- Email: [email protected]
Comparison of fasting and postprandial plasma lipoproteins in subjects with and without coronary heart disease
Comparison of Fasting and Postprandial Plasma Lipoproteins in Subjects With and Without Coronary Heart Disease Ernst J. Schaefer, MD, Marie C. Audelin, MD, BSc, Judith R. McNamara, MT, Paulesh K. Shah, BSc, Timothy Tayler, BSc, Jennifer A. Daly, BSc, Joi L. Augustin, MS, Leo J. Seman, MD, PhD, and Joel J. Rubenstein, MD
RD,
Plasma lipoprotein levels, including remnant-like particle (RLP) cholesterol and RLP triglycerides, were assessed in fasting (12 hours) and postprandial (PP) (4 hours after a fat-rich meal) states in 88 patients with coronary heart disease (CHD) and 88 controls. All lipoproteins were assessed by direct methods. We hypothesized that patients with CHD would have greater percent increases in their triglyceride levels, RLP cholesterol, and RLP triglycerides, in response to a fat-rich meal. In the fasting state, triglycerides, RLP cholesterol, RLP triglycerides, and lowdensity lipoprotein (LDL) cholesterol levels were all significantly higher in cases versus controls by 51%, 35%, 39%, and 40%, respectively. These levels were 57%, 37%, 64%, and 37% higher in the PP state, respectively. Mean high-density lipoprotein (HDL) cholesterol values were 27% lower in cases in both the fasting and PP states. After eating, triglycerides, RLP cholesterol, and RLP triglycerides increased 64%, 71%, and 290% in
controls, respectively, whereas in cases these levels increased by 71%, 94%, and 340%, respectively (all p <0.0001). Percent increases in the PP state were not significantly different in cases versus controls. Following the fat-rich meal, LDL and HDL cholesterol decreased by 5% and 4% in controls, and by 7% and 6% in patients, with no significant difference in percent changes between groups. Fasting values correlated very highly with PP values for all parameters (all p <0.0001). Our data indicate that although patients with CHD have higher fasting and PP levels of triglycerides, RLP cholesterol, and RLP triglycerides than controls, the response (percent increase) to a fat-rich meal is comparable in both groups. Thus, a feeding challenge is not essential for assessment of these lipoproteins. Moreover, it is not necessary to obtain a fasting sample to assess direct LDL and HDL cholesterol. 䊚2001 by Excerpta Medica, Inc. (Am J Cardiol 2001;88:1129 –1133)
t has been well documented that triglyceride levels are an independent risk factor for coronary heart Idisease (CHD) after adjustment for other predictors,
lipoproteins, from the plasma, leaving behind partially metabolized triglyceride-rich lipoproteins of liver and intestinal origin. After precipitation, levels of cholesterol and triglycerides in the supranatant fraction, which contains these partially metabolized triglyceride-rich lipoproteins, can be measured.11–14 This study assessed plasma lipoprotein levels (including RLP), in CHD cases and controls in the fasting and postprandial (PP) states. Our hypothesis was that patients with CHD would have greater percent increases in their levels of triglycerides, RLP cholesterol, and RLP triglycerides than controls, in response to a fat-rich meal.
including high-density lipoprotein (HDL) cholesterol, especially in woman.1 Patients who have elevated remnant levels, which are formed after the metabolism of triglyceride-rich lipoproteins, also have an increased risk of CHD.2,3 Such patients may have type III hyperlipoproteinemia, or other more common states, such as familial dyslipidemia, diabetes mellitus, and renal disease.4 –10 A new assay has been developed to measure the remnant-like particle (RLP). It uses monoclonal antibodies specific to apolipoprotein (apo)A-I and apoB to remove low-density lipoprotein (LDL) and HDL, and newly formed triglyceride-rich From the Lipid and Heart Disease Prevention Program, New England Medical Center and Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, and Cardiology Division, Newton Wellesley Hospital, Newton, Massachusetts. This study was supported by contracts from Parke Davis/Pfizer, Morris Plains, New Jersey; Otsuka America Pharmaceutical, Inc., Rockville, Maryland; Schering-Plough Corporation, Kenilworth, New Jersey; grants HL 39326 and HL 57477 from the National Institutes of Health, Bethesda, Maryland; and contract 533K06-5-10 from the US Department of Agriculture Research Service, Washington, DC. Manuscript received May 2, 2001; revised manuscript received and accepted July 13, 2001. Address for reprints: Ernst J. Schaefer, MD, Lipid Metabolism Laboratory, Tufts University, 711 Washington St., Boston, Massachusetts 02111. E-mail: [email protected]. ©2001 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 88 November 15, 2001
METHODS
Study design and population: This is a substudy of a placebo-controlled randomized trial with a crossover design, aimed to assess the effect of statins on fasting and PP lipids in patients with CHD. For the main trial, 103 patients with CHD were recruited primarily from outpatient clinics (cardiology and lipid) of 2 medical centers from the Boston area, as well as by newspaper advertising. Of the 103 patients enrolled in the main trial, 88 also agreed to undergo a fat-rich meal challenge after an 8-week washout period and were included in the present substudy. For the present study, to compare with these cases, 88 control subjects matched for age (5-year age group) and sex, and free 0002-9149/01/$–see front matter PII S0002-9149(01)02047-1
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of clinical evidence of CHD, were enrolled provided that they met study entry criteria. Control subjects were recruited by newspaper advertising in the Boston area. Study subjects were predominantly Caucasians residing in the Boston area, mean age was 62 years, and 15% of the group were women. Inclusion and exclusions criteria: Patients were considered for participation in the main trial if they had documented CHD, based on either a history of myocardial infarction, coronary artery bypass grafting surgery or percutaneous coronary intervention, a coronary angiogram documenting significant narrowing (⬎50%) of ⱖ1 coronary arteries, or a positive stress test with nuclear imaging. Patients with CHD had to be free of any clinical event for a period of ⱖ6 months before enrollment and they had to agree to discontinuation of any lipid-lowering medication for a period of 6 weeks before the beginning of the study. Exclusion criteria were as stated in the protocol of the original main trial and were: age⬍20 or ⬎75 years; if female: premenopausal state or use of estrogen replacement therapy (to minimize potential confounders); plasma triglyceride levels ⬎400 mg/dl (4.5 mol/L); plasma LDL cholesterol values ⱕ130 mg/dl (3.4 mol/L); diabetes mellitus requiring insulin therapy or uncontrolled diabetes (hemoglobin A1c ⬎10%); severe obesity (body mass index ⬎35); active liver disease or hepatic dysfunction; acute or chronic pancreatitis; uncontrolled primary hypothyroidism, nephrotic syndrome, or renal dysfunction (blood urea nitrogen ⱖ30 mg/dl (10.5 mmol/L); creatinine ⱖ2.5 mg/dl (220 mol/L); or creatinine clearance ⬍30 ml/min. The same exclusion criteria were applied to control subjects; however, those with LDL cholesterol ⬍130 mg/dl were also accepted into the study. Subjects were maintained on all their nonlipid-lowering medications throughout the study, including  blockers and thiazide diuretics. Protocol: All subjects were studied after an overnight fast (12 hours) and 4 hours after a fat-rich meal. The meal was obtained from a popular restaurant chain (McDonald’s) and consisted of 2 sausages, 2 eggs, and 2 muffins (880 calories, 500 calories or 57% from fat, 180 calories or 20% from saturated fat, 510 mg of cholesterol). Information about smoking, height, weight, and systolic and diastolic blood pressures was collected at the time of first examination. Participants reporting smoking ⱖ1 cigarette per day during the preceding year were classified as smokers. Body mass index was calculated as weight in kilograms divided by the square of height in meters. Hypertension was defined as systolic or diastolic blood pressure ⬎140 or ⬎90 mm Hg, respectively, or use of antihypertensive medication. Diabetes was defined as fasting blood glucose ⬎125 mg/dl (6.85 mmol/L) or use of hypoglycemic medication. The Human Investigation Committee of the New England Medical Center and Tufts University approved the protocol, and informed consent was obtained from all subjects. 1130 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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Lipoproteins and remnant-like particle analysis:
Blood was drawn from each subject after a 12-hour overnight fast and 4 hours after a fat-rich meal. Plasma was separated by centrifugation (2,500 rpm, 4°C, 20 minutes). Plasma lipoprotein (total cholesterol, triglyceride, and HDL cholesterol) concentrations were measured fresh, using standard enzymatic methods, as previously described.15 LDL cholesterol was measured directly using a homogenous reagent kit obtained from Genzyme Diagnostics (Cambridge Massachusetts). Coefficients of variations were ⬍10%.16 RLP isolation was based on removal of apoA-I-containing particles (HDL) and most apoBcontaining particles (LDL, nascent very LDL [VLDL], and nascent chylomicrons), using an immunoseparation technique (Japan Immunoresearch Laboratories, Takasaki, Japan) as previously described,11–14 which has been shown to leave particles characteristic of VLDL remnants and chylomicron remnants in the unbound fraction.17,18 Monoclonal antibodies to apoA-I and apoB, which do not recognize partially hydrolyzed, apoE-enriched lipoprotein remnants, were immobilized on agarose gel. RLP cholesterol and RLP triglyceride concentrations were measured in plasma aliquots and stored at ⫺80°C until analysis. Thawed plasma was incubated with the gel for 2 hours, after which the gel, containing the bound (non-RLP) lipoproteins, was precipitated with lowspeed centrifugation (5 minutes, 135 g). RLP cholesterol and RLP triglyceride levels were then measured in the unbound supernates on an Abbott Spectrum CCx analyzer (Abbott Diagnostics, Irving, Texas), using 2-reagent enzymatic, colorimetric assays containing a sensitive chromophore (Kyowa Medex, Tokyo, Japan). Precision studies yielded a between-run RLP cholesterol coefficient of variation of 9.1% at 0.18 mmol/L (7 mg/dl) and 7.3% at 0.62 mmol/L (24 mg/dl). Between-run RLP triglyceride coefficients of variation were 8.3% at 0.25 mmol/L (22 mg/dl) and 5.0% at 1.23 mmol/L (109 mg/dl).13 Statistical analysis: Categorical variables are reported as frequency (in percent), and continuous variables as mean ⫾ SD. To compare categorical variables between both groups, we used a Fisher’s exact test. A 2-sample t test analysis was performed for continuous variables. When these variables were not normally distributed, they were log-transformed (total triglycerides, RLP cholesterol, RLP triglycerides). To assess the difference between fasting and PP values in each group, we used a paired t test analysis. Finally, for evaluation of the correlation between fasting and PP values for each lipoprotein category, we used a Pearson correlation coefficient test.
RESULTS Baseline characteristics of the study subjects are listed in the Table 1. Both groups were comparable for age, sex, height, and weight. There was a small but significant difference in body mass index, with patients having higher values than controls. Hypertension was also more prevalent among patients than controls, as was diabetes. Correlations between liNOVEMBER 15, 2001
in both the fasting and PP states (Table 3). After a fat-rich meal, total cholesterol and LDL cholesterol CHD were slightly reduced for controls and cases. However, the percent reductions in levels of total cholesCharacteristics 0 (n ⫽ 88) ⫹ (n ⫽ 88) p Value terol and LDL cholesterol were similar for both Age (yrs) (mean ⫾ SD) 62.0 ⫾ 8.6 61.8 ⫾ 9.4 NA groups. The patients with CHD also had lower HDL Women 15% 15% NA cholesterol values than the controls in both the fasting Diabetes mellitus 0% 5.7% 0.06 and the fed state, but had similar percent reductions in Systemic hypertension 30.9% 57.2% 0.01 Cigarette smokers 20.0% 17.2% NS the PP state. Height (cm) 173.9 ⫾ 9.1 171.6 ⫾ 9.1 NS For patients with established CHD, mean triglyc(mean ⫾ SD) eride levels were 51% higher in the fasting state; after Weight (kg) 79.4 ⫾ 15.2 81.2 ⫾ 12.8 NS a fat-rich meal these levels were 57% higher com(mean ⫾ SD) Body mass index 26.2 ⫾ 4.2 27.5 ⫾ 3.3 0.04 pared with levels in controls. The magnitude of the (mean ⫾ SD) difference was similar in both the fasting and fed states. The fat-rich meal caused a 64% increase in triglyceride levels in the control subjects and a 71% increase in the subjects with CHD. Between each TABLE 2 Correlation Between Fasting and Postprandial Values of Lipoproteins group the percent differences were not significantly different. RLP cholesterol levels were 35% higher in Controls Patients patients than in controls in the fasting state, and 37% Lipoprotein Pearson p Value Pearson p Value higher in the fed state. As for total triglycerides, Total cholesterol 0.9795 ⬍0.0001 0.9842 ⬍0.0001 differences in RLP cholesterol levels between patients LDL cholesterol 0.9501 ⬍0.0001 0.9709 ⬍0.0001 and controls were similar in the fasting and the fed HDL cholesterol 0.9875 ⬍0.0001 0.9651 ⬍0.0001 states. After a fat-rich meal, mean RLP cholesterol Triglycerides 0.9040 ⬍0.0001 0.8385 ⬍0.0001 increased by 71% in controls and 94% in patients. RPL cholesterol 0.7450 ⬍0.0001 0.9387 ⬍0.0001 Although the percent increase was somewhat greater RLP triglycerides 0.5294 ⬍0.0001 0.6166 ⬍0.0001 in cases, it was not statistically different from that in controls. With regard to RLP triglycerides, patients TABLE 3 Fasting and Postprandial Lipoprotein Concentrations for Coronary Heart had 39% and 64% higher values in Disease Patients and Controls the fasting state and the fed state, CHD respectively. The fat-rich meal resulted in a 290% increase in RLP Lipoprotein 0 (n ⫽ 88) ⫹ (n ⫽ 88) Difference (%) triglyceride values in the controls Fasting total cholesterol (⫾SD) (mg/dl) 215 ⫾ 45 280 ⫾ 70 ⫹30§ § and a 340% increase in the patients. 4-hr PP total cholesterol (⫾SD) (mg/dl) 211 ⫾ 45 272 ⫾ 66 ⫹29 As for total triglycerides and RLP PP change (%) ⫺2㛳 ⫺3¶ ⫺1* Fasting LDL cholesterol (⫾ SD) (mg/dl) 135 ⫾ 46 188 ⫾ 60 ⫹40§ cholesterol, the response of RLP tri4-hr PP LDL cholesterol (⫾ SD) (mg/dl) 127 ⫾ 42 174 ⫾ 57 ⫹37§ glycerides to the fat-rich meal was PP change (%) ⫺5¶ ⫺7¶ ⫺2* greater in patients than in controls; § Fasting HDL cholesterol (⫾ SD) (mg/dl) 51 ⫾ 15 37 ⫾ 9 ⫺27 but once again, the difference be4-hr PP HDL cholesterol (⫾ SD) (mg/dl) 48 ⫾ 14 35 ⫾ 9 ⫺27§ PP change (%) ⫺4¶ ⫺6¶ ⫺2† tween 2 groups was not statistically Fasting triglycerides (⫾ SD) (mg/dl) 130 ⫾ 82 196 ⫾ 84 ⫹51§ significant. 4-hr PP triglycerides (⫾ SD) (mg/dl) 207 ⫾ 119 325 ⫾ 136 ⫹57§ It should be noted that there was a ¶ ¶ PP change (%) ⫹64 ⫹71 ⫹8* wide degree of variability in these Fasting RLP cholesterol (⫾ SD) (mg/dl) 7.7 ⫾ 3.6 10.4 ⫾ 11.2 ⫹35‡ parameters, because the SDs are 4-hr PP RLP cholesterol (⫾ SD) (mg/dl) 12.5 ⫾ 6.2 17.1 ⫾ 11.9 ⫹37§ PP change (%) ⫹71¶ ⫹94¶ ⫹23* quite large. There was a wide variFasting RLP triglycerides (⫾ SD) (mg/dl) 28 ⫾ 33 39 ⫾ 26 ⫹39§ ability in the PP responses for RLP 4-hr PP RLP triglycerides (⫾ SD) (mg/dl) 87 ⫾ 65 143 ⫾ 86 ⫹64§ triglycerides and total triglycerides, ¶ ¶ PP change (%) ⫹290 ⫹340 ⫹50* as can be seen in Figure 1, where *p ⫽ NS; †p ⬍0.05; ‡p ⬍0.01; §p ⬍0.0001 (2-sample t test); 㛳p ⬍0.001; ¶p ⬍0.0001 (paired t test). percent changes from baseline to the PP state are shown for total triglycerides and RLP values. There was poprotein levels in the fasting and fed states are listed less variability for RLP cholesterol, with the exception in Table 2. Fasting triglycerides, RLP cholesterol, and of one extreme outlier in each group. RLP triglyceride levels were highly and significantly correlated with fed values in patients and controls. DISCUSSION Total cholesterol, LDL cholesterol, and HDL cholesIn the present study patients with CHD had higher terol values in the fasting state were also very highly fasting and PP levels of total triglycerides, RLP chocorrelated with corresponding values in the fed state in lesterol, and RLP triglycerides, as well as higher levboth groups. els of total and LDL cholesterol, and they had lower Patients with CHD had significantly higher levels levels of HDL cholesterol. However, although percent of total cholesterol and LDL cholesterol than controls changes in the PP state were slightly greater in paTABLE 1 Baseline Characteristics of the Study Subjects
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a modest increase in hepatic VLDL apoB-100 secretion along with an increase in VLDL triglycerides. In this process, there is less conversion of VLDL apoB-100 to LDL apoB-100 via lipolysis. However, LDL apoB100 mass does not change because there is a simultaneous decrease in LDL apoB-100 catabolism, presumably due to the increased delivery of dietary cholesterol to the liver in the fed state and downregulation of LDL receptor activity.22–25 Moreover, with the influx of triglyceride-rich lipoprotein from the intestine and subsequent lipolysis of triglycerides, there is a transfer of cholesterol ester from LDL and HDL to these particles through the action of cholesterol ester transfer protein. This results in a modest decrease in LDL and HDL cholesterol in the fed state compared with the PP state.22 Remnant-like cholesterol and RLP triglycerides have also been shown by other investigators to increase significantly after a fat-rich meal.26 Moreover, the composition of these particles has been characterized, and their levels are increased in patients with various forms of hypertriglyceridemia, FIGURE 1. Variability in PP responses of RLP cholesterol, RLP triglycerides, and total especially in patients with type III hytriglyceride levels. perlipoproteinemia.4 – 6,27,28 It has been documented that both PP hytients than controls, these changes were not statisti- pertriglyceridemia and elevated remnant lipoprocally different. Moreover, in cases and controls, the teins significantly impair endothelium-dependent fasting values of lipoproteins correlated very highly vasomotor function.29 –31 Kugiyama et al2 recently with the PP values. These results indicate that al- demonstrated that RLP cholesterol and RLP triglycthough the levels of PP triglycerides and constituents eride measurements appear to provide for a more of triglyceride-rich lipoproteins are elevated in pa- precise assessment of CHD risk than plasma total tients with CHD compared with controls, an evalua- triglyceride levels. We have also reported that RLP tion in the fasting state appears sufficient for adequate cholesterol was an independent CHD risk factor in assessment of such lipoproteins, without the need of a women participating in the Framingham Offspring fat-rich meal challenge. Additionally, the results dem- Study and it was superior to triglycerides in this reonstrate that it is not necessary to obtain a fasting gard.3 Moreover, these particles have been associated sample for the measurement of total cholesterol, LDL with increased risk of sudden death in patients with cholesterol, and HDL cholesterol. CHD.32 In our view, remnants are atherogenic parti19,20 Our data are consistent with recent reviews, cles along with LDL cholesterol and lipoprotein(a), which indicate that elevated PP triglyceride levels are and elevated values should become targets for therapy associated with CHD. The results recently reported by to adequately prevent CHD. Given the present findthe investigators of the Atherosclerosis Risk in Com- ings, it seems reasonable to target such therapy based munity Study,21 indicating that fasting triglyceride on the fasting levels of these particles. values are highly correlated with PP triglyceride values and are by far their strongest predictor, also agree with our own results. We already demonstrated that 1. Hokanson JE, Austin MA. Plasma triglyceride level as a risk factor for after a meal rich in both saturated fat and cholesterol, cardiovascular disease independent of high-density lipoprotein cholesterol level: there are substantial increases in apoB-48 (intestinal a meta-analysis of population-based prospective studies. J Cardiovasc Risk origin) containing particles (fourfold), and a 20% in- 1996;3:213–219. 2. Kugiyama K, Doi H, Takazoe K, Kawano H, Soejima H, Mizuno Y, Tsunoda crease in apoB-100 (liver origin) within the triglycer- R, Sakamoto T, Nakano T, Nakajima K, et al. Remnant lipoprotein levels in ide-rich lipoprotein fraction. Associated with the in- fasting serum predict coronary events in patients with coronary artery disease. Circulation 1999;99:2858–2860. flux of lipid in the fed state, there is increased delivery 3. McNamara JR, Shah PK, Nakajima K, Cupples LA, Wilson PWF, Ordovas of fatty acids and cholesterol to the liver, resulting in JM, Schaefer EJ. Remnant-like particle (RLP) cholesterol is an independent 1132 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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cardiovascular disease risk factor in women: results from the Framingham Heart Study. Atherosclerosis 2000;154:229–237. 4. Morganroth J, Levy RY, Frederickson DS. The biochemical, clinical and genetic features of type III hyperlipoproteinemia. Ann Intern Med 1975;82:158– 174. 5. Kane JP, Chen GC, Hamilton RL, Hardman DA, Malloy MJ, Havel RJ. Remnants of lipoproteins of intestinal and hepatic origin in familial dysbetalipoproteinemia. Atherosclerosis 1983;3:47–56. 6. Brewer HB Jr, Zech LA, Gregg RE, Schwartz D, Schaefer EJ. NIH conference. Type III hyperlipoproteinemia: diagnosis, molecular defects, pathology, and treatment. Ann Int Med 1983;98:623–640. 7. Genest J Jr, Bard JM, Fruchard JC, Ordovas JM, Wilson PWF, Schaefer EJ. Plasma apolipoprotein (a), A-I, A-II, B, E, and C-III containing particles in men with premature coronary artery disease. Atherosclerosis 1991;90:149–157. 8. Genest J Jr, Martin-Munley S, McNamara JR, Ordovas JM, Jenner J, Myers RM, Wilson PWF, Schaefer EJ. Prevalence of familial lipoprotein disorders in patients with premature coronary artery disease. Circulation 1992;85:2025–2033. 9. Shimizu H, Mori M, Saito T. An increase of serum remnant-like particles in non-insulin-dependent diabetic patients with microalbuminuria. Clin Chim Acta 1993;22:191–196. 10. Nestel PJ, Fidge NH, Tan MH. Increased lipoprotein-remnant formation in chronic renal failure. N Engl J Med 1982;307:329–333. 11. Campos E, Nakajima K, Tanaka A, Havel RJ. Properties of an apolipoprotein E-enriched fraction of triglyceride-rich lipoproteins isolated from human blood plasma with a monoclonal antibody to apolipoprotein B-100. J Lipid Res 1992; 33:369–380. 12. Nakajima K, Saito T, Tamura A, Suzuki M, Nakano T, Adachi M, Tanaka A, Tada N, Nakamura H, Campos E, Havel RJ. Cholesterol in remnant-like lipoproteins in human serum using monoclonal anti apo B-100 and anti apo A-I immunoaffinity mixed gels. Clin Chim Acta 1993;223:53–71. 13. McNamara JR, Shah PK, Nakajima K, Cupples LA, Wilson PWF, Ordovas JM, Schaefer EJ. Remnant lipoprotein cholesterol and triglyceride: reference ranges from the Framingham Heart Study. Clin Chem 1998;44:1224–1232. 14. Leary ET, Wang T, Baker DJ, Cilla DD, Zhong J, Warnick GR, Nakajima K, Havel RJ. Evaluation of an immunoseparation method for quantitative measurement of remnant-like particle-cholesterol in serum and plasma. Clin Chem 1998; 44:2490–2498. 15. McNamara JR, Schaefer EJ. Automated enzymatic standardized lipid analyses for plasma and lipoprotein fractions. Clin Chim Acta 1987;166:1–8. 16. McNamara JR, Cole TG, Contois JH, Ferguson CA, Schaefer EJ. Immunoseparation method for measuring low-density lipoprotein cholesterol directly from serum evaluated. Clin Chem 1995;41:232–240. 17. Mjos OD, Faergeman O, Hamilton RL, Havel RJ. Characterization of remnants produced during the metabolism of triglyceride-rich lipoproteins of blood plasma and intestinal lymph in the rat. J Clin Invest 1975;56:603–615. 18. Pagnan A, Havel RJ, Kane JP, Kotite L. Characterization of human very low density lipoproteins containing two electrophoretic populations: double prebeta
lipoproteinemia and primary dysbetalipoproteinemia. J Lipid Res 1977;18:613– 622. 19. Karpe F. Postprandial lipoprotein metabolism and atherosclerosis. J Intern Med 1999;246:341–355. 20. Cohn JS. Postprandial lipemia: emerging evidence for atherogenicity of remnant lipoproteins. Can J Cardiol 1998;14(suppl B):18B–27B. 21. Sharrett AR, Heiss LE, Chambless E, Boerwinkle SA, Coady AR, Folsom AR, Patsh W. Metabolic, and lifestyle determinants of postprandial lipemia differ from those of fasting triglycerides. Arterioscler Thromb Vasc Biol 2001;21:275– 281. 22. Cohn JS, McNamara JR, Cohn SD, Ordovas JM, Schaefer EJ. Postprandial plasma lipoprotein changes in human subjects of different ages. J Lipid Res 1988;28:469–478. 23. Cohn JS, Johnson EJ, Millar JS, Cohn SD, Milne RW, Marcel YL, Russell RM, Schaefer EJ. The contribution of apoB-48 and apoB-100 triglyceride-rich to post-prandial increases in plasma triglycerides and retinyl esters. J Lipid Res 1993;34:2033–2040. 24. Cohn JS, McNamara JR, Cohn SD, Ordovas JM, Schaefer EJ. Plasma apolipoprotein changes in the triglyceride-rich lipoprotein fractions of human subjects fed a fat-rich meal. J Lipid Res 1988;29:925–936. 25. Cohn JS, Wagner DA, Cohn SD, Millar JS, Schaefer EJ. The measurements of very low density lipoproteins and low density lipoprotein apoB-100 and high density lipoprotein apoA-I in human subjects using deuterated leucine: effect of fasting and feeding. J Clin Invest 1990;85:804–811. 26. Tanaka A, Tomie N, Nakano T, Nakajima K, Yui K, Tamura M, Numano F. Measurements of postprandial remnant-like particles (RLPs) following a fat loading test. Clin Chim Acta 1998;275:43–52. 27. Marcoux C, Tremblay M, Nakajima K, Davignon J, Cohn JS. Characterization of remnant-like particles isolated by immunoaffinity gel by the plasma of type III and type V hyperlipoproteinemic patients. J Lipid Res 1999;40:636–647. 28. Marcoux C, Tremblay M, Fredenrich A, Jacques H, Nakajima K, Davignon J, Cohn JS. Plasma remnant-like particles lipid and apolipoprotein levels in normolipidemic and hyperlipidemic subjects. Atherosclerosis 1998;139:161–171. 29. Vogel RA, Coretti MC, Plotnick GD. Effect of a single high-fat meal on endothelial function in healthy subjects. Am J Cardiol 1997;79:350–354. 30. Doi H, Kugiyama K, Ohgushi M, Sugiyama S, Matsumura T, Ohta Y, Nakano T, Nakajima K, Yasue H. Remnant of chylomicron and very low-density lipoprotein impair endothelium-dependant vasorelaxation. Atherosclerosis 1998;137: 341–349. 31. Kugiyama K, Doi H, Motoyama T, Soejima H, Misumi K, Kawano H, Nakagawa O, Yoshimura M, Ogawa H, Matsumura T, et al. Association of remnant lipoprotein levels with impairment of endothelium-dependant vasomotor function in human coronary arteries. Circulation 1998;97:2519–2526. 32. Takeichi S, Yakawa N, Nakajima Y, Osawa M, Saito T, Seto Y, Nakano T, Saniabadi AR, Adachi M, Wang T, Nakajima K. Association of plasma triglyceride-rich lipoprotein remnant with coronary atherosclerosis in cases of sudden cardiac death. Atherosclerosis 1999;142:309–315.
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RD,
Plasma lipoprotein levels, including remnant-like particle (RLP) cholesterol and RLP triglycerides, were assessed in fasting (12 hours) and postprandial (PP) (4 hours after a fat-rich meal) states in 88 patients with coronary heart disease (CHD) and 88 controls. All lipoproteins were assessed by direct methods. We hypothesized that patients with CHD would have greater percent increases in their triglyceride levels, RLP cholesterol, and RLP triglycerides, in response to a fat-rich meal. In the fasting state, triglycerides, RLP cholesterol, RLP triglycerides, and lowdensity lipoprotein (LDL) cholesterol levels were all significantly higher in cases versus controls by 51%, 35%, 39%, and 40%, respectively. These levels were 57%, 37%, 64%, and 37% higher in the PP state, respectively. Mean high-density lipoprotein (HDL) cholesterol values were 27% lower in cases in both the fasting and PP states. After eating, triglycerides, RLP cholesterol, and RLP triglycerides increased 64%, 71%, and 290% in
controls, respectively, whereas in cases these levels increased by 71%, 94%, and 340%, respectively (all p <0.0001). Percent increases in the PP state were not significantly different in cases versus controls. Following the fat-rich meal, LDL and HDL cholesterol decreased by 5% and 4% in controls, and by 7% and 6% in patients, with no significant difference in percent changes between groups. Fasting values correlated very highly with PP values for all parameters (all p <0.0001). Our data indicate that although patients with CHD have higher fasting and PP levels of triglycerides, RLP cholesterol, and RLP triglycerides than controls, the response (percent increase) to a fat-rich meal is comparable in both groups. Thus, a feeding challenge is not essential for assessment of these lipoproteins. Moreover, it is not necessary to obtain a fasting sample to assess direct LDL and HDL cholesterol. 䊚2001 by Excerpta Medica, Inc. (Am J Cardiol 2001;88:1129 –1133)
t has been well documented that triglyceride levels are an independent risk factor for coronary heart Idisease (CHD) after adjustment for other predictors,
lipoproteins, from the plasma, leaving behind partially metabolized triglyceride-rich lipoproteins of liver and intestinal origin. After precipitation, levels of cholesterol and triglycerides in the supranatant fraction, which contains these partially metabolized triglyceride-rich lipoproteins, can be measured.11–14 This study assessed plasma lipoprotein levels (including RLP), in CHD cases and controls in the fasting and postprandial (PP) states. Our hypothesis was that patients with CHD would have greater percent increases in their levels of triglycerides, RLP cholesterol, and RLP triglycerides than controls, in response to a fat-rich meal.
including high-density lipoprotein (HDL) cholesterol, especially in woman.1 Patients who have elevated remnant levels, which are formed after the metabolism of triglyceride-rich lipoproteins, also have an increased risk of CHD.2,3 Such patients may have type III hyperlipoproteinemia, or other more common states, such as familial dyslipidemia, diabetes mellitus, and renal disease.4 –10 A new assay has been developed to measure the remnant-like particle (RLP). It uses monoclonal antibodies specific to apolipoprotein (apo)A-I and apoB to remove low-density lipoprotein (LDL) and HDL, and newly formed triglyceride-rich From the Lipid and Heart Disease Prevention Program, New England Medical Center and Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, and Cardiology Division, Newton Wellesley Hospital, Newton, Massachusetts. This study was supported by contracts from Parke Davis/Pfizer, Morris Plains, New Jersey; Otsuka America Pharmaceutical, Inc., Rockville, Maryland; Schering-Plough Corporation, Kenilworth, New Jersey; grants HL 39326 and HL 57477 from the National Institutes of Health, Bethesda, Maryland; and contract 533K06-5-10 from the US Department of Agriculture Research Service, Washington, DC. Manuscript received May 2, 2001; revised manuscript received and accepted July 13, 2001. Address for reprints: Ernst J. Schaefer, MD, Lipid Metabolism Laboratory, Tufts University, 711 Washington St., Boston, Massachusetts 02111. E-mail: [email protected]. ©2001 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 88 November 15, 2001
METHODS
Study design and population: This is a substudy of a placebo-controlled randomized trial with a crossover design, aimed to assess the effect of statins on fasting and PP lipids in patients with CHD. For the main trial, 103 patients with CHD were recruited primarily from outpatient clinics (cardiology and lipid) of 2 medical centers from the Boston area, as well as by newspaper advertising. Of the 103 patients enrolled in the main trial, 88 also agreed to undergo a fat-rich meal challenge after an 8-week washout period and were included in the present substudy. For the present study, to compare with these cases, 88 control subjects matched for age (5-year age group) and sex, and free 0002-9149/01/$–see front matter PII S0002-9149(01)02047-1
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of clinical evidence of CHD, were enrolled provided that they met study entry criteria. Control subjects were recruited by newspaper advertising in the Boston area. Study subjects were predominantly Caucasians residing in the Boston area, mean age was 62 years, and 15% of the group were women. Inclusion and exclusions criteria: Patients were considered for participation in the main trial if they had documented CHD, based on either a history of myocardial infarction, coronary artery bypass grafting surgery or percutaneous coronary intervention, a coronary angiogram documenting significant narrowing (⬎50%) of ⱖ1 coronary arteries, or a positive stress test with nuclear imaging. Patients with CHD had to be free of any clinical event for a period of ⱖ6 months before enrollment and they had to agree to discontinuation of any lipid-lowering medication for a period of 6 weeks before the beginning of the study. Exclusion criteria were as stated in the protocol of the original main trial and were: age⬍20 or ⬎75 years; if female: premenopausal state or use of estrogen replacement therapy (to minimize potential confounders); plasma triglyceride levels ⬎400 mg/dl (4.5 mol/L); plasma LDL cholesterol values ⱕ130 mg/dl (3.4 mol/L); diabetes mellitus requiring insulin therapy or uncontrolled diabetes (hemoglobin A1c ⬎10%); severe obesity (body mass index ⬎35); active liver disease or hepatic dysfunction; acute or chronic pancreatitis; uncontrolled primary hypothyroidism, nephrotic syndrome, or renal dysfunction (blood urea nitrogen ⱖ30 mg/dl (10.5 mmol/L); creatinine ⱖ2.5 mg/dl (220 mol/L); or creatinine clearance ⬍30 ml/min. The same exclusion criteria were applied to control subjects; however, those with LDL cholesterol ⬍130 mg/dl were also accepted into the study. Subjects were maintained on all their nonlipid-lowering medications throughout the study, including  blockers and thiazide diuretics. Protocol: All subjects were studied after an overnight fast (12 hours) and 4 hours after a fat-rich meal. The meal was obtained from a popular restaurant chain (McDonald’s) and consisted of 2 sausages, 2 eggs, and 2 muffins (880 calories, 500 calories or 57% from fat, 180 calories or 20% from saturated fat, 510 mg of cholesterol). Information about smoking, height, weight, and systolic and diastolic blood pressures was collected at the time of first examination. Participants reporting smoking ⱖ1 cigarette per day during the preceding year were classified as smokers. Body mass index was calculated as weight in kilograms divided by the square of height in meters. Hypertension was defined as systolic or diastolic blood pressure ⬎140 or ⬎90 mm Hg, respectively, or use of antihypertensive medication. Diabetes was defined as fasting blood glucose ⬎125 mg/dl (6.85 mmol/L) or use of hypoglycemic medication. The Human Investigation Committee of the New England Medical Center and Tufts University approved the protocol, and informed consent was obtained from all subjects. 1130 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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Lipoproteins and remnant-like particle analysis:
Blood was drawn from each subject after a 12-hour overnight fast and 4 hours after a fat-rich meal. Plasma was separated by centrifugation (2,500 rpm, 4°C, 20 minutes). Plasma lipoprotein (total cholesterol, triglyceride, and HDL cholesterol) concentrations were measured fresh, using standard enzymatic methods, as previously described.15 LDL cholesterol was measured directly using a homogenous reagent kit obtained from Genzyme Diagnostics (Cambridge Massachusetts). Coefficients of variations were ⬍10%.16 RLP isolation was based on removal of apoA-I-containing particles (HDL) and most apoBcontaining particles (LDL, nascent very LDL [VLDL], and nascent chylomicrons), using an immunoseparation technique (Japan Immunoresearch Laboratories, Takasaki, Japan) as previously described,11–14 which has been shown to leave particles characteristic of VLDL remnants and chylomicron remnants in the unbound fraction.17,18 Monoclonal antibodies to apoA-I and apoB, which do not recognize partially hydrolyzed, apoE-enriched lipoprotein remnants, were immobilized on agarose gel. RLP cholesterol and RLP triglyceride concentrations were measured in plasma aliquots and stored at ⫺80°C until analysis. Thawed plasma was incubated with the gel for 2 hours, after which the gel, containing the bound (non-RLP) lipoproteins, was precipitated with lowspeed centrifugation (5 minutes, 135 g). RLP cholesterol and RLP triglyceride levels were then measured in the unbound supernates on an Abbott Spectrum CCx analyzer (Abbott Diagnostics, Irving, Texas), using 2-reagent enzymatic, colorimetric assays containing a sensitive chromophore (Kyowa Medex, Tokyo, Japan). Precision studies yielded a between-run RLP cholesterol coefficient of variation of 9.1% at 0.18 mmol/L (7 mg/dl) and 7.3% at 0.62 mmol/L (24 mg/dl). Between-run RLP triglyceride coefficients of variation were 8.3% at 0.25 mmol/L (22 mg/dl) and 5.0% at 1.23 mmol/L (109 mg/dl).13 Statistical analysis: Categorical variables are reported as frequency (in percent), and continuous variables as mean ⫾ SD. To compare categorical variables between both groups, we used a Fisher’s exact test. A 2-sample t test analysis was performed for continuous variables. When these variables were not normally distributed, they were log-transformed (total triglycerides, RLP cholesterol, RLP triglycerides). To assess the difference between fasting and PP values in each group, we used a paired t test analysis. Finally, for evaluation of the correlation between fasting and PP values for each lipoprotein category, we used a Pearson correlation coefficient test.
RESULTS Baseline characteristics of the study subjects are listed in the Table 1. Both groups were comparable for age, sex, height, and weight. There was a small but significant difference in body mass index, with patients having higher values than controls. Hypertension was also more prevalent among patients than controls, as was diabetes. Correlations between liNOVEMBER 15, 2001
in both the fasting and PP states (Table 3). After a fat-rich meal, total cholesterol and LDL cholesterol CHD were slightly reduced for controls and cases. However, the percent reductions in levels of total cholesCharacteristics 0 (n ⫽ 88) ⫹ (n ⫽ 88) p Value terol and LDL cholesterol were similar for both Age (yrs) (mean ⫾ SD) 62.0 ⫾ 8.6 61.8 ⫾ 9.4 NA groups. The patients with CHD also had lower HDL Women 15% 15% NA cholesterol values than the controls in both the fasting Diabetes mellitus 0% 5.7% 0.06 and the fed state, but had similar percent reductions in Systemic hypertension 30.9% 57.2% 0.01 Cigarette smokers 20.0% 17.2% NS the PP state. Height (cm) 173.9 ⫾ 9.1 171.6 ⫾ 9.1 NS For patients with established CHD, mean triglyc(mean ⫾ SD) eride levels were 51% higher in the fasting state; after Weight (kg) 79.4 ⫾ 15.2 81.2 ⫾ 12.8 NS a fat-rich meal these levels were 57% higher com(mean ⫾ SD) Body mass index 26.2 ⫾ 4.2 27.5 ⫾ 3.3 0.04 pared with levels in controls. The magnitude of the (mean ⫾ SD) difference was similar in both the fasting and fed states. The fat-rich meal caused a 64% increase in triglyceride levels in the control subjects and a 71% increase in the subjects with CHD. Between each TABLE 2 Correlation Between Fasting and Postprandial Values of Lipoproteins group the percent differences were not significantly different. RLP cholesterol levels were 35% higher in Controls Patients patients than in controls in the fasting state, and 37% Lipoprotein Pearson p Value Pearson p Value higher in the fed state. As for total triglycerides, Total cholesterol 0.9795 ⬍0.0001 0.9842 ⬍0.0001 differences in RLP cholesterol levels between patients LDL cholesterol 0.9501 ⬍0.0001 0.9709 ⬍0.0001 and controls were similar in the fasting and the fed HDL cholesterol 0.9875 ⬍0.0001 0.9651 ⬍0.0001 states. After a fat-rich meal, mean RLP cholesterol Triglycerides 0.9040 ⬍0.0001 0.8385 ⬍0.0001 increased by 71% in controls and 94% in patients. RPL cholesterol 0.7450 ⬍0.0001 0.9387 ⬍0.0001 Although the percent increase was somewhat greater RLP triglycerides 0.5294 ⬍0.0001 0.6166 ⬍0.0001 in cases, it was not statistically different from that in controls. With regard to RLP triglycerides, patients TABLE 3 Fasting and Postprandial Lipoprotein Concentrations for Coronary Heart had 39% and 64% higher values in Disease Patients and Controls the fasting state and the fed state, CHD respectively. The fat-rich meal resulted in a 290% increase in RLP Lipoprotein 0 (n ⫽ 88) ⫹ (n ⫽ 88) Difference (%) triglyceride values in the controls Fasting total cholesterol (⫾SD) (mg/dl) 215 ⫾ 45 280 ⫾ 70 ⫹30§ § and a 340% increase in the patients. 4-hr PP total cholesterol (⫾SD) (mg/dl) 211 ⫾ 45 272 ⫾ 66 ⫹29 As for total triglycerides and RLP PP change (%) ⫺2㛳 ⫺3¶ ⫺1* Fasting LDL cholesterol (⫾ SD) (mg/dl) 135 ⫾ 46 188 ⫾ 60 ⫹40§ cholesterol, the response of RLP tri4-hr PP LDL cholesterol (⫾ SD) (mg/dl) 127 ⫾ 42 174 ⫾ 57 ⫹37§ glycerides to the fat-rich meal was PP change (%) ⫺5¶ ⫺7¶ ⫺2* greater in patients than in controls; § Fasting HDL cholesterol (⫾ SD) (mg/dl) 51 ⫾ 15 37 ⫾ 9 ⫺27 but once again, the difference be4-hr PP HDL cholesterol (⫾ SD) (mg/dl) 48 ⫾ 14 35 ⫾ 9 ⫺27§ PP change (%) ⫺4¶ ⫺6¶ ⫺2† tween 2 groups was not statistically Fasting triglycerides (⫾ SD) (mg/dl) 130 ⫾ 82 196 ⫾ 84 ⫹51§ significant. 4-hr PP triglycerides (⫾ SD) (mg/dl) 207 ⫾ 119 325 ⫾ 136 ⫹57§ It should be noted that there was a ¶ ¶ PP change (%) ⫹64 ⫹71 ⫹8* wide degree of variability in these Fasting RLP cholesterol (⫾ SD) (mg/dl) 7.7 ⫾ 3.6 10.4 ⫾ 11.2 ⫹35‡ parameters, because the SDs are 4-hr PP RLP cholesterol (⫾ SD) (mg/dl) 12.5 ⫾ 6.2 17.1 ⫾ 11.9 ⫹37§ PP change (%) ⫹71¶ ⫹94¶ ⫹23* quite large. There was a wide variFasting RLP triglycerides (⫾ SD) (mg/dl) 28 ⫾ 33 39 ⫾ 26 ⫹39§ ability in the PP responses for RLP 4-hr PP RLP triglycerides (⫾ SD) (mg/dl) 87 ⫾ 65 143 ⫾ 86 ⫹64§ triglycerides and total triglycerides, ¶ ¶ PP change (%) ⫹290 ⫹340 ⫹50* as can be seen in Figure 1, where *p ⫽ NS; †p ⬍0.05; ‡p ⬍0.01; §p ⬍0.0001 (2-sample t test); 㛳p ⬍0.001; ¶p ⬍0.0001 (paired t test). percent changes from baseline to the PP state are shown for total triglycerides and RLP values. There was poprotein levels in the fasting and fed states are listed less variability for RLP cholesterol, with the exception in Table 2. Fasting triglycerides, RLP cholesterol, and of one extreme outlier in each group. RLP triglyceride levels were highly and significantly correlated with fed values in patients and controls. DISCUSSION Total cholesterol, LDL cholesterol, and HDL cholesIn the present study patients with CHD had higher terol values in the fasting state were also very highly fasting and PP levels of total triglycerides, RLP chocorrelated with corresponding values in the fed state in lesterol, and RLP triglycerides, as well as higher levboth groups. els of total and LDL cholesterol, and they had lower Patients with CHD had significantly higher levels levels of HDL cholesterol. However, although percent of total cholesterol and LDL cholesterol than controls changes in the PP state were slightly greater in paTABLE 1 Baseline Characteristics of the Study Subjects
