H. Robert Superko, MD a,b, *, Kaspar K. Berneis, MD c, Paul T. Williams, PhD d, Manfredi Rizzo, MD d,e, and Peter D.

Transcription

1 Gemfibrozil Reduces Small Low-Density Lipoprotein More in Normolipemic Subjects Classified as Low-Density Lipoprotein Pattern B Compared With Pattern A H. Robert Superko, MD a,b, *, Kaspar K. Berneis, MD c, Paul T. Williams, PhD d, Manfredi Rizzo, MD d,e, and Peter D. Wood, PhD, DSc f We tested the hypothesis that gemfibrozil has a differential effect on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) subclass distributions and postprandial lipemia that is different in subjects classified as having LDL subclass pattern A or LDL pattern B who do not have a classic lipid disorder. Forty-three normolipemic subjects were randