Cholesterol Feeding Increases C-Reactive Protein and Serum Amyloid A Levels in Lean Insulin-Sensitive Subjects
http://www.100md.com
《循环学杂志》
the Department of Medicine (L.R.T., K.D.O., R.H.K., B.R., B.F., M.H.W., S.E.K., A.C.) University of Washington, Seattle
the Veterans Administration Puget Sound Health Care System (S.E.K.), Seattle, Wash. L.R.T. is currently at the Department of Medicine, University of Kentucky, Lexington.
Abstract
Background— Inflammatory markers associated with elevated cardiovascular risk are increased by cholesterol feeding in animal models. However, whether dietary cholesterol increases inflammatory marker levels in humans is not known.
Methods and Results— C-reactive protein (CRP), serum amyloid A (SAA), and lipoprotein levels were compared in 201 healthy subjects on an American Heart Association–National Cholesterol Education Program step 1 diet at baseline and after addition of 4 eggs per day for 4 weeks. Subjects were classified a priori into 3 groups based on their body mass index (BMI) and insulin sensitivity index (SI): lean insulin sensitive (LIS), mean±SEM BMI, 23.2±0.3 kg/m2, and SI, 6.7±0.3x10–4min–1/(μU/mL), n=66; lean insulin resistant (LIR), BMI, 24.5±0.2 kg/m2 and SI, 2.9±0.1x10–4min–1/(μU/mL), n=76; or obese insulin resistant (OIR), BMI, 31.4±0.5 kg/m2 and SI, 2.1±0.1x10–4min–1/(μU/mL), n=59. Insulin resistance and obesity each were associated with increased baseline levels of both CRP (P for trend, <0.001) and SAA (P for trend=0.015). Egg feeding was associated with significant increases in both CRP and SAA in the LIS group (both P<0.01) but not in the LIR or OIR groups. Egg feeding also was associated with a significant increase in non-HDL cholesterol (P<0.001) in LIS subjects; however, there was no correlation between the change in non-HDL cholesterol or changes in either CRP or SAA in this group.
Conclusions— A high-cholesterol diet leads to significant increases in both inflammatory markers and non-HDL cholesterol levels in insulin-sensitive individuals but not in lean or obese insulin-resistant subjects.
Key Words: cholesterol ; C-reactive protein ; insulin resistance ; lipoproteins ; serum amyloid A
Introduction
Inflammation is a hallmark of atherosclerosis. Associations exist between increased risk of cardiovascular events and elevated levels of inflammatory markers such as C-reactive protein (CRP)1–4 and serum amyloid A (SAA).5–7 In addition, both CRP and SAA levels are increased with obesity and with insulin resistance.8–14
Although a high intake of dietary cholesterol also is associated with increased cardiovascular risk,15,16 the effect of dietary cholesterol on plasma lipid levels varies substantially17 and is influenced by the presence of lipid disorders,18,19 as well as by obesity and insulin resistance.20 Dietary cholesterol also increases SAA in hyperlipidemic mice.21 However, it is not known whether dietary fat and cholesterol affect CRP and SAA levels in humans or whether the response is influenced by obesity and/or insulin resistance. Therefore, we studied the effects of egg feeding on CRP and SAA levels in a cohort of healthy, community-dwelling individuals stratified according to their degree of obesity and insulin resistance. We report here the surprising finding that high dietary cholesterol, in the form of 4 eggs per day added to the diet, increased CRP and SAA levels in lean, insulin-sensitive only subjects but not in obese or lean insulin-resistant subjects.
Methods
Subjects
Subjects (n=201) had participated previously in a study examining the effect of insulin resistance and/or obesity on lipid and lipoprotein responses to egg feeding.20 As described previously, all subjects had baseline measurements of height; weight; waist-hip ratio (WHR); fasting plasma levels of glucose, insulin, cholesterol (total, LDL, and HDL), triglycerides, and apolipoprotein B; abdominal and subcutaneous fat (from a computed tomographic image taken at the level of the umbilicus); and insulin sensitivity index (SI) (with use of the minimal model of glucose kinetics from the results of a frequently sampled, tolbutamide-modified, intravenous, glucose-tolerance test.20 The study was approved by the University of Washington Human Subjects Review Committee, and all subjects gave written, informed consent.
Subjects were divided into 3 a priori–defined groups: lean insulin-sensitive (LIS); lean insulin-resistant (LIR); or obese insulin-resistant (OIR). Body mass index (BMI) was used to classify patients as lean (BMI<27.5 kg/m2) or obese (BMI27.5 kg/m2). The BMI cut point was based on criteria used in the National Health and Nutrition Examination Survey II, which did not include the current "overweight" category. Thus, both lean and obese groups of the present study contain some individuals who would be classified as overweight under current criteria. Baseline measurement of SI was used to classify patients as insulin sensitive [SI4.2 (10–4 min–1 (μU/mL)] or insulin resistant [SI4.2x10–4 min–1/(μU/mL)]. SI measurements were not repeated at the end of the study.
Diets
All subjects were counseled on the National Cholesterol Education Program step 1 diet, and compliance was evaluated periodically. Subjects ingested 0, 2, or 4 eggs/d for 4-week periods, in random order, with each intervention period separated by a 4-week "washout" period during which the subjects consumed only the National Cholesterol Education Program step 1 diet.20 Maintenance of stable weight was emphasized. Subjects were given individual frozen daily portions of egg preparation (homogenized natural eggs). The 4-egg preparation consisted of 68 g egg yolk, 20 g Egg Beaters egg substitute, and 20 g water and provided 253 kcal, 13.4 g protein, 21 g fat, 871 mg cholesterol, and 6.5 g saturated fat. Three-day food records were collected at the beginning and end of each intervention period, and the records were analyzed to demonstrate consistency of dietary intake throughout the study. The present study evaluated measurements from before and after the 4 egg/d visits.
Inflammatory Marker Assays
CRP (high-sensitivity assay) and SAA levels were determined on deeply frozen (–80°C), not previously thawed samples by a nephelometric method. Interleukin (IL)-1;, IL-6, IL-8, and tumor necrosis factor (TNF)- were measured simultaneously with a multiplex bead system on a Luminex analyzer. Subjects with values too low to be detected by the assays were assigned a value of 50% of the assay’s lowest detectable value (for CRP, 0.08 mg/L; for SAA, 0.4 mg/L; for IL-1;, 1.6 pg/mL; and for TNF-, 1.6 pg/mL; no values for IL-6 or IL-8 were below the lower limit of detection).
Statistics
Data are presented as mean±SEM unless otherwise specified. CRP, SAA, and cytokines are presented as medians and interquartile ranges. Tests for significant differences among the 3 groups were performed by 2-way ANOVA evaluating the effect of group and sex, with pairwise multiple comparisons made with the Holm-Sidak method. The effects of egg feeding on various parameters were analyzed by paired t test when the variables were satisfactory for parametric tests or by Wilcoxon signed-rank tests. Lipid values were normally distributed. Because CRP and SAA values were not normally distributed, regression analyses with these variables were performed with logarithmically (natural) transformed values. Four subjects (all in OIR) had baseline CRP values >10 mg/L, and 5 subjects (1 each in LIS and LIR and 3 in OIR) had subsequent CRP values >10 mg/L; CRP values >10 mg/L generally are considered to indicate clinically relevant inflammation. However, because exclusion of these subjects did not affect results, all subjects were included in analyses. All figures depict raw (untransformed) data. The strength of associations of CRP and SAA (dependent variables) with lipoprotein variables (independent variables) was evaluated by linear regression on each variable separately, unadjusted for covariates. Data were considered significant at (2 sided) P<0.05.
Results
Subject Characteristics
The present study includes 201 subjects who completed the 4 egg/d intervention (Table). The LIS group was slightly younger than the LIR group. By definition, body weight and BMI were greatest in the OIR group. There was an incremental increase in subcutaneous fat, intra-abdominal fat, and WHR among the 3 groups, with the greatest increment in the OIR group over the LIR group. Insulin sensitivity, by definition, was greatest in the LIS group, although there was a small but significant difference between the LIR and OIR groups. Non-HDL cholesterol and triglycerides were significantly lower in the LIS group compared with either the LIR or OIR group (Table). HDL cholesterol was highest in the LIS group, but HDL also was higher in the LIR group compared with the OIR group (Table).20 Significant effects of sex were found for weight, WHR, subcutaneous and intra-abdominal fat areas, glucose, CRP, SAA, and HDL. However, there were no significant interactions between group and sex for CRP or SAA; thus, the observed differences in CRP and SAA among groups are not accounted for by sex effects. The data for age, BMI, subcutaneous and intra-abdominal fat areas, and non-HDL and HDL cholesterol levels did have significant interactions between group and sex. However, the differences in mean values for these factors (age, BMI, subcutaneous and intra-abdominal fat areas, and non-HDL and HDL cholesterol levels) among the different groups remained significant after allowing for the effects of sex.
Subject Characteristics
Insulin resistance was associated with higher CRP levels (P<0.001 for LIS versus LIR), as was obesity (P<0.001 for LIR versus OIR and for LIS versus OIR; Table), as has been reported previously.8–14 Similarly, insulin resistance and obesity were associated with higher SAA levels (P<0.001 for LIS versus OIR). Although there was a trend toward higher levels of cytokines with increased insulin resistance and obesity, there were no significant differences for any cytokine measures between groups (P values [1-way ANOVA]: IL-1;, 0.27; IL-6, 0.10; IL-8, 0.46; and TNF-, 0.14).
Egg Feeding and Changes in CRP and SAA
CRP and SAA levels from before and after addition of 4 eggs/d to the diet were compared. Although baseline levels of both CRP and SAA were higher in the LIR and OIR groups than in the LIS group, egg feeding was associated with a significant increase in levels of both CRP and SAA in the LIS group alone (Figure 1). Within the LIS group and for all subjects, the change in CRP was highly correlated with the change in SAA (LIS group only, r=0.955, P<0.001; all subjects combined, r=0.754, P<0.001). Body weight did not change with egg feeding (P=NS; data not shown).
Egg Feeding and Changes in Lipoproteins
Egg feeding increased non-HDL cholesterol only in the LIS group (Figure 2). As has been reported previously for cholesterol feeding,20,22–24 egg feeding was associated with significant increases in HDL cholesterol in all 3 groups. Although triglycerides tended to decrease slightly in all 3 groups, none of these changes reached statistical significance.
Lack of Correlation Between Changes in CRP or SAA and Changes in Lipoproteins
Because egg feeding led to significant increases in CRP and SAA as well as lipid variables in the LIS group, regression analysis was performed to evaluate whether individual changes in non-HDL cholesterol predicted changes in CRP or SAA levels. However, within the LIS group, the change in non-HDL cholesterol was not correlated with changes in either CRP (r=–0.04, P=0.77) or SAA (r=–0.03, P=0.82). Also, although egg feeding was associated with significant increases in HDL cholesterol in all 3 groups, there were no correlations between changes in CRP and changes in HDL cholesterol in any group (LIS, r=–0.001, P=0.99; LIR, r=0.002, P=0.99; and OIR, r=–0.12, P=0.35). Similarly, there were no significant correlations between changes in SAA and changes in HDL cholesterol in any group (LIS, r=0.05, P=0.71; LIR, r=0.13, P=0.26; and OIR: r=–0.09, P=0.51).
Discussion
In this study, we found that among healthy, community-dwelling individuals, egg feeding was associated with significant increases in CRP and SAA levels in LIS subjects. In addition, egg feeding was associated with a significant increase in non-HDL cholesterol in the same subjects. Moreover, because the change in non-HDL cholesterol was not correlated with a change in either CRP or SAA, a lack of change in non-HDL cholesterol could not be taken to indicate a lack of increase in either of these 2 inflammatory markers.
Previous studies have shown relations between CRP and obesity and, taken together, they generally suggest that CRP is largely determined by BMI and/or total fat mass.8,25–27 Insulin sensitivity also has been shown to be independently associated with CRP.28 Our data confirm that a higher BMI and reduced insulin sensitivity are associated with higher CRP levels and also demonstrate that a higher BMI and reduced insulin sensitivity are associated with higher levels of SAA.
The present study found that in subjects who were nonobese and insulin sensitive, egg feeding had effects on inflammatory markers similar to those seen in mice with genetic dyslipidemias29 and dietary cholesterol feeding.21 In contrast, egg feeding did not have a significant effect on CRP or SAA levels in human subjects who were obese and/or insulin resistant, ie, groups whose inflammatory marker levels were already elevated at baseline. In addition, the effect on lipoprotein levels of adding dietary cholesterol was much less pronounced in obese and/or insulin-resistant subjects. Although this finding may seem surprising, previous studies have demonstrated that cholesterol absorption may be inhibited in patients who are either obese or insulin resistant.30,31 In addition, a recent study of weight loss diets found that among obese women randomized to a high-fat, cholesterol-rich, Atkins-type diet, CRP and SAA did not rise but rather decreased in proportion to the amount of weight lost.32 Obesity is associated with hypersecretion of biliary cholesterol,33,34 which may inhibit dietary cholesterol absorption through competition for micellar solubilization and membrane uptake.35 Thus, we hypothesize that decreased cholesterol absorption associated with obesity may account, in part, for the relative lack of an adverse effect of dietary cholesterol on inflammatory markers and lipoprotein levels in the LIR and OIR groups.
Similar to what has been found in previous studies, dietary cholesterol was associated with increased HDL cholesterol, which has been associated with a lower cardiovascular disease risk in epidemiological studies. However, it is not clear that increases in HDL cholesterol induced by dietary cholesterol feeding are necessarily beneficial. First, many studies have demonstrated that high dietary cholesterol intake is associated with increased cardiovascular risk, despite the ability of dietary cholesterol to increase HDL cholesterol. Second, dietary cholesterol increases SAA levels, at least in LIS subjects, and SAA is carried on HDL cholesterol particles.36,37 Several recent studies have demonstrated that increased SAA levels are associated with increased cardiovascular risk.1,5,6 Moreover, in vitro and animal studies have demonstrated that SAA-containing HDL binds more avidly to arterial wall proteoglycans21,29 and that SAA levels are correlated with atherosclerotic lesion size in LDL receptor–deficient mice.21 Thus, at least some of the HDL cholesterol increase seen with dietary cholesterol feeding is caused by an increase in particles that contain SAA, a protein associated with increased atherosclerosis.
Unlike some other authors,13,27,38 we failed to find any relation between cytokines (IL-1;, IL-6, IL-8, and TNF-) and measures of obesity or insulin resistance. We did not find a relation between these cytokines and CRP or SAA, nor did circulating cytokine levels increase with cholesterol feeding. This may in part be related to sample size. Alternatively, it may be that the effect of dietary cholesterol on inflammatory markers is independent of circulating cytokines.
It is interesting to note that, as reported previously for LDL cholesterol levels, the LIS group had the most significant changes in non-HDL cholesterol levels with egg feeding.20 However, we were unable to demonstrate a correlation between individual changes in non-HDL cholesterol and changes in either CRP or SAA in LIS subjects. LIS subjects showed greater adverse effects of egg feeding with respect to a number of markers of cardiovascular risk compared with LIR or OIR subjects, although the levels attained were not as high as the baseline levels in LIR or OIR subjects. These findings are consistent with the hypothesis that in LIS subjects, a diet high in cholesterol might induce inflammatory stress similar to that observed with abdominal obesity and insulin resistance.
Acknowledgments
This work was supported in part by a medical school grant from Merck; the Medical Research Service of the Department of Veterans Affairs; a grant from the American Egg Board; and National Institutes of Health grants DK02456, DK17047, and DK35816 from the National Institute of Diabetes, Digestive, and Kidney Diseases; HL30086 from the National Heart, Lung, and Blood Institute; and RR00037 from the National Center for Research Resources. The authors thank Margaret Mayes for her expert technical assistance and Dr John Brunzell for his thoughtful comments during the preparation of this manuscript.
Disclosure
Alan Chait received a grant from the Merck Medical School grant program, and Robert Knopp and Steven Kahn received a grant from the American Egg Board to support this work. The study sponsors had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the manuscript for publication.
Footnotes
Presented in part at the 76th Scientific Sessions of the American Heart Association, Orlando, Fla, November 12, 2003, and published in abstract form (Circulation. 2003;108[suppl IV]:IV-784).
References
Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1997; 336: 973–979.
Kuller LH, Tracy RP, Shaten J, Meilahn EN. Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study: Multiple Risk Factor Intervention Trial. Am J Epidemiol. 1996; 144: 537–547.
Koenig W, Sund M, Frohlich M, Fischer HG, Lowel H, Doring A, Hutchinson WL, Pepys MB. C-reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men: results from the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992. Circulation. 1999; 99: 237–242.
Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002; 347: 1557–1565.
Delanghe JR, Langlois MR, De Bacquer D, Mak R, Capel P, Van Renterghem L, De Backer G. Discriminative value of serum amyloid A and other acute-phase proteins for coronary heart disease. Atherosclerosis. 2002; 160: 471–476.
Johnson BD, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, Shaw LJ, Pepine CJ, Sharaf B, Merz CNB, Sopko G, Olson MB, Reis SE. Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women: the National Heart, Lung, and Blood Institute-sponsored Women’s Ischemia Syndrome Evaluation (WISE). Circulation. 2004; 109: 726–732.
Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med. 2004; 350: 1387–1397.
Visser M, Bouter LM, McQuillan GM, Wener MH, Harris TB. Elevated C-reactive protein levels in overweight and obese adults. JAMA. 1999; 282: 2131–2135.
Uhlar CM, Whitehead AS. Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem. 1999; 265: 501–523.
Frohlich M, Imhof A, Berg G, Hutchinson WL, Pepys MB, Boeing H, Muche R, Brenner H, Koenig W. Association between C-reactive protein and features of the metabolic syndrome: a population-based study. Diabetes Care. 2000; 23: 1835–1839.
Lemieux I, Pascot A, Prud’homme D, Almeras N, Bogaty P, Nadeau A, Bergeron J, Despres JP. Elevated C-reactive protein: another component of the atherothrombotic profile of abdominal obesity. Arterioscler Thromb Vasc Biol. 2001; 21: 961–967.
McLaughlin T, Abbasi F, Lamendola C, Liang L, Reaven G, Schaaf P, Reaven P. Differentiation between obesity and insulin resistance in the association with C-reactive protein. Circulation. 2002; 106: 2908–2912.
Escobar-Morreale HF, Villuendas G, Botella-Carretero JI, Sancho J, San Millan JL. Obesity, and not insulin resistance, is the major determinant of serum inflammatory cardiovascular risk markers in pre-menopausal women. Diabetologia. 2003; 46: 625–633.