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a modest increase in hepatic VLDL apoB-100 secretion along with an increase in VLDL triglycerides. In this process, there is less conversion of VLDL apoB-100 to LDL apoB-100 via lipolysis. However, LDL apoB100 mass does not change because there is a simultaneous decrease in LDL apoB-100 catabolism, presumably due to the increased delivery of dietary cholesterol to the liver in the fed state and downregulation of LDL receptor activity.22–25 Moreover, with the influx of triglyceride-rich lipoprotein from the intestine and subsequent lipolysis of triglycerides, there is a transfer of cholesterol ester from LDL and HDL to these particles through the action of cholesterol ester transfer protein. This results in a modest decrease in LDL and HDL cholesterol in the fed state compared with the PP state.22 Remnant-like cholesterol and RLP triglycerides have also been shown by other investigators to increase significantly after a fat-rich meal.26 Moreover, the composition of these particles has been characterized, and their levels are increased in patients with various forms of hypertriglyceridemia, FIGURE 1. Variability in PP responses of RLP cholesterol, RLP triglycerides, and total especially in patients with type III hytriglyceride levels. perlipoproteinemia.4 – 6,27,28 It has been documented that both PP hytients than controls, these changes were not statisti- pertriglyceridemia and elevated remnant lipoprocally different. Moreover, in cases and controls, the teins significantly impair endothelium-dependent fasting values of lipoproteins correlated very highly vasomotor function.29 –31 Kugiyama et al2 recently with the PP values. These results indicate that al- demonstrated that RLP cholesterol and RLP triglycthough the levels of PP triglycerides and constituents eride measurements appear to provide for a more of triglyceride-rich lipoproteins are elevated in pa- precise assessment of CHD risk than plasma total tients with CHD compared with controls, an evalua- triglyceride levels. We have also reported that RLP tion in the fasting state appears sufficient for adequate cholesterol was an independent CHD risk factor in assessment of such lipoproteins, without the need of a women participating in the Framingham Offspring fat-rich meal challenge. Additionally, the results dem- Study and it was superior to triglycerides in this reonstrate that it is not necessary to obtain a fasting gard.3 Moreover, these particles have been associated sample for the measurement of total cholesterol, LDL with increased risk of sudden death in patients with cholesterol, and HDL cholesterol. CHD.32 In our view, remnants are atherogenic parti19,20 Our data are consistent with recent reviews, cles along with LDL cholesterol and lipoprotein(a), which indicate that elevated PP triglyceride levels are and elevated values should become targets for therapy associated with CHD. The results recently reported by to adequately prevent CHD. Given the present findthe investigators of the Atherosclerosis Risk in Com- ings, it seems reasonable to target such therapy based munity Study,21 indicating that fasting triglyceride on the fasting levels of these particles. values are highly correlated with PP triglyceride values and are by far their strongest predictor, also agree with our own results. We already demonstrated that 1. Hokanson JE, Austin MA. Plasma triglyceride level as a risk factor for after a meal rich in both saturated fat and cholesterol, cardiovascular disease independent of high-density lipoprotein cholesterol level: there are substantial increases in apoB-48 (intestinal a meta-analysis of population-based prospective studies. J Cardiovasc Risk origin) containing particles (fourfold), and a 20% in- 1996;3:213–219. 2. Kugiyama K, Doi H, Takazoe K, Kawano H, Soejima H, Mizuno Y, Tsunoda crease in apoB-100 (liver origin) within the triglycer- R, Sakamoto T, Nakano T, Nakajima K, et al. Remnant lipoprotein levels in ide-rich lipoprotein fraction. Associated with the in- fasting serum predict coronary events in patients with coronary artery disease. Circulation 1999;99:2858–2860. flux of lipid in the fed state, there is increased delivery 3. McNamara JR, Shah PK, Nakajima K, Cupples LA, Wilson PWF, Ordovas of fatty acids and cholesterol to the liver, resulting in JM, Schaefer EJ. Remnant-like particle (RLP) cholesterol is an independent 1132 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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