Title: U-shape relationship between change in dietary cholesterol absorptionand plasma lipoprotein responsiveness and evidence for extreme interindividualvariation in dietary cholesterol absorption in humans
Abstract: A possible relationship between change in dietary cholesterol absorptionand plasma lipoprotein responsiveness was examined in 18 normal subjects fed lowfat low cholesterol, high fat low cholesterol, and high fat high cholesteroldiets. For the group, neither dietary cholesterol nor dietary fat affected thepercentage dietary cholesterol absorption, whreas dietary cholesterol intakeraised total and LDL-C and dietary fat raised total, LDL, and HDL-C. On a fixeddiet there was approximately a 2-fold variation among subjects in percentagedietary cholesterol absorption. Subjects also varied in response to dietarycholesterol and fat with regard to dietary cholesterol absorption and plasmalipoprotein responsiveness. There was a U-shaped parabolic relationship betweendietary cholesterol-induced percent change in LDL-C and the change in percentagedietary cholesterol absorption (R2 = 0.62,P = 0.005). A similar but weaker relationship characterizedthe responsiveness of HDL-C (R2 = 0.38,P = 0.05). For the group, increased cholesterol intakeraised dietary cholesterol mass absorption from 1.6 to 4.6 mg/kg per day, butthe range of increase was from 1 to 4.7 mg/kg per day. Increased fat intake alsoaffected dietary cholesterol mass absorption with most subjects displaying astrong inverse relationship between fat intake and mass absorption(r = −0.77, P < 0.003). Insummary: i) the percentage change in dietary cholesterolabsorption in response to dietary cholesterol does appear to regulate dietresponsiveness of LDL and HDL-C, and ii) the large variabilityin percent absorption and changes in percentage and mass absorption in responseto dietary cholesterol suggest the presence of genetically determineddifferences among individuals in the regulation of dietary cholesterolabsorption.—Sehayek, E., C. Nath, T. Heinemann, M. McGee, C. E. Seidman, P.Samuel, and J. L. Breslow. U-shape relationship between change in dietarycholesterol absorption and plasma lipoprotein responsiveness and evidence forextreme interindividual variation in dietary cholesterol absorption in humans.J. Lipid Res. 1998. 39: 2415–2422. A possible relationship between change in dietary cholesterol absorptionand plasma lipoprotein responsiveness was examined in 18 normal subjects fed lowfat low cholesterol, high fat low cholesterol, and high fat high cholesteroldiets. For the group, neither dietary cholesterol nor dietary fat affected thepercentage dietary cholesterol absorption, whreas dietary cholesterol intakeraised total and LDL-C and dietary fat raised total, LDL, and HDL-C. On a fixeddiet there was approximately a 2-fold variation among subjects in percentagedietary cholesterol absorption. Subjects also varied in response to dietarycholesterol and fat with regard to dietary cholesterol absorption and plasmalipoprotein responsiveness. There was a U-shaped parabolic relationship betweendietary cholesterol-induced percent change in LDL-C and the change in percentagedietary cholesterol absorption (R2 = 0.62,P = 0.005). A similar but weaker relationship characterizedthe responsiveness of HDL-C (R2 = 0.38,P = 0.05). For the group, increased cholesterol intakeraised dietary cholesterol mass absorption from 1.6 to 4.6 mg/kg per day, butthe range of increase was from 1 to 4.7 mg/kg per day. Increased fat intake alsoaffected dietary cholesterol mass absorption with most subjects displaying astrong inverse relationship between fat intake and mass absorption(r = −0.77, P < 0.003). Insummary: i) the percentage change in dietary cholesterolabsorption in response to dietary cholesterol does appear to regulate dietresponsiveness of LDL and HDL-C, and ii) the large variabilityin percent absorption and changes in percentage and mass absorption in responseto dietary cholesterol suggest the presence of genetically determineddifferences among individuals in the regulation of dietary cholesterolabsorption. —Sehayek, E., C. Nath, T. Heinemann, M. McGee, C. E. Seidman, P.Samuel, and J. L. Breslow. U-shape relationship between change in dietarycholesterol absorption and plasma lipoprotein responsiveness and evidence forextreme interindividual variation in dietary cholesterol absorption in humans.J. Lipid Res. 1998. 