the increase inplasma cholesterol in subjects on high compared to low cholesterol diet.Data of the present study did not show any significant influence ofplasma cholesterol concentration upon cholesterol synthesis (Figures 10-16). The mechanisms involved in the regulation of cholesterol synthesis inresponse to changes in plasma cholesterol levels are still unclear. It isproposed that regulation of cholesterol synthesis may be a relativelypassive component of overall regulatory process, and its suppression occursonly when plasma cholesterol levels are not normally controlled by othermechanisms such as decreased cholesterol absorption from the gut, increasedbile acid synthesis and excretion (Jones et a/. 1993b). As a result, wepostulate that plasma cholesterol level in response to dietary cholesterolis primarily governed by other regulatory mechanisms including alterations79of the intestinal absorption efficiency, LDL-receptor activity, secretionof cholesterol into bile, and hepatic conversion of cholesterol into bileacid. The response of plasma cholesterol concentration to cholesterolsynthesis is only secondary to the failure of these compensatingmechanisms.805.6 ConclusionsMechanism of cholesterol homeostasis was investigated by identifyingdifferences in dietary cholesterol level and endogenous cholesterolproduction. These factors may contribute to variations in plasmacholesterol concentration which are closely associated with the developmentof CHD.Plasma cholesterol concentration was clearly increased in response toan increase of dietary cholesterol. In the subjects on a high cholesteroldiet, plasma cholesterol was increased by 13% (P = 0.002) as compared tothe low cholesterol diet. The threshold and ceiling amount of dietarycholesterol, however, could not be determined due to an inadequate numberof levels of cholesterol in the experimental diets. Although there was anobservable increase of plasma cholesterol in response to change of dietarycholesterol from low to medium, the magnitude of increase in plasmacholesterol tended to flatten out when dietary cholesterol was changed frommedium to high. Prospectively, graded cholesterol diets utilizing anincreased number of at least six cholesterol levels are needed to welldefine the threshold and ceiling amount of dietary cholesterol.During the initial 12 hour postdose interval, deuterium incorporationfrom plsama to RBC cholesterol was delayed in all three cholesterol diets.This lag in deuterium enrichment in RBC was significantly different in highcompared with low and medium cholesterol diets. Therefore, use of RBCdeuterium uptake results may lead to an underestimation of cholesterolsynthesis over the initial 12 hour interval when cholesterol intake ishigh. 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N Eng J Med 282:1128-1138, 1179-1183, 1241-1249, 1970Dietschy JM: Regulation of cholesterol metabolism in man andin other species. Klin Wochesnschr 62:338-345, 1984Dietschy JM and Spady DK: Measurement of rates of cholesterolsynthesis using tritiated water. J lipid Res 25:1469-1476,1984Erichson BA, Coots RH, Mattson FH et al.: The effect ofpartial hydrogenation of dietary fats, and of the ratio ofpolyunsaturated to saturated fatty acids, and of dietarycholesterol upon plasma lipids in man. J Clin Invest43:2017-2025, 1964Everson GT, McKinley C and Kern FJ: Mechanisms of gallstoneformation in women. Effects of exogenous estrogen(Premarin) and dietary cholesterol on hepatic lipidmetabolism. J din Invest 87:237-246, 1991Ferezou J, Rautreau J, Coste T, Gouffrier E and Chevallier F:Cholesterol turnover in human plasma lipoproteins: Studieswith stable and radioactive isotopes. Am J Clin Nutr36:235-244, 1982Flaim E, Ferreri L, Thye F, Hill J and Ritchey S: Plasma lipidand lipoprotein cholesterol concentrations in adult