Leinonen E, Hurt-Camejo E, Wiklund O, Hulten LM, Hiukka A, Taskinen MR. Insulin resistance and adiposity correlate with acute-phase reaction and soluble cell adhesion molecules in type 2 diabetes. Atherosclerosis. 2003; 166: 387–394.
Keys A, Aravanis C, Vanbuchem FSP, Blackburn H, Buzina R, Djordjevic BS, Dontas AS, Fidanza F, Karvonen MJ, Kimura N, Menotti A, Nedeljkovic S, Puddu V, Punsar S, Taylor HL. The diet and all-causes death rate in the 7 Countries Study. Lancet. 1981; 2: 58–61.
Marmot MG, Syme SL, Kagan A, Kato H, Cohen JB, Belsky J. Epidemiologic studies of coronary heart disease and stroke in Japanese men living in Japan, Hawaii and California: prevalence of coronary and hypertensive heart disease and associated risk factors. Am J Epidemiol. 1975; 102: 514–525.
Jacobs DR Jr, Anderson JT, Hannan P, Keys A, Blackburn H. Variability in individual serum cholesterol response to change in diet. Arteriosclerosis. 1983; 3: 349–356.
Ordovas JM, Schaefer EJ. Genetic determinants of plasma lipid response to dietary intervention: the role of the APOA1/C3/A4 gene cluster and the APOE gene. Br J Nutr. 2000; 83 (suppl 1): S127–S136.
Knopp RH, Walden CE, Retzlaff BM, Mccann BS, Dowdy AA, Albers JJ, Gey GO, Cooper MN. Long-term cholesterol-lowering effects of 4 fat-restricted diets in hypercholesterolemic and combined hyperlipidemic men: the Dietary Alternatives Study. JAMA. 1997; 278: 1509–1515.
Knopp RH, Retzlaff B, Fish B, Walden C, Wallick S, Anderson M, Aikawa K, Kahn SE. Effects of insulin resistance and obesity on lipoproteins and sensitivity to egg feeding. Arterioscler Thromb Vasc Biol. 2003; 23: 1437–1443.
Lewis KE, Kirk EA, McDonald TO, Wang S, Wight TN, O’Brien KD, Chait A. Increase in serum amyloid A evoked by dietary cholesterol is associated with increased atherosclerosis in mice. Circulation. 2004; 110: 540–545.
Slater GG, Alfinslater RB. Effect of dietary cholesterol on plasma cholesterol, HDL cholesterol and triglycerides in human subjects. Fed Proc. 1978; 37: 630.
Lichtenstein AH, Ausman LM, Carrasco W, Jenner JL, Ordovas JM, Schaefer EJ. Hypercholesterolemic effect of dietary cholesterol in diets enriched in polyunsaturated and saturated fat: dietary cholesterol, fat saturation, and plasma lipids. Arterioscler Thromb. 1994; 14: 168–175.
Ginsberg HN, Karmally W, Siddiqui M, Holleran S, Tall AR, Blaner WS, Ramakrishnan R. Increases in dietary cholesterol are associated with modest increases in both LDL and HDL cholesterol in healthy young women. Arterioscler Thromb Vasc Biol. 1995; 15: 169–178.
Hak AE, Stehouwer CD, Bots ML, Polderman KH, Schalkwijk CG, Westendorp IC, Hofman A, Witteman JC. Associations of C-reactive protein with measures of obesity, insulin resistance, and subclinical atherosclerosis in healthy, middle-aged women. Arterioscler Thromb Vasc Biol. 1999; 19: 1986–1991.
Ford ES. Body mass index, diabetes, and C-reactive protein among US adults. Diabetes Care. 1999; 22: 1971–1977.
Connelly PW, Hanley AJ, Harris SB, Hegele RA, Zinman B. Relation of waist circumference and glycemic status to C-reactive protein in the Sandy Lake Oji-Cree. Int J Obes Relat Metab Disord. 2003; 27: 347–354.
Festa A, D’Agostino R, Howard G, Mykkanen L, Tracy RP, Haffner SM. Chronic subclinical inflammation as part of the insulin resistance syndrome: the Insulin Resistance Atherosclerosis Study (IRAS). Circulation. 2000; 102: 42–47.
O’Brien KD, McDonald TO, Kunjathoor V, Eng K, Knopp EA, Lewis K, Lopez R, Kirk EA, Chait A, Wight TN, deBeer FC, LeBoeuf RC. Serum amyloid A and lipoprotein retention in murine models of atherosclerosis. Arterioscler Thromb Vasc Biol. 2005; 25: 785–790.
Miettinen TA, Kesaniemi YA. Cholesterol absorption: regulation of cholesterol synthesis and elimination and within-population variations of serum cholesterol levels. Am J Clin Nutr. 1989; 49: 629–635.
Simonen P, Gylling H, Howard AN, Miettinen TA. Introducing a new component of the metabolic syndrome: low cholesterol absorption. Am J Clin Nutr. 2000; 72: 82–88.
O’Brien KD, Brehm BJ, Seeley RJ, Bean J, Wener MH, Daniels S, D’Alessio DA. Diet-induced weight loss is associated with decreases in plasma serum amyloid A and C-reactive protein independent of dietary macronutrient composition in obese subjects. J Clin Endocrinol Metab. 2005; 90: 2244–2249.
Bennion LJ, Grundy SM. Effects of obesity and caloric intake on biliary lipid metabolism in man. J Clin Invest. 1975; 56: 996–1011.
Shaffer EA, Small DM. Biliary lipid secretion in cholesterol gallstone disease: the effect of cholecystectomy and obesity. J Clin Invest. 1977; 59: 828–840.
Ros E. Intestinal absorption of triglyceride and cholesterol: dietary and pharmacological inhibition to reduce cardiovascular risk. Atherosclerosis. 2000; 151: 357–379.
Kisilevsky R, Subrahmanyan L. Serum amyloid A changes high density lipoprotein’s cellular affinity: a clue to serum amyloid A’s principal function. Lab Invest. 1992; 66: 778–785.
Malle E, De Beer FC. Human serum amyloid A (SAA) protein: a prominent acute-phase reactant for clinical practice. Eur J Clin Invest. 1996; 26: 427–435.
Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased adipose tissue expression of tumor necrosis factor- in human obesity and insulin resistance. J Clin Invest. 1995; 95: 2409–2415.(Lisa R. Tannock, MD; Kevi)
the Veterans Administration Puget Sound Health Care System (S.E.K.), Seattle, Wash. L.R.T. is currently at the Department of Medicine, University of Kentucky, Lexington.
Abstract
Background— Inflammatory markers associated with elevated cardiovascular risk are increased by cholesterol feeding in animal models. However, whether dietary cholesterol increases inflammatory marker levels in humans is not known.
Methods and Results— C-reactive protein (CRP), serum amyloid A (SAA), and lipoprotein levels were compared in 201 healthy subjects on an American Heart Association–National Cholesterol Education Program step 1 diet at baseline and after addition of 4 eggs per day for 4 weeks. Subjects were classified a priori into 3 groups based on their body mass index (BMI) and insulin sensitivity index (SI): lean insulin sensitive (LIS), mean±SEM BMI, 23.2±0.3 kg/m2, and SI, 6.7±0.3x10–4min–1/(μU/mL), n=66; lean insulin resistant (LIR), BMI, 24.5±0.2 kg/m2 and SI, 2.9±0.1x10–4min–1/(μU/mL), n=76; or obese insulin resistant (OIR), BMI, 31.4±0.5 kg/m2 and SI, 2.1±0.1x10–4min–1/(μU/mL), n=59. Insulin resistance and obesity each were associated with increased baseline levels of both CRP (P for trend, <0.001) and SAA (P for trend=0.015). Egg feeding was associated with significant increases in both CRP and SAA in the LIS group (both P<0.01) but not in the LIR or OIR groups. Egg feeding also was associated with a significant increase in non-HDL cholesterol (P<0.001) in LIS subjects; however, there was no correlation between the change in non-HDL cholesterol or changes in either CRP or SAA in this group.
Conclusions— A high-cholesterol diet leads to significant increases in both inflammatory markers and non-HDL cholesterol levels in insulin-sensitive individuals but not in lean or obese insulin-resistant subjects.
Key Words: cholesterol ; C-reactive protein ; insulin resistance ; lipoproteins ; serum amyloid A
Introduction
Inflammation is a hallmark of atherosclerosis. Associations exist between increased risk of cardiovascular events and elevated levels of inflammatory markers such as C-reactive protein (CRP)1–4 and serum amyloid A (SAA).5–7 In addition, both CRP and SAA levels are increased with obesity and with insulin resistance.8–14
Although a high intake of dietary cholesterol also is associated with increased cardiovascular risk,15,16 the effect of dietary cholesterol on plasma lipid levels varies substantially17 and is influenced by the presence of lipid disorders,18,19 as well as by obesity and insulin resistance.20 Dietary cholesterol also increases SAA in hyperlipidemic mice.21 However, it is not known whether dietary fat and cholesterol affect CRP and SAA levels in humans or whether the response is influenced by obesity and/or insulin resistance. Therefore, we studied the effects of egg feeding on CRP and SAA levels in a cohort of healthy, community-dwelling individuals stratified according to their degree of obesity and insulin resistance. We report here the surprising finding that high dietary cholesterol, in the form of 4 eggs per day added to the diet, increased CRP and SAA levels in lean, insulin-sensitive only subjects but not in obese or lean insulin-resistant subjects.
Methods
Subjects
Subjects (n=201) had participated previously in a study examining the effect of insulin resistance and/or obesity on lipid and lipoprotein responses to egg feeding.20 As described previously, all subjects had baseline measurements of height; weight; waist-hip ratio (WHR); fasting plasma levels of glucose, insulin, cholesterol (total, LDL, and HDL), triglycerides, and apolipoprotein B; abdominal and subcutaneous fat (from a computed tomographic image taken at the level of the umbilicus); and insulin sensitivity index (SI) (with use of the minimal model of glucose kinetics from the results of a frequently sampled, tolbutamide-modified, intravenous, glucose-tolerance test.20 The study was approved by the University of Washington Human Subjects Review Committee, and all subjects gave written, informed consent.
Subjects were divided into 3 a priori–defined groups: lean insulin-sensitive (LIS); lean insulin-resistant (LIR); or obese insulin-resistant (OIR). Body mass index (BMI) was used to classify patients as lean (BMI<27.5 kg/m2) or obese (BMI27.5 kg/m2). The BMI cut point was based on criteria used in the National Health and Nutrition Examination Survey II, which did not include the current "overweight" category. Thus, both lean and obese groups of the present study contain some individuals who would be classified as overweight under current criteria. Baseline measurement of SI was used to classify patients as insulin sensitive [SI4.2 (10–4 min–1 (μU/mL)] or insulin resistant [SI4.2x10–4 min–1/(μU/mL)]. SI measurements were not repeated at the end of the study.
Diets
All subjects were counseled on the National Cholesterol Education Program step 1 diet, and compliance was evaluated periodically. Subjects ingested 0, 2, or 4 eggs/d for 4-week periods, in random order, with each intervention period separated by a 4-week "washout" period during which the subjects consumed only the National Cholesterol Education Program step 1 diet.20 Maintenance of stable weight was emphasized. Subjects were given individual frozen daily portions of egg preparation (homogenized natural eggs). The 4-egg preparation consisted of 68 g egg yolk, 20 g Egg Beaters egg substitute, and 20 g water and provided 253 kcal, 13.4 g protein, 21 g fat, 871 mg cholesterol, and 6.5 g saturated fat. Three-day food records were collected at the beginning and end of each intervention period, and the records were analyzed to demonstrate consistency of dietary intake throughout the study. The present study evaluated measurements from before and after the 4 egg/d visits.
Inflammatory Marker Assays
CRP (high-sensitivity assay) and SAA levels were determined on deeply frozen (–80°C), not previously thawed samples by a nephelometric method. Interleukin (IL)-1;, IL-6, IL-8, and tumor necrosis factor (TNF)- were measured simultaneously with a multiplex bead system on a Luminex analyzer. Subjects with values too low to be detected by the assays were assigned a value of 50% of the assay’s lowest detectable value (for CRP, 0.08 mg/L; for SAA, 0.4 mg/L; for IL-1;, 1.6 pg/mL; and for TNF-, 1.6 pg/mL; no values for IL-6 or IL-8 were below the lower limit of detection).