39: 2415–2422. Atherosclerotic cardiovascular disease is the number one public health problem inthe United States, and by the year 2020 it is predicted this will be true world wide.This disease is a complex genetic disease with many genes involved and importantgene–environment interactions. Epidemiological, clinical, and animal studieshave clearly established an important role for dietary cholesterol and saturated fat inatherosclerosis susceptibility. Numerous studies have shown that increased consumptionof cholesterol and saturated fat are associated with increased plasma levels of LDLcholesterol and increased risk of cardiovascular diseases, whereas low dietarycholesterol and low saturated fat have the opposite effect (1Sonnenberg L.M. Posner B.M. Belanger A.J. Cupples L.A. D'Agostino R.B. Dietary predictors of serum cholesterol in men: theFramingham cohort population.J. Clin.Epidemiol. 1992; 45: 413-418Google Scholar). However, it has been repeatedly observed that there is greatinterindividual variation in plasma lipoprotein responsiveness to dietary cholesteroland saturated fat. This variation is presumably genetic but the specific genes involvedare largely unknown. It has been previously shown in humans that for each 100 mg/day increase indietary cholesterol the mean plasma cholesterol level rises 7 mg/dl, but someindividuals are unresponsive and have even decreasing cholesterol levels, while othersshow an exaggerated response, with increases of more than 2-fold the mean (2Beynen A.C. Katan M.B. VanZutphen L.F. Hypo- and hyperresponders: individual differences in theresponse of serum cholesterol concentration to changes indiet.Adv. Lipid Res. 1987; 22: 115-171Google Scholar, 3Katan M.B. Beynen A.C. Characteristics of human hypo- and hyperresponders to dietarycholesterol.Am. J.Epidemiol. 1987; 125: 387-399Google Scholar, 4Beynen A.C. Katan M.B. Reproducibility of the variations between humans in theresponse of serum cholesterol to cessation of eggconsumption.Atherosclerosis. 1985; 57: 19-31Google Scholar, 5Katan M.B. Beynen A.C. deVries J.H. Nobels A. Existence of consistent hypo- andhyperresponders to dietary cholesterol in man.Am. J. Epidemiol. 1986; 123: 221-234Google Scholar, 6Katan M.B. Berns M.A. Glatz J.F. Knuiman J.T. Nobels A. deVries J.H. Congruence of individual responsiveness to dietarycholesterol and to saturated fat in humans.J. Lipid Res. 1988; 29: 883-892Google Scholar).In human studies, increasing saturated fat intake also increases cholesterol levels withsimilar interindividual variability (7Grundy S.M. Denke M.A. Dietary influences on serum lipids andlipoproteins.J. LipidRes. 1990; 31: 1149-1172Google Scholar, 8Denke M.A. Grundy S.M. Individual responses to a cholesterol-lowering diet in 50 menwith moderate hypercholesterolemia.Arch.Intern. Med. 1994; 154: 317-325Google Scholar). In human studies, it has been suggested that theability to down-regulate endogenous cholesterol synthesis in response to a dietarychallenge limits an individual's plasma lipoprotein responsiveness (9Mistry P. Miller N.E. Laker M. Hazzard W.R. Lewis B. Individual variation in the effects ofdietary cholesterol on plasma lipoproteins and cellular cholesterolhomeostasis in man. Studies of low density lipoprotein receptor activity and3-hydroxy-3-methylglutaryl coenzyme A reductase activity in bloodmononuclear cells.J. Clin.Invest. 1981; 67: 493-502Google Scholar, 10McNamara D.J. Kolb R. Parker T.S. Batwin H. Samuel P. Brown C.D. Ahrens Jr., E.H. Heterogeneity of cholesterol homeostasis inman. Response to changes in dietary fat quality and cholesterolquantity.J. Clin. Invest. 