malesconsuming normal and high cholesterol diets undercontrolled conditions. Am J din Nutr 34:1103-1108, 1981Goodman DS, Noble RP and Dell RB: Three pool model of thelong-term turnover of plasma cholesterol in man. J LipidRes 14:178-188, 1973Goodman DS, Smith FR, Seplowitz AH, Ramakrishnan R and DellRB: Prediction of the parameters of whole body cholesterolmetabolism in humans. J Lipid Res 21:699-713, 1980Goodman DS, Deckelbaum RJ, Palmer RH, Dell RB, Ramakrishnan R,Delpre G et a/.: Cholesterol turnover and metabolism intwo patients with abetalipoproteinemia. J Lipid Res24:1605-1611, 1983Grande F, Anderson JT, Chlouverakis C et al.: Effect ofdietary cholesterol on man's serum lipids. J Nutr 87:52-62, 1965Grundy SM, Ahrens EHJ and Davignon J: The interaction ofcholesterol absorption and cholesterol synthesis in man. J85Lipid Res 10:304-315, 1969Grundy S, Barrett-Connor E, Rudel L, Miettinen T and SpectorA: Workshop on the impact of dietary cholesterol on plasmalipoproteins and atherogenesis. Arteriosclerosis 8:95-101,1988Gylling H and Miettinen TA: Cholesterol absorption andsynthesis related to low density lipoprotein metabolismduring varying cholesterol intake in men with differentapoE phenotypes. J Lipid Res 33:1361-1371, 1992Hagerman JS and Gould RG: The in vitro interchange ofcholesterol synthesis between plasma and red cells. ProcSoc Exp Biol Med 78:329-332, 1951Hegsted DM, McGandy RB, Meyers ML et al.: Quantitative effectsof dietary fat on serum cholesterol in man. Am J din Nutr17:281-295, 1965Hopkins PN: Effect of dietary cholesterol on serumcholesterol: a meta-analysis and review. Am J Clin Nutr55:1060-1070, 1992Jacobs DR, Anderson JT, Hannan P, Keys A and Blackburn H:Variability in individual serum cholesterol response tochange in diet. Arteriosclerosis 3:349-356, 1983Jagannathan SN, Connor WE, Baker WH and Bhattacharyya AK: Theturnover of cholesterol in human atherosclerotic arteries.J din Invest 54:366-377, 1974Jolliffe N and Alpert E: The "performance index" as a methodfor estimating effectiveness of reducing regimens.Postgraduate Medicine 9:106-115, 1950Jones PJH, Scanu AM and Schoeller DA: Plasma cholesterolsynthesis using deuterated water in humans: Effect ofshort-term food restriction. J Lab din Med 111:627-633,1988Jones PJH, Illingworth DR and Leitch CA: Correspondencebetween plasma mevalonic acid levels and deuterium uptakein measuring human cholesterol synthesis. Europ J dinInvest 22:609-613, 1992Jones PJH, Leitch CA, Li ZC and Connor WE: Human cholesterolsynthesis measurement using deuterated water: theoreticaland procedural considerations. Arteriosclerosis &Thrombosis 13:247-253, 1993aJones PJH, Main BF and Frohlich JJ: Response of cholesterolsynthesis to cholesterol feeding in men with differentapolipoprotein E genotypes. Metabolism 42:1-9, 1993b86Katan MB and Beynen AC: Hypo- and hyperresponders: Individualdifferences in the response of serum cholesterolconcentration to changes in diet, in Paoloetti R,Kritchevsky D (eds): Advances in Lipid Research, vol 22,San Diego, CA, Academic Press, pp115-171, 1987Kern FJ: Normal plasma cholesterol in an 88-year-old man whoeats 25 eggs a day. Mechanisms of adaptation. N Engl J Med324:896-899, 1991Keys A, Anderson JT, Mickelson 0 et al.: Diet and serumcholesterol in man: lack of effect of dietary cholesterol.J Nutr 59:39-56, 1956Keys A, Anderson JT and Grande F: Serum cholesterol responseto changes in the diet. II The effect of cholesterol inthe diet. Metabolism 14:759-765, 1965Kopito RR and Brunengraber H: (R)-mevalonate excretion inhuman and rat urines. Proc Natl Acad Sci USA 77:5738-5740,1980Kopito RR, Weinstock SB et al.: Metabolism of plasmamevalonate in rats and humans. J Lipid Res 23:577-583,1982Kummerow FA, Kim Y, Pollard J et al.: The influence of eggconsumption on the serum cholesterol level in