Statistics
Data are presented as mean±SEM unless otherwise specified. CRP, SAA, and cytokines are presented as medians and interquartile ranges. Tests for significant differences among the 3 groups were performed by 2-way ANOVA evaluating the effect of group and sex, with pairwise multiple comparisons made with the Holm-Sidak method. The effects of egg feeding on various parameters were analyzed by paired t test when the variables were satisfactory for parametric tests or by Wilcoxon signed-rank tests. Lipid values were normally distributed. Because CRP and SAA values were not normally distributed, regression analyses with these variables were performed with logarithmically (natural) transformed values. Four subjects (all in OIR) had baseline CRP values >10 mg/L, and 5 subjects (1 each in LIS and LIR and 3 in OIR) had subsequent CRP values >10 mg/L; CRP values >10 mg/L generally are considered to indicate clinically relevant inflammation. However, because exclusion of these subjects did not affect results, all subjects were included in analyses. All figures depict raw (untransformed) data. The strength of associations of CRP and SAA (dependent variables) with lipoprotein variables (independent variables) was evaluated by linear regression on each variable separately, unadjusted for covariates. Data were considered significant at (2 sided) P<0.05.
Results
Subject Characteristics
The present study includes 201 subjects who completed the 4 egg/d intervention (Table). The LIS group was slightly younger than the LIR group. By definition, body weight and BMI were greatest in the OIR group. There was an incremental increase in subcutaneous fat, intra-abdominal fat, and WHR among the 3 groups, with the greatest increment in the OIR group over the LIR group. Insulin sensitivity, by definition, was greatest in the LIS group, although there was a small but significant difference between the LIR and OIR groups. Non-HDL cholesterol and triglycerides were significantly lower in the LIS group compared with either the LIR or OIR group (Table). HDL cholesterol was highest in the LIS group, but HDL also was higher in the LIR group compared with the OIR group (Table).20 Significant effects of sex were found for weight, WHR, subcutaneous and intra-abdominal fat areas, glucose, CRP, SAA, and HDL. However, there were no significant interactions between group and sex for CRP or SAA; thus, the observed differences in CRP and SAA among groups are not accounted for by sex effects. The data for age, BMI, subcutaneous and intra-abdominal fat areas, and non-HDL and HDL cholesterol levels did have significant interactions between group and sex. However, the differences in mean values for these factors (age, BMI, subcutaneous and intra-abdominal fat areas, and non-HDL and HDL cholesterol levels) among the different groups remained significant after allowing for the effects of sex.
Subject Characteristics
Insulin resistance was associated with higher CRP levels (P<0.001 for LIS versus LIR), as was obesity (P<0.001 for LIR versus OIR and for LIS versus OIR; Table), as has been reported previously.8–14 Similarly, insulin resistance and obesity were associated with higher SAA levels (P<0.001 for LIS versus OIR). Although there was a trend toward higher levels of cytokines with increased insulin resistance and obesity, there were no significant differences for any cytokine measures between groups (P values [1-way ANOVA]: IL-1;, 0.27; IL-6, 0.10; IL-8, 0.46; and TNF-, 0.14).
Egg Feeding and Changes in CRP and SAA
CRP and SAA levels from before and after addition of 4 eggs/d to the diet were compared. Although baseline levels of both CRP and SAA were higher in the LIR and OIR groups than in the LIS group, egg feeding was associated with a significant increase in levels of both CRP and SAA in the LIS group alone (Figure 1). Within the LIS group and for all subjects, the change in CRP was highly correlated with the change in SAA (LIS group only, r=0.955, P<0.001; all subjects combined, r=0.754, P<0.001). Body weight did not change with egg feeding (P=NS; data not shown).
Egg Feeding and Changes in Lipoproteins
Egg feeding increased non-HDL cholesterol only in the LIS group (Figure 2). As has been reported previously for cholesterol feeding,20,22–24 egg feeding was associated with significant increases in HDL cholesterol in all 3 groups. Although triglycerides tended to decrease slightly in all 3 groups, none of these changes reached statistical significance.
Lack of Correlation Between Changes in CRP or SAA and Changes in Lipoproteins
Because egg feeding led to significant increases in CRP and SAA as well as lipid variables in the LIS group, regression analysis was performed to evaluate whether individual changes in non-HDL cholesterol predicted changes in CRP or SAA levels. However, within the LIS group, the change in non-HDL cholesterol was not correlated with changes in either CRP (r=–0.04, P=0.77) or SAA (r=–0.03, P=0.82). Also, although egg feeding was associated with significant increases in HDL cholesterol in all 3 groups, there were no correlations between changes in CRP and changes in HDL cholesterol in any group (LIS, r=–0.001, P=0.99; LIR, r=0.002, P=0.99; and OIR, r=–0.12, P=0.35). Similarly, there were no significant correlations between changes in SAA and changes in HDL cholesterol in any group (LIS, r=0.05, P=0.71; LIR, r=0.13, P=0.26; and OIR: r=–0.09, P=0.51).
Discussion
In this study, we found that among healthy, community-dwelling individuals, egg feeding was associated with significant increases in CRP and SAA levels in LIS subjects. In addition, egg feeding was associated with a significant increase in non-HDL cholesterol in the same subjects. Moreover, because the change in non-HDL cholesterol was not correlated with a change in either CRP or SAA, a lack of change in non-HDL cholesterol could not be taken to indicate a lack of increase in either of these 2 inflammatory markers.
Previous studies have shown relations between CRP and obesity and, taken together, they generally suggest that CRP is largely determined by BMI and/or total fat mass.8,25–27 Insulin sensitivity also has been shown to be independently associated with CRP.28 Our data confirm that a higher BMI and reduced insulin sensitivity are associated with higher CRP levels and also demonstrate that a higher BMI and reduced insulin sensitivity are associated with higher levels of SAA.