1987; 79: 1729-1739Google Scholar). Inanimal studies, evidence has been provided that species fed a low cholesterol diet andhave high hepatic cholesterol synthesis, which can down-regulate synthesis in responseto cholesterol feeding, are less responsive to dietary challenge than species in whichhepatic cholesterol synthesis is low (11Spady D.K. Woollett L.A. Dietschy J.M. Regulation of plasma LDL-cholesterol levels by dietarycholesterol and fatty acids.Annu. Rev.Nutr. 1993; 13: 355-381Google Scholar). Thusmetabolic processes, and the genes that control them, that regulate individualdifferences in endogenous cholesterol synthesis may be fundamental to understandingplasma lipoprotein responsiveness to dietary challenge. Another metabolic process that could influence diet responsiveness is theabsorption of dietary cholesterol from the intestine, as this represents the firstobligatory step that allows dietary cholesterol to exert its metabolic effects. Afterabsorption, dietary cholesterol is transported by chylomicrons and their remnants mainlyto the liver where it can directly influence lipoprotein production and removal pathways(7Grundy S.M. Denke M.A. Dietary influences on serum lipids andlipoproteins.J. LipidRes. 1990; 31: 1149-1172Google Scholar, 11Spady D.K. Woollett L.A. Dietschy J.M. Regulation of plasma LDL-cholesterol levels by dietarycholesterol and fatty acids.Annu. Rev.Nutr. 1993; 13: 355-381Google Scholar). Individual differences in absorption could influence lipoproteinresponsiveness in this manner. Differences in dietary cholesterol absorption could alsoaccount for differences in the endogenous cholesterol synthesis proposed in human andanimal studies to account for plasma lipoprotein responsiveness. The current study was undertaken to determine the relationship, if any, betweendietary cholesterol absorption and plasma lipoprotein responsiveness. Eighteen normalsubjects were fed low fat low cholesterol, high fat low cholesterol, and high fat highcholesterol diets and the percentage dietary cholesterol absorption and plasmalipoprotein responsiveness were measured. The percentage change in dietary cholesterolabsorption in response to dietary cholesterol was found to be highly correlated with thepercent change in LDL and HDL cholesterol levels and the relationships were bestdescribed by U-shaped parabolic curves. Documentation was also provided for largeindividual differences in i) dietary cholesterol absorption andii) the dietary cholesterol affect on percentage dietarycholesterol absorption and dietary cholesterol mass absorption. These differencessuggest genetic variation among humans in the regulation of dietary cholesterolabsorption. Eighteen normal volunteers were recruited through advertisements postedat The Rockefeller University and neighboring institutions or through collegeundergraduate work–study programs. There were no exclusions based ongender, race, or ethnic background. Subjects had normal thyroid, renal, andliver function tests and no systemic diseases by history or physicalexamination. None of the subjects were smokers or on any medication, includingbirth control pills. There were 10 males and 8 females varying in age from 19 to60 years (mean ± SD of 30.3 ± 13.3) with body mass indices (BMI)from 17.0 to 27.3 (mean ± SD of 23.2 ± 2.9). The distribution ofapoE phenotypes was E3/3, 44.4%; E4/3, 22.2%; E3/2, 22.2%; E4/4, 5.6%; and E2/2,5.6%. All subjects were normolipidemic upon initial screening with lipid andlipoprotein levels between 10th and 90th percentile for age and sex based onLipid Research Clinic data (12Department of Health and HumanServices National Institutes ofHealth The LipidResearch Clinic population studies data book.Theprevalence study. I. National Institutes ofHealth, Bethesda,MD1980Google Scholar). The subjects were studied on the inpatient unit of The RockefellerUniversity Hospital and encouraged to continue their usual physical activity.The study design was a randomized crossover trial of three isocaloric, naturalfood diets that differed in dietary fat and/or cholesterol. The diets, asdescribed below, were: low fat low cholesterol (LFLC), high fat low cholesterol(HFLC), and high fat high cholesterol (HFHC). Each metabolic diet was consumedfor 3 weeks. The diets were randomly assigned to each subject with every set ofthree subjects in Latin squares balanced for sequence, so that each dietfollowed the others twice, with an equal number of subjects on each diet. Allsubjects completed the study with no dropouts. Fasting lipoprotein profiles wereobtained four times in the third week of each diet period. We have previouslyshown that under metabolic ward conditions, similar to the ones in this study,changes in dietary fat