humansubjects. Am J Clin Nutr 30:664-673, 1977Langer T, Strober W and Levy RI: The metabolism of low densitylipoprotein in familial type II hyperlipo-proteinemia. Jdin Invest 51:1528-1536, 1972Lieberman S and Samuel P: Determination of total bodycholesterol: Input-output analysis versus compartmentalanalysis. In: Lipoprotein Kinetics and Modeling, edited byBerman M, Grundy SM and Howard G. Academic Press Inc., NewYork. pp. 331-336, 1982Lin DS and Connor WE: The long-term effects of dietarycholesterol upon the plasma lipids,lipoproteins,cholesterol absorption, and the sterolbalance in man: The demonstration of feedback inhibitionof cholesterol biosynthesis and increased bile acidexcretion. J Lipid Res 21:1042-1052, 1981Lipid Research Clinic Program manual of laboratory operations.Vol. 1. Bethesda, MD: Clinics Program. National Institutesof Health, 1974. [DHEW publications (NIH) 75-628].Lipid Research Clinics Program. The Lipid Research ClinicsCoronary Primary Prevention Trial results. II. The87relationship of reduction in incidence of coronary heartdisease to cholesterol lowing. JAMA 251:365-374, 1984Liu GCK, Ahrens EH Jr, Schreibman PH, Samuel P, McNamara DJand Crouse R: Measurement of cholesterol synthesis in manby isotope kinetics of squalene. Proc Natl Acad Sci USA72:4612-4616, 1975London IM and Schwartz H: Erythrocyte cholesterol : themetabolic behavior of the cholesterol of humanerythrocytes. J din Invest 32:1248-1252, 1953Mahley RW, Innerarity TL, Bersot TP et al.: Alteration inhuman high-density lipoproteins with or without increasedplasma cholesterol, induced by diets high in cholesterol.Lancet 11:807-809, 1978Mattson FH, Erickson BA and Klingman AM: Effect of dietarycholesterol on serum cholesterol in man. Am J din Nutr25:589-594, 1972McMurry MP, Connor WE, Lin DS, Cerqueira MT and Connor SL: Theabsorption of cholesterol and the sterol balance in theTaraumara Indians of Mexico fed cholesterol-free and highcholesterol diets. Am J din Nutr 41:1289-1298, 1985McNamara DJ, Kolb R, Parker TS et a/.: Heterogeneity ofcholesterol homeostasis in man, Response to changes indietary fat quality and cholesterol quantity. J dinInvest 79:1729-1739, 1987Messinger WJ, Porosowska Y and Steele JM: Effect of feedingegg yolk and cholesterol on serum cholesterol levels. ArchIntern Med 86:189-195, 1950Miettinen TA: Diurnal variation of cholesterol precursorssqualene and methyl sterols in human plasma lipoproteins.J Lipid Res 23:466-473, 1982Miettinen TA and Kesaniemi YA: Cholesterol absorption:regulation of cholesterol synthesis and elimination andwithin-population variations of serum levels. Am J ClinNutr 49:629-635, 1989Mistry P, Miller NE, Laker M, Hazzard WR and Lewis B:Individual variation in the effects of dietary cholesterolon plasma lipoproteins and cellular cholesterolhomeostasis in man. Studies of low density lipoproteinreceptor activity and 3-hydroxy-3-methylglutaryl coenzymeA reductase activity in blood mononuclearcells. J ClinInvest 67:493-502, 1981Nestel PJ, Schreibman PH and Ahrens EH: Cholesterol metabolismin human obesity. J din Invest 52:2389-2397, 197388Nestel PJ and Kudchodkar B: Plasma squalene as an index ofcholesterol synthesis. Clin Sci Mol Med 49:621-624, 1975Nestel PJ and Poyser A: Changes in cholesterol synthesis andexcretion when cholesterol intake is increased. Metabolism25:1591-1599, 1976Newman HAI and Zilversmit DB: Quantitative aspects ofcholesterol flux in rabbit atherosclerosis lesions. J BiolChem 237:2078-2084, 1962Norum KR, Berg T, Helgerud P and Drevon CA: Transport ofcholesterol. Phys Rev 63:1343-1419, 1983Packard CJ, McKinney L, Carr K et a/.: Cholesterol feedingincreases low density lipoprotein synthesis. J din Invest72:45-51, 1983Parker TS, McNamara DJ, Brown CD, Garrigan 0, Kolb R, Batwin Het al.: Mevalonic acid in human plasma: relationship