The present study found that in subjects who were nonobese and insulin sensitive, egg feeding had effects on inflammatory markers similar to those seen in mice with genetic dyslipidemias29 and dietary cholesterol feeding.21 In contrast, egg feeding did not have a significant effect on CRP or SAA levels in human subjects who were obese and/or insulin resistant, ie, groups whose inflammatory marker levels were already elevated at baseline. In addition, the effect on lipoprotein levels of adding dietary cholesterol was much less pronounced in obese and/or insulin-resistant subjects. Although this finding may seem surprising, previous studies have demonstrated that cholesterol absorption may be inhibited in patients who are either obese or insulin resistant.30,31 In addition, a recent study of weight loss diets found that among obese women randomized to a high-fat, cholesterol-rich, Atkins-type diet, CRP and SAA did not rise but rather decreased in proportion to the amount of weight lost.32 Obesity is associated with hypersecretion of biliary cholesterol,33,34 which may inhibit dietary cholesterol absorption through competition for micellar solubilization and membrane uptake.35 Thus, we hypothesize that decreased cholesterol absorption associated with obesity may account, in part, for the relative lack of an adverse effect of dietary cholesterol on inflammatory markers and lipoprotein levels in the LIR and OIR groups.
Similar to what has been found in previous studies, dietary cholesterol was associated with increased HDL cholesterol, which has been associated with a lower cardiovascular disease risk in epidemiological studies. However, it is not clear that increases in HDL cholesterol induced by dietary cholesterol feeding are necessarily beneficial. First, many studies have demonstrated that high dietary cholesterol intake is associated with increased cardiovascular risk, despite the ability of dietary cholesterol to increase HDL cholesterol. Second, dietary cholesterol increases SAA levels, at least in LIS subjects, and SAA is carried on HDL cholesterol particles.36,37 Several recent studies have demonstrated that increased SAA levels are associated with increased cardiovascular risk.1,5,6 Moreover, in vitro and animal studies have demonstrated that SAA-containing HDL binds more avidly to arterial wall proteoglycans21,29 and that SAA levels are correlated with atherosclerotic lesion size in LDL receptor–deficient mice.21 Thus, at least some of the HDL cholesterol increase seen with dietary cholesterol feeding is caused by an increase in particles that contain SAA, a protein associated with increased atherosclerosis.
Unlike some other authors,13,27,38 we failed to find any relation between cytokines (IL-1;, IL-6, IL-8, and TNF-) and measures of obesity or insulin resistance. We did not find a relation between these cytokines and CRP or SAA, nor did circulating cytokine levels increase with cholesterol feeding. This may in part be related to sample size. Alternatively, it may be that the effect of dietary cholesterol on inflammatory markers is independent of circulating cytokines.
It is interesting to note that, as reported previously for LDL cholesterol levels, the LIS group had the most significant changes in non-HDL cholesterol levels with egg feeding.20 However, we were unable to demonstrate a correlation between individual changes in non-HDL cholesterol and changes in either CRP or SAA in LIS subjects. LIS subjects showed greater adverse effects of egg feeding with respect to a number of markers of cardiovascular risk compared with LIR or OIR subjects, although the levels attained were not as high as the baseline levels in LIR or OIR subjects. These findings are consistent with the hypothesis that in LIS subjects, a diet high in cholesterol might induce inflammatory stress similar to that observed with abdominal obesity and insulin resistance.
Acknowledgments
This work was supported in part by a medical school grant from Merck; the Medical Research Service of the Department of Veterans Affairs; a grant from the American Egg Board; and National Institutes of Health grants DK02456, DK17047, and DK35816 from the National Institute of Diabetes, Digestive, and Kidney Diseases; HL30086 from the National Heart, Lung, and Blood Institute; and RR00037 from the National Center for Research Resources. The authors thank Margaret Mayes for her expert technical assistance and Dr John Brunzell for his thoughtful comments during the preparation of this manuscript.
Disclosure
Alan Chait received a grant from the Merck Medical School grant program, and Robert Knopp and Steven Kahn received a grant from the American Egg Board to support this work. The study sponsors had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the manuscript for publication.
Footnotes
Presented in part at the 76th Scientific Sessions of the American Heart Association, Orlando, Fla, November 12, 2003, and published in abstract form (Circulation. 2003;108[suppl IV]:IV-784).
References
Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1997; 336: 973–979.
Kuller LH, Tracy RP, Shaten J, Meilahn EN. Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study: Multiple Risk Factor Intervention Trial. Am J Epidemiol. 1996; 144: 537–547.
Koenig W, Sund M, Frohlich M, Fischer HG, Lowel H, Doring A, Hutchinson WL, Pepys MB. C-reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men: results from the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992. Circulation. 1999; 99: 237–242.
Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002; 347: 1557–1565.
Delanghe JR, Langlois MR, De Bacquer D, Mak R, Capel P, Van Renterghem L, De Backer G. Discriminative value of serum amyloid A and other acute-phase proteins for coronary heart disease. Atherosclerosis. 2002; 160: 471–476.
Johnson BD, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, Shaw LJ, Pepine CJ, Sharaf B, Merz CNB, Sopko G, Olson MB, Reis SE. Serum amyloid A as a predictor of coronary artery disease and cardiovascular outcome in women: the National Heart, Lung, and Blood Institute-sponsored Women’s Ischemia Syndrome Evaluation (WISE). Circulation. 2004; 109: 726–732.
Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med. 2004; 350: 1387–1397.
Visser M, Bouter LM, McQuillan GM, Wener MH, Harris TB. Elevated C-reactive protein levels in overweight and obese adults. JAMA. 1999; 282: 2131–2135.
Uhlar CM, Whitehead AS. Serum amyloid A, the major vertebrate acute-phase reactant. Eur J Biochem. 1999; 265: 501–523.
Frohlich M, Imhof A, Berg G, Hutchinson WL, Pepys MB, Boeing H, Muche R, Brenner H, Koenig W. Association between C-reactive protein and features of the metabolic syndrome: a population-based study. Diabetes Care. 2000; 23: 1835–1839.
Lemieux I, Pascot A, Prud’homme D, Almeras N, Bogaty P, Nadeau A, Bergeron J, Despres JP. Elevated C-reactive protein: another component of the atherothrombotic profile of abdominal obesity. Arterioscler Thromb Vasc Biol. 2001; 21: 961–967.
McLaughlin T, Abbasi F, Lamendola C, Liang L, Reaven G, Schaaf P, Reaven P. Differentiation between obesity and insulin resistance in the association with C-reactive protein. Circulation. 2002; 106: 2908–2912.
Escobar-Morreale HF, Villuendas G, Botella-Carretero JI, Sancho J, San Millan JL. Obesity, and not insulin resistance, is the major determinant of serum inflammatory cardiovascular risk markers in pre-menopausal women. Diabetologia. 2003; 46: 625–633.