and cholesterol result in a new steady state in plasmalipoprotein levels by day 15 with no drift between days 15 and 25 (13Denke M.A. Breslow J.L. Effects of a low fat diet with and without intermittentsaturated fat and cholesterol ingestion on plasma lipid, lipoprotein, andapolipoprotein levels in normal volunteers.J. Lipid Res. 1988; 29: 963-969Google Scholar). The study protocol was approved by theInstitutional Review Board of The Rockefeller University, and each subjectsigned an informed consent prior to the study. Meals were prepared by the nutrition staff of The Rockefeller UniversityHospital Clinical Research Center. The diets consisted of common ingredients ofknown composition listed in the USDA Handbook 8 (14United States Department ofAgriculture, Agricultural ResearchService, . 1990. US Department ofAgriculture Nutrient Data Base for Standard Reference, release 9.Hyattsville, MD.Google Scholar), and the foods were weighed to the nearest 0.1 gram. A 2-dayrotating menu was provided throughout each study period. Breakfast and dinnerwere routinely consumed at the Clinical Research Center and lunches were usuallypacked for convenience. Subjects were instructed not to eat any foods other thanthe metabolic diets provided and all foods served had to be consumed by 8pm on the same day. The physicians and research nutritionistscommunicated with each subject daily to encourage compliance. The initialcaloric requirement for each subject was estimated according to theHarris-Benedict equation with an adjustment for physical activity (15Harris J.A. Benedict F.G. Abiometric study of basal metabolism in man. Carnegie Institute of Washington. JBLippincott, Philadelphia1919Google Scholar). The caloric requirements ranged from2300 to 3500 kcal, (mean ± SD of 2789 ± 362). Subjects were keptin a metabolic steady state with no significant changes in weight or physicalactivity during the study. The composition of the diets is shown inTable 1. The LFLC diet conformedto the American Heart Association Phase II Diet and contained 60% carbohydrate,15% protein, 25% fat (26% saturated, 40% monounsaturated and 34% polyunsaturatedfatty acids), and 80 mg cholesterol/1000 kcal/day, the equivalent of 0.04%weight/weight (w/w) dietary cholesterol. The HFLC diet was characterized by 42%carbohydrates, 15% protein, 43% fat (44% saturated, 40% monounsaturated and 16%polyunsaturated fatty acids), and 80 mg cholesterol/1000 kcal/day (0.04% w/wdietary cholesterol). The HFHC diet was identical to the HFLC diet except forcholesterol content of 200 mg/1000 kcal per day that is equivalent to 0.1% w/w dietarycholesterol. The compositions of the diets were verified by chemical analysis ofcomposites of each day of the three metabolic diets by Hazelton Laboratories(Madison, WI).TABLE 1Diet compositionLFLCHFLCHFHCCarbohydrates (%)604242Protein (%)151515Fat (%)254343Fatty acids compositionSaturated (%)264444Monounsaturated (%)404040Polyunsaturated (%)341616P/S ratio1.50.350.35Cholesterol (mg/1000 kcal/d)8080200 Open table in a new tab In the third week of each diet period (days 16, 17, 19, and 20) fourfasting plasma samples anticoagulated with EDTA were obtained after a 12-hovernight fast for lipid and lipoprotein measurements. The values used foranalysis were the average of these four measurements for each subject on eachdiet. Total cholesterol and triglycerides were determined by enzymatic methodsusing Boehringer Mannheim reagents. HDL-cholesterol was determined afterprecipitation of non-HDL-C by dextran sulfate (16Warnick G.R. Benderson J. Albers J.J. Dextran sulfate-Mg2+ precipitation procedure forquantitation of high-densitylipoprotein cholesterol.Clin. Chem. 1982; 28: 1379-1388Google Scholar). LDL-C plus HDL-C was determined on the infranatant afterairfuge ultracentrifugation (Beckman Instruments). LDL-C was the differencebetween the infranatant and HDL-C value. VLDL-C was the difference between totaland the infranatant cholesterol. Total and HDL-cholesterol values werestandardized by the Lipid Standardization Program of the Centers for DiseaseControl and Prevention supported by the National Heart, Lung, and BloodInstitute (17Clinical ChemistryStandardization Section Lipid Standardization Programs of the Center for DiseaseControl. Center