ofconcentration and circadian rhythm to cholesterolsynthesis rates in man. Proc Natl Acad Sci USA 79:3037-3041, 1982Parker TS, McNamara DJ, Brown CD, Kolb R, EH Ahrens Jr, AlbertAW et al.: Plasma mevalonate as a measure of cholesterolsynthesis in man. J din Invest 74:795-804, 1984Pace N and Rathbun EN: Studies on body composition. III. Thebody water and chemically combined nitrogen content inrelation to fat content. J Biol Chem 158:685-691, 1945Peterson DW: High precision mass spectrometric hydrogenisotope ratio measurements. Ph.D. Thesis, IndianaUniversity, 1979Porter MW, Yamanaka W, Carlson SD et a/.: Effect of dietaryegg on serum cholesterol and triglyceride of human males.Am J din Nutr 30:490-495, 1977Rittenberg D and Schoenheimer R: Deuterium as an indicator inthe study of intermediary metabolism. J Biol Chem 121:235-251, 1937Schoeller DA, Van Santen E, Peterson DW, Dietz W, Jaspan J andKlein PD: Total body water measurement in humans with 180and 2H water. Am J din Nutr 33:2686-2693, 1980Schoeller DA, Peterson DW and Hayes JM: Double-comparisonmethod for mass spectrometric determination hydrogenisotopic abundances. Anal Chem 55:827-832, 1983Schwartz CC: Cholesterol kinetics and modeling: introduction.89In: Lipoprotein Kinetics and Modelina, edited by Berman M,Grundy SM and Howard G. Academic Press Inc., New York. pp.309-312, 1982Schwartz CC, Zech LA, VandenBroek JM and Cooper PS:Cholesterol kinetics in subjects with bile fistula:Positive relationship between size of the bile acidprecursor pool and bile acid synthetic rate. J din Invest91:923-938, 1993Slater G, Mead J, Dhopeshwarkar G et a/.: Plasma cholesteroland triglyceride in men with added eggs in the diet. NutrDept Intl 14:249-259, 1976Spady D and Dietschy J: Sterol synthesis in vivo in 18 tissuesof the squirrel, monkey, guinea pig, rabbit, hamster andrat. J Lipid Res 24:363-475, 1983Stamler J, Wentworth D and Neaton J: Is the relationshipbetween serum cholesterol and risk of death from coronaryheart disease continuous and graded? JANA 256:2823-2828,1986Steiner A and Domanki B: Dietary hypercholesterolemia. Am JMed Sci 201:820-824, 1941Steiner A, Howard EJ and Akgun S: Importance of dietarycholesterol in man. JAMA 181:186-190, 1962Taylor CB, Mikkelson B, Anderson JA and Forman DT: Human serumcholesterol synthesis measured with the deuterium label.Arch Path 81:213-231, 1966The Pooling Project Research Group: Relationship of bloodpressure, serum cholesterol, smoking habit, relativeweight and ECG abnormalities to incidence of majorcoronary events: Final report of the pooling projectresearch group. J Chron Dis 31:201-306, 1978Turley S and West C: Effect of cholesterol and cholestyraminefeeding and of fasting on sterol synthesis in liver,ileum, and lung of the guinea pig. Lipids 11:571-577, 1976Turley S, Spady D and Dietschy J: Alteration of the degree ofmanipulation of the pools of preformed and newlysynthesized cholesterol. Gastroen 84:253-264, 1983Wells VM and Bronte-Stewart B: Egg yolk and serum cholesterollevels. Brit Med J 1:577-581, 1963Wilkinson L.. SYSTAT: The system for statistics. Evanston, IL:SYSTAT, Inc., 1990Wong WW, Hachey DL, Feste A et al.: Measurement of in vivo90cholesterol synthesis from 2820: a rapid procedure for theisolation, combustion, and isotopic assay of erythrocytecholesterol. J Lipid Res 32:1049-1056, 1991Zar JH: Multiple hypotheses. In: Biostatistical analysis. 