Leinonen E, Hurt-Camejo E, Wiklund O, Hulten LM, Hiukka A, Taskinen MR. Insulin resistance and adiposity correlate with acute-phase reaction and soluble cell adhesion molecules in type 2 diabetes. Atherosclerosis. 2003; 166: 387–394.
Keys A, Aravanis C, Vanbuchem FSP, Blackburn H, Buzina R, Djordjevic BS, Dontas AS, Fidanza F, Karvonen MJ, Kimura N, Menotti A, Nedeljkovic S, Puddu V, Punsar S, Taylor HL. The diet and all-causes death rate in the 7 Countries Study. Lancet. 1981; 2: 58–61.
Marmot MG, Syme SL, Kagan A, Kato H, Cohen JB, Belsky J. Epidemiologic studies of coronary heart disease and stroke in Japanese men living in Japan, Hawaii and California: prevalence of coronary and hypertensive heart disease and associated risk factors. Am J Epidemiol. 1975; 102: 514–525.
Jacobs DR Jr, Anderson JT, Hannan P, Keys A, Blackburn H. Variability in individual serum cholesterol response to change in diet. Arteriosclerosis. 1983; 3: 349–356.
Ordovas JM, Schaefer EJ. Genetic determinants of plasma lipid response to dietary intervention: the role of the APOA1/C3/A4 gene cluster and the APOE gene. Br J Nutr. 2000; 83 (suppl 1): S127–S136.
Knopp RH, Walden CE, Retzlaff BM, Mccann BS, Dowdy AA, Albers JJ, Gey GO, Cooper MN. Long-term cholesterol-lowering effects of 4 fat-restricted diets in hypercholesterolemic and combined hyperlipidemic men: the Dietary Alternatives Study. JAMA. 1997; 278: 1509–1515.
Knopp RH, Retzlaff B, Fish B, Walden C, Wallick S, Anderson M, Aikawa K, Kahn SE. Effects of insulin resistance and obesity on lipoproteins and sensitivity to egg feeding. Arterioscler Thromb Vasc Biol. 2003; 23: 1437–1443.
Lewis KE, Kirk EA, McDonald TO, Wang S, Wight TN, O’Brien KD, Chait A. Increase in serum amyloid A evoked by dietary cholesterol is associated with increased atherosclerosis in mice. Circulation. 2004; 110: 540–545.
Slater GG, Alfinslater RB. Effect of dietary cholesterol on plasma cholesterol, HDL cholesterol and triglycerides in human subjects. Fed Proc. 1978; 37: 630.
Lichtenstein AH, Ausman LM, Carrasco W, Jenner JL, Ordovas JM, Schaefer EJ. Hypercholesterolemic effect of dietary cholesterol in diets enriched in polyunsaturated and saturated fat: dietary cholesterol, fat saturation, and plasma lipids. Arterioscler Thromb. 1994; 14: 168–175.
Ginsberg HN, Karmally W, Siddiqui M, Holleran S, Tall AR, Blaner WS, Ramakrishnan R. Increases in dietary cholesterol are associated with modest increases in both LDL and HDL cholesterol in healthy young women. Arterioscler Thromb Vasc Biol. 1995; 15: 169–178.
Hak AE, Stehouwer CD, Bots ML, Polderman KH, Schalkwijk CG, Westendorp IC, Hofman A, Witteman JC. Associations of C-reactive protein with measures of obesity, insulin resistance, and subclinical atherosclerosis in healthy, middle-aged women. Arterioscler Thromb Vasc Biol. 1999; 19: 1986–1991.
Ford ES. Body mass index, diabetes, and C-reactive protein among US adults. Diabetes Care. 1999; 22: 1971–1977.
Connelly PW, Hanley AJ, Harris SB, Hegele RA, Zinman B. Relation of waist circumference and glycemic status to C-reactive protein in the Sandy Lake Oji-Cree. Int J Obes Relat Metab Disord. 2003; 27: 347–354.
Festa A, D’Agostino R, Howard G, Mykkanen L, Tracy RP, Haffner SM. Chronic subclinical inflammation as part of the insulin resistance syndrome: the Insulin Resistance Atherosclerosis Study (IRAS). Circulation. 2000; 102: 42–47.
O’Brien KD, McDonald TO, Kunjathoor V, Eng K, Knopp EA, Lewis K, Lopez R, Kirk EA, Chait A, Wight TN, deBeer FC, LeBoeuf RC. Serum amyloid A and lipoprotein retention in murine models of atherosclerosis. Arterioscler Thromb Vasc Biol. 2005; 25: 785–790.
Miettinen TA, Kesaniemi YA. Cholesterol absorption: regulation of cholesterol synthesis and elimination and within-population variations of serum cholesterol levels. Am J Clin Nutr. 1989; 49: 629–635.
Simonen P, Gylling H, Howard AN, Miettinen TA. Introducing a new component of the metabolic syndrome: low cholesterol absorption. Am J Clin Nutr. 2000; 72: 82–88.
O’Brien KD, Brehm BJ, Seeley RJ, Bean J, Wener MH, Daniels S, D’Alessio DA. Diet-induced weight loss is associated with decreases in plasma serum amyloid A and C-reactive protein independent of dietary macronutrient composition in obese subjects. J Clin Endocrinol Metab. 2005; 90: 2244–2249.
Bennion LJ, Grundy SM. Effects of obesity and caloric intake on biliary lipid metabolism in man. J Clin Invest. 1975; 56: 996–1011.
Shaffer EA, Small DM. Biliary lipid secretion in cholesterol gallstone disease: the effect of cholecystectomy and obesity. J Clin Invest. 1977; 59: 828–840.
Ros E. Intestinal absorption of triglyceride and cholesterol: dietary and pharmacological inhibition to reduce cardiovascular risk. Atherosclerosis. 2000; 151: 357–379.
Kisilevsky R, Subrahmanyan L. Serum amyloid A changes high density lipoprotein’s cellular affinity: a clue to serum amyloid A’s principal function. Lab Invest. 1992; 66: 778–785.
Malle E, De Beer FC. Human serum amyloid A (SAA) protein: a prominent acute-phase reactant for clinical practice. Eur J Clin Invest. 1996; 26: 427–435.
Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased adipose tissue expression of tumor necrosis factor- in human obesity and insulin resistance. J Clin Invest. 1995; 95: 2409–2415.(Lisa R. Tannock, MD; Kevi)