for Environmental Health, Center for Disease Control,Department of Health and Human Services, Atlanta1985Google Scholar). Apolipoprotein Egenotyping was determined according to Hixson and Vernier (18Hixson J.E. Vernier D.T. Restriction isotyping of human apolipoprotein E by geneamplification and cleavage with HhaI.J.Lipid Res. 1990; 31: 545-548Google Scholar). Cholesterol absorption was determined by the isotope ratio method asdescribed by Zilversmit and Hughes (19Zilversmit D.B. Hughes L.B. Validation of a dual-isotope plasma ratio method formeasurement of cholesterol absorption in rats.J. Lipid Res. 1974; 15: 465-473Google Scholar)and modified for human studies by Samuel, Crouse, and Ahrens (20Samuel P. Crouse J.R. Ahrens Jr., E.H. Evaluation of an isotope ratio method formeasurement of cholesterol absorption in man.J. Lipid Res. 1978; 19: 82-93Google Scholar). Briefly, on the third week of eachdietary period (day 16) subjects were fasted overnight and radiolabeledcholesterol was administered intravenously and orally between 8 and 10am. For intravenous administration, [1,2-3H]cholesteroldissolved in 1 ml of ethanol was suspended in 150 ml of saline and immediatelyinfused. Residual radioactivity in the infusion set was measured after tolueneextraction and subtracted from the total radioactivity to calculate theadministered dose. For oral administration, [4-14C] cholesteroldissolved in 1 ml of ethanol was mixed with 5 ml of milk in a glass beaker andimmediately ingested. Subsequently, another 5 ml of milk was added to the beakerand this too was ingested. Residual radioactivity remaining in the beaker wasmeasured by ethanol extraction and the net amount administered was determined.Dosage of radioactivity varied from 1 to 2 μCi of [1,2-3H]-and [4-14C]cholesterol per assay. Radioactivity was measured in aBeckman LS 5000TD model scintillation counter (Beckman Instruments Co.,Fullerton, CA) with automatic quench compensation after Compton spectrummeasurement. Plasma 3H and 14C labels were determined onmorning blood samples drawn on days 18, 19, 20, and 21 of each diet. Percentcholesterol absorption was calculated using the equation: %absorption=C14∕H3ratio×intravenousH3dosedpmoralC14dosedpm×100 Mass absorption of dietary cholesterol was calculated by multiplying thedaily cholesterol intake by the percent cholesterol absorption and expressed asmg cholesterol/kg body weight per day. The study was designed as a three-treatment, three-period crossovertrial with no wash-out periods between treatments. Sample size was calculatedduring the design phase of the trial, using a power of 80% and a significancelevel of 0.05. During sample size calculations, it was assumed that there wouldbe no carryover effects from one diet to the next. Data analysis was performedusing a computer model in Excel 7.0 (Microsoft®, 1996), which usedformulas for the analysis of crossover studies found in Fleiss (21Fleiss J.L. The crossover study.in: The Design and Analysis of Clinical Experiments. John Wiley & Sons, New York, NY1986: 263-290Google Scholar). Data were examined for the amount ofvariation attributable to subjects, periods, treatment effects, carryovereffects, and residual effects for each variable of interest under study (TC, TG,VLDL-C, LDL-C, HDL-C, and percentage dietary cholesterol absorption). There wereno significant carryover effects for any of the variables except HDL-C. ForHDL-C, the variation due to crossover effects resulted in an F-ratio of 11.58with 2 and 30 degrees of freedom. This corresponds to a P valueless than 0.001 indicating evidence for the presence of some carryover effectsfor HDL-C. For variables in which there was evidence for a treatment effect butno evidence of a carryover effect, pairwise differences between treatments wereexamined using Tukey's HSD. The relationship between cholesterol absorption and plasma lipoproteinresponsiveness was investigated using a polynomial regression model. Themultiple coefficient of determination (R2) was usedas a measure of the goodness of fit of each model. Pairwise dot plots were alsoused to examine changes in important parameters between treatments. All dataanalyses were done in SPSS 7.5 on a Gateway 2000 E-3000 computer running theWindows 95 operating