2ndedition. pp. 162-184, 1984Appendix 1. Lipid Studies Volunteer Information FormDateNameLast^First^MI^(Maiden Name)Birthday  Age ^ Sex (Circle) M FPhone (home) ^ (work) ^ message ^Address^ .Street^City^State^ZipOHSU Clinic Card #  Social Security # -State/Country of Birth ^ Mather's Maiden Name ^ .Typical Work Schedule ^Days^Hours^OccupationDo 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)LunchDinnerDo 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 ^92Exercise Regimen:^Type^Minutes/Session^Sessions/WeekMarital 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 yourcholesterol 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^[] NoPlease list any other health problems:(Include major illnesses and/or surgeries along with approximate dates)Are you taking any medicines? [] Yes^[] NoIf yes, please indicate below:Name of medication Dose For how longDiuretic (water pill)Diabetes medicationMedication to lowercholesterol and/ortriglyceridesThyroid medicationHormonesBirth control pillsBlood pressure medication^93Aspirin or Anti-flammatory^AnticoagulatesOthers (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 aphysician 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 thereany times in the next six months that you would not want to beinvolved 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 familymember such as grandfather, sister, brother, aunt, or uncle:YOU^MOTHER^FATHER^OTHER FAMILYMEMBER(SPECIFY)HIGH CHOLESTEROLHIGH TRIGLYCERIDESTROKEHIGH BLOOD PRESSUREHEART ATTACKANGINADIABETESOTHER HEARTPROBLEMS (SPECIFY)GALL BLADDERPROBLEMSARTERIOSCLEROSISOF LEGS (HARDENINGOF ARTERIES)LIVER DISEASEKIDNEY DISEASEHYPOTHYROIDISMALCOHOL ABUSEMILDLY OVERWEIGHTEXTREMELY OVERWEIGHTPlease check how you feel about the following foods:FRUITS^ VEGETABLESYUM OK YUK^ YUM OK YUKFresh:^ Cookedapples beans:bananas frozen/cannedgrapefruit^ greenoranges kidneybroccolicarrotsFresh (seasonal):^cauliflowercantaloupe^ cabbagegrapes mushroomshoneydew melon onionsnectarines^ peaspeaches tomatoespineapplesplums^ Raw:strawberries^ broccolitangerine cauliflowerwatermelon cabbage95kiwi^ carrotsceleryFrozen: cucumberspeaches^ green peppersblackberries lettuce, icebergblueberries mushroomsstrawberries^ onionsspinachCanned: tomatoespruneapplesauce^ vegetable juicecranberry sauce tomatopeaches V-8 (vegetable)pearspineapples^ PROTEIN FOODSYUM OK YUKOthers:raisinshoneyjellyJuices:pineappleapplecranberrygrapegrapefruitorangeBREADS AND GRAINYUM OK YUKCereal, hotcream of wheatoatmealCereal, coldall brancorn flakespuffed wheatrice krispiesraisin branwheat iesToast, Roll, Breadfrench breadwhole wheat breadwhite breadbranola breadenglish muffinwheatberry breadbagelbanana muffinturkeychickenhambeefyogurt, plainfishporkeggbeatersegg white1% cottage cheesecountdown cheeseENTREES (fat-free, low salt) I'LL TRY IT^YUKMeatless chiliw/beansMacaroni & cheese(fat free)Tomato soupvegetable soupMeatlessSpaghetti SauceStir fry veggiesin orientalBaked potatow/mock sour crmTetrazzini:noodles inwhite sauce w/optional peasand mushroomsSALADS & DRESSINGS96blueberry bran muf^ YUM OK YUKpocket breadOther Carbohydrate Choicesbread stickscorn tortillastaco chipspotatoesrice cakesbrown ricewhite ricespaghettimacaronicornangel cakesherbetmixed green saladmacaroni saladbean saladpotato saladlow caloriesfrench dressingno oil italiandressingAppendix 2. Consent and Instruction Form"Dietary Cholesterol in Graded Amounts: Threshold to Ceiling Effects uponPlasma Lipoproteins, Apoproteins and LDL Turnover"^ , herewith agree to serve as a subject in theinvestigation entitled "Dietary Cholesterol in Graded Amounts: Threshold toCeiling 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., DonLin, M.S..I. PurposeThe aim is to study the effects of different and precise amounts of dietarycholesterol upon blood fat levels.II. ProceduresThe procedures in which I will participate are:a. Eating Research Diets. There will be three separate dietary periods eachlasting four weeks. Between each dietary period, I will be required toreturn to my typical home diet for at least four weeks. The study dietswill consist of whole mixed foods (meats, grains, vegetables, dietaryproducts, fruits) and will be provided as three meals with snacks. Inaddition, a formula or foods as custard or cookies containing varyingamounts of cholesterol will be provided. I agree to eat all of my mealsprovided by the study and to eat nothing else during the study. Iunderstand that I may drink coffee, tea and sugar-free beverages and chewsugar-free gum, but not consume alcohol because of its excess calories andother 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 dietaryperiods.b. Prestudy Evaluations. Prior to beginning of the study, my blood will bedrawn on three separate occasions to establish my baseline cholesterollevel. I agree to an initial medical history and examination. Initial bloodpressure, height, and weight measurements will be taken.c. Daily Maintenance. During the dietary periods, body weight will bemeasured daily and blood pressure will be measured twice a week. I will beasked 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 anLDL turnover, three times during this study. A radioactive isotope (25microcuries of 1251) will be given each time. The radiation exposure lieswithin the limit of experimental radiation exposure considered acceptableby the Radiological Health Section of the Oregon State Board of Health.Although no radiation dose has been proven to be entirely safe, the dose towhich you will be exposed has not shown to cause cancer or other problems.98e. Assessment of cholesterol Synthesis by Administration of "Tagged" Water(Deuterium Labeled Water). Deuterium is a heavy isotope of hydrogen andthus pose no radiation hazard or toxicity with the small amount given, Whenconsumed, it is incorporated in the body's water and can be used to tracean individual's cholesterol metabolism. On the last day of each of thedietary periods, I will be asked to spend the day at the Clinical ResearchCenter for this test. At about 8 AM, I will drink approximately 1 ounce ofthe "tagged" water to begin the test and then for the reminder of the 24hour period I will use the "tagged" water provided to me for making anybeverages or for any water that I wish to drink. Blood samples will bedrawn initially and at 6, 12, and 24 hours after the first dose of taggedwater. I will consume the :esearch diet normally provided to me on thistest day; however, the meal times will be fixed. In addition, I will beasked to limit my exercise during this 24 hour period.f. Venipuncture or Blood Sampling. Blood will be drawn three times before Ibegin consuming the study diet to evaluate my baseline cholesterol. Duringeach of the dietary periods blood will be drawn 27 times including theblood sampling for the LDL and deuterated water procedures.The total amount of blood drawn for the three dietary periods will beapproximately 1 1/3 cups for each dietary period. I understand that myblood count will be monitored so that this amount of blood sampling willnot cause anemia over the lengthy time period of the study.g. 24 Hour Urine Collection. I agree to collect all urine for 24 hours: twotimes before the first diet period and two times during each of the threedietary periods.I will be instructed on the methods to collect the 24 hour urine samplesand these samples will be used to assess the effects of the different dietson cholesterol synthesis as well as determine the urinary excretion ofradioactivity following the injection of radioiodinated lipoproteins.III. CostI understand that during the course of the study, the food and the chargesincurred by the blood tests necessary to evaluate the study will be free ofcharge. I will receive a small payment of $2.50 per day at successfulcompletion of the entire study. This study will involve approximately 14weeks (22 weeks including break period) of my time during which I will beinconvenienced as little as possible.IV. RiskVenipuncture causes modest discomfort and may be associated with bruisingand rarely infection