system. The percentage absorption of dietary cholesterol for each subject on thedifferent diets is shown in Fig.1. For the group, neither dietary cholesterol nor dietary fatsignificantly altered the percentage dietary cholesterol absorption. It is important tonote, however, that regardless of diet type, the individuals within the groupdiffered markedly in the percentage dietary cholesterol absorption. For example, onthe LFLC diet percentage dietary cholesterol absorption varied from 36 to 74%. Nosignificant relationships were found between apoE genotype and cholesterolabsorption rates. Individual changes in percentage dietary cholesterol absorption in responseto dietary cholesterol are shown in Fig.2. The responsiveness of percentage dietary cholesterolabsorption varied markedly among individuals with some subjects increasing, somemaintaining, and others decreasing their values in response to dietary cholesterol.Similar variability characterized the responsiveness of percentage dietarycholesterol absorption to dietary fat (data not shown). For the group as a whole, the plasma lipid and lipoprotein levels on eachdiet and the isolated effects of increasing dietary cholesterol (HFHC–HFLC)and dietary fat (HFLC–LFLC) are shown in Table 2. Dietary cholesterol increased LDL-C levelsby 11.6 ± 14.9 mg/dl, whereas dietary fat increased LDL-C 6.8 ± 7.3mg/dl. There was no significant effect of dietary cholesterol on HDL-C levels, butincreased dietary fat increased HDL-C 5.2 ± 5.3 mg/dl. Finally, dietarycholesterol and dietary fat did not significantly change triglyceride or VLDL-Clevels. Thus dietary cholesterol and dietary fat were shown to have differenteffects on the plasma lipoprotein pattern.TABLE 2Effect of diets and isolated effects of dietary fat and dietarycholesterol on plasma lipids and lipoprotein levelsLFLCHFLCHFHCHFLC-LFLC (Fat)HFHC-HFLC (Cholesterol)mg/dlTC153.6 ± 19.2166.6 ± 24.1177.7 ± 25.6bP < 0.01 versus LFLC.12.9 ± 9.111.7 ± 18.7TG80.2 ± 26.884.0 ± 27.877.5 ± 23.2VLDL-C19.3 ± 7.419.3 ± 8.718.7 ± 5.9LDL-C87.9 ± 18.394.7 ± 21.9106.1 ± 23.2aP < 0.05 versus HFLC.,bP < 0.01 versus LFLC.6.8 ± 7.311.6 ± 14.9HDL-C46.9 ± 11.953.2 ± 13.052.1 ± 13.55.2 ± 5.31.2 ± 4.4Value are given as mean ± SD. (HFLC-LFLC) and (HFHC-HFLC) arethe changes in lipid/lipoprotein levels in response to dietary fatand dietary cholesterol, respectively.a P < 0.05 versus HFLC.b P < 0.01 versus LFLC. Open table in a new tab Value are given as mean ± SD. (HFLC-LFLC) and (HFHC-HFLC) arethe changes in lipid/lipoprotein levels in response to dietary fatand dietary cholesterol, respectively. Individual changes in LDL-C levels in response to dietary cholesterol areshown in Fig. 2. Like dietary cholesterolabsorption, the LDL-C responsiveness varied markedly among individuals with somesubjects increasing, some maintaining, and others decreasing their LDL-C in responseto dietary cholesterol. Similar variability characterized the responsiveness ofLDL-C to dietary fat (data not shown). Next, possible relationships were sought between percentage dietarycholesterol absorption and LDL-C levels. During none of the diet periods was there arelationship between these variables (data not shown). However, there are manyfactors that might influence LDL-C levels aside from dietary cholesterol absorptionand these might obscure a possible relationship. Therefore, to minimize the effect of other variables, a relationship was sought between dietarycholesterol-induced changes in percentage dietary cholesterol absorption and percentchange in LDL-C levels. To accomplish this, for each subject the value of LDL-C onthe HFLC diet was subtracted from the values on the HFHC diet and the difference wasexpressed as percent change after normalization to the value on the HFLC diet.Individual changes in cholesterol absorption were calculated by subtracting thepercentage absorption on HFLC from the percentage absorption on HFHC diet. As shownin Fig. 3, for the 18 subjects inthe study, the plot of dietary cholesterol-induced change in percentage dietarycholesterol absorption versus percent change in LDL-C level reveals a non-linearrelationship. A U-shaped parabolic curve, which best describes the relationship,indicates a high coefficient of multiple determination between the variables(R2 = 0.62, P = 0.005). This suggests that almosttwo-thirds of the variation in the dietary cholesterol-induced change in LDL-C isexplained by the dietary cholesterol-induced change in percentage dietarycholesterol absorption. Further analysis of this relationship suggests twopopulations of individuals. Analyzing the data for the top and bottom half of thedietary cholesterol-induced changes in percentage dietary cholesterol absorption, asshown in the Fig. 3 insets, revealedsignificant linear correlations in the top and bottom groups, r =0.71; P < 0.04 and r = −0.82;P < 0.007, respectively. To determine whether theU-shaped curve is influenced by outliers, we removed the subject with a 50% change in LDL-C and the subjectthat reduced his LDL-C by over 10%. The new analysis resulted in R2 value of 0.46 and P = 0.019, suggesting that theU-shape relationship is not heavily influenced by these outliers. Analysis ofdietary cholesterol-induced change in percentage dietary cholesterol absorptionversus percent change in HDL-C levels also revealed a parabolic relationship, butthis was weaker with an R2 = 0.38, P = 0.05 explaining a smaller proportion ofthe variation in HDL-C responsiveness (Fig.4). In addition, the dietary cholesterol-induced changes inLDL-C and HDL-C were correlated (r = 0.48, P< 0.04, data not shown). Finally, no significant relationships were foundbetween dietary fat-induced changes in percentage dietary cholesterol absorption anddietary fat-induced changes in LDL-C or HDL-C levels.Fig. 4Relationships of changes in HDL-C and cholesterol absorption rates inresponse to dietary cholesterol. The parabolic relationship wascharacterized by R2 = 0.38 and P = 0.05.View Large Image Figure ViewerDownload (PPT) Differences among individuals in the mass of dietary cholesterol absorbed(mg dietary cholesterol/kg body weight per day) under similar dietary constraintswere also examined. This was assessed by multiplying the cholesterol in the diet(mg/kcal per day) by the daily kcal ingested by the percentage dietary cholesterolabsorption. As shown in Fig. 5A,on the HFLC diet an average of 1.7 ± 0.4 mg cholesterol/kg per day wasabsorbed with a range from 1 to 2.6 mg cholesterol/kg per day. On the HFHC diet, anaverage of 4.6 ± 1.2 mg dietary cholesterol/kg per day was absorbed with arange from 2 to 6.6 mg dietary cholesterol/kg per day. In switching from the HFLC tothe HFHC diet in different individuals, the range of increase in cholesterolabsorption was from 1 to 4.7 mg dietary cholesterol/kg per day. Dietary fat alsoaffected the mass absorption of dietary cholesterol. As shown in Fig. 5B, most individuals decreased but somemaintained and others increased their dietary cholesterol mass absorption. Moreover,the subgroup of 13 subjects that responded to dietary fat by decreased dietarycholesterol mass absorption was characterized by a strong and inverse relationshipbetween fat intake (gm/day) and the decrease in dietary cholesterol absorbed per kgbody weight per day (r = −0.77, P< 0.003; Fig. 6). Theseresults suggest independent effects of dietary cholesterol and dietary fat ondietary cholesterol mass absorption and remarkable interindividual variation in dietary cholesterol mass absorption in responseto dietary fat and dietary cholesterol.Fig. 6Relationships of dietary cholesterol mass absorption to fat intake.Displayed are the values in a subset of 13 subjects that responded todietary cholesterol by decreased dietary cholesterol mass absorption(r = −0.77, P <0.003).View Large Image Figure ViewerDownload (PPT) In the current study we have shown: i) markedinterindividual variability in the effect of dietary cholesterol on percentagedietary cholesterol absorption and LDL-C levels; ii) a relationshipbetween dietary cholesterol-induced changes in the percentage of dietary cholesterolabsorbed and changes in LDL-C and HDL-C levels, best characterized by U-shapedparabolic curves; iii) dietary cholesterol and dietary fatindependently affect dietary cholesterol mass absorption; iv) insubjects that decreased their dietary cholesterol mass absorption in response todietary fat, the decrease was strongly related to their fat intake; and v)the increase in dietary cholesterol mass absorption i