at the venipuncture site and the possibility of clotsin the vein or the occurrence of small scars. Collection of a 24 hour urinesample involves my inconvenience but is without risk.Although I may not benefit directly from the study, the informationcontained may improve the understanding and treatment of patients withdisorders of cholesterol metabolism.99V. ConfidentialityI understand that neither my name nor my identity will be used forpublication or publicity purposes.I understand that I am free not to participate, and I may withdraw fromparticipation in this study at any time, and it will in no way affect myrelationship with the Oregon Health Sciences University.I understand that Drs. Connor, Illingworth, Ullmann and Lauren Hatcher willanswer at any time during the study, all questions I might have about thestudy or procedures. If I have any questions or if there is an emergency, Imay call Dr. Connor at 494-8005 (work) or 226-0529 (home).VI. LiabilityI 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 amparticipating, to compensate or provide medical treatment for humansubjects in the event the research results in physical injury. The OregonHealth Sciences University, as an agency of the state, is covered by theState Liability Fund. If I suffer any injury from the research project,compensation would be available to me only if I establish that the injuryoccurred through the fault of the University, its officers or employees. IfI have further questions, I may call Michael D. Baird, M.D. at (503)494-8014.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)^(Date)^(Time)(Signature of Witness)^(Date)^(Time)(Signature of Investigator)^(Date)^(Time)100Appendix 3. A Sample of Three Day Food RecordsYou have been asked to keep food records for the research study you areparticipating in. The information these records will supply is veryimportant to the study, as it tells us about the composition of the foodsyou typically eat. What you typically eat can affect the way you mayrespond to the study diet or supplement. A dietitian will tell which daysto write down all the food and beverages that you eat. The following is alist of points to be kept in mind as you keep your food record:1. Record BRAND NAMES where applicable. This is especiallyimportant for foods containing fat. Bring in the label ifpossible.2. Specify if foods are COOKED or RAW.3. For COOKED foods specify METHOD of PREPARATION. Example -whether meat is fried, broiled, baked, etc.. Identifyfats 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 ofmeat, amount of mayonnaise, mustard, lettuce, cheese,etc..6. Record AMOUNT of foods. Use measuring cups and measuringspoons or copy the weight from the food package ifpossible. Record dimensions (in inches) if weights ormeasures are not available.7. Note AMOUNT of foods NOT consumed. Example - bone andfats in meat.Example:Time/^Food^ AmountLocation07:30 scrambled eggs,home scrambled with Parkay stick marg.toast - Branola breadParkay marg. on toastorange juice - from frozen conc.10:00 coffee with creamcaf.^real creamdonut - glazed yeast donut 5"12:00 sandwich: whitebreadhome turkey wafer thin slices2 whole large eggs2 teaspoons2 slices2 teaspoons8 oz8 oz1 tablespoon1 whole2 slices1/2 of 3 oz pkg101mayonnaise "Lite" Best FoodsCoke - no iceapple large 4" diameters18:00 baked chicken breast (no skin)rest. mashed potatoesgreen beans seasoned with marg.Beer 12 oz Lite Budweiser22:00 milk - 2%home cake - chocolate from Pillsbury,made with whole eggs andWesson oil1 tablespoon1 - 12 oz can1 whole1 medium1/2 cup1/2 cup1 whole12 oz1 - 4x4x2" square102Appendix 4. Calculation of Caloric Intake of Selected SubjectsConsuming North American DietEstimated 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:^kcalAppendix 4 (cont'd) Estimation of food factor% aboveBMR^Classification^Groups of individuals20%^limited activity^bed-restanorexia nervosa30%^minimum activity^up and about inactivewomen > 50 years old40%^limited activity^most in-patient womenmen > 50 years old50%^limited to normal most in-patient menactivity^most out-patient women60%^normal lifestyle^most out-patient men70%^heavy work^physical laborersthose who schedule daily rigorousphysical exercise e.g. 1 hr/dayFood FactorFood Factor Food Factor kcalskcalskcalsSelected eucaloric level:Explanation:Appendix 4 (cont'd) Selected daily caloric allowance of thesubjectsSubject Body surface* BMR*^Food factor Food allowancearea (m2)^(kcal/day) ^(kcal/day)1^1.59^1420^1.4^20002^2.00^1950^1.8 36003^1.85^1720^1.7^30004^1.72^1600^1.6 25005^1.76^1620^1.7^28006^1.65^1520^1.6 24007^1.65^1540^1.4^22008^2.12^2100^1.8 3800* determined from reference Jolliffe and Alpert 1950.Appendix 5. Randomization Scheme for Graded CholesterolProtocolScheme#^ Phase orderFirst^Second^Third(Dietary cholesterol level of phase, mg/day)A 50 350 650B 50 650 350C 350 50 650D 350 50 650E 650 50 350F 650 350 50Appendix 6. Deuterated Water Test Schedule Sample FormVolunteer Name:Diet Phase: Unit #:Schedule for consumption of deuterated water on ^day, 199_, atAM.^grams deuterated water to be consumed. Container to be rinsed with50 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 vertigowith the deuterated water.Actual time deuterated water was consume ^ AM.0 hr 20 cc EDTA6 hr 20 cc EDTA12 hr 20 cc EDTA24 hr 20 cc EDTASend 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 drawnthe next morning. (Tubes to be labeled 0, 6, 12, or 24 hr).In addition to beverages which are a part of the research diet, volunteershould consume, throughout the day, 2000 g of distill water containing 2.4grams deuterated water. This water can be used for making decaffeinatedcoffee and/or tea and Crystal Lite beverage. Total amount of coffee or teaconsumed should be 3 cups or less. No other beverages, including carbonatedbeverages, are allowed.Dieticians to keep a record of dietary intake -- menu used and time ofmeals and snack(s). Meal/snack times and menu used to be the same on eachdiet phase.107Appendix 7. Doses of Deuterated WaterSubject Total body water^Estimation method^D20(kg)^ (g) 1 32.9 BIA 19.72 51.0 BIA 30.63 40.6 BIA 24.44 41.0 body weight x 0.6 24.65 43.4 body weight x 0.6 26.16 40.0 BIA 24.07 38.8 body weight x 0.6 23.38 54.0 BIA 32.4Appendix 8. Deuterium Enrichment of Plasma and RBC FreeCholesterol From Baseline Plasma Water atVarious Timepoints Among Three ExperimentalDietsSubjectCodeFree cholesterol 2H/1H relative to SMOW^Plasma^ RBC6hr^12hr 24hr^6hr^12hr(°/oo)24hrLow diet1 21.6 -17.0 174.4 24.4 11.8 91.82 49.0 79.1 131.1 7.2 45.8 96.73 10.3 2.2 -14.0 30.5 43.2 51.64 59.3 78.7 121.1 14.8 27.6 91.95 34.6 80.6 185.7 10.3 53.2 139.66 38.9 52.3 105.8 20.1 26.1 110.27 81.6 111.0 148.1 14.4 31.9 97.08 5.2 18.0 90.2 -45.2 -11.6 70.8Mean 32.2 50.6 117.8 9.6 28.5 93.7SEM 11.7 15.9 22.0 8.3 7.4 9.2Medium diet1 33.2 158.4 15.9 50.7 148.22 43.8 97.1 156.4 16.0 137.73 42.2 43.7 74.5 63.0 49.5 85.94 15.2 27.8 139.2 9.2 3.6 36.35 13.3 54.6 83.7 27.9 49.3 142.66 36.6 50.4 95.6 3.6 30.6 47.97 18.8 25.4 78.8 -20.9 44.1 101.38 10.2 40.1 84.5 30.6 44.5 105.3Mean 26.7 48.4 108.9 18.2 38.9 100.7SEM 4.9 9.1 12.8 8.5 6.4 15.0High diet1 27.2 55.8 152.3 5.1 29.9 120.22 34.9 91.9 175.2 13.3 43.1 133.23 57.7 78.4 118.3 -37.2 -18.3 31.04 1.1 15.1 80.8 4.5 7.8 57.25 141.9 117.5 118.5 58.5 42.7 119.76 83.3 56.8 45.3 -25.9 2.8 85.57 44.4 41.7 79.0 7.4 26.3 62.98 23.5 22.9 34.0 -11.6 -5.5 46.9Mean 51.7a 60.0b 100.4 1.8 16.1 82.1SEM 15.5 12.3 17.5 10.2 8.0 13.6a Significantly different from that of RBC in the highdiet (P = 0.010).b Significantly different from that of RBC in the highdiet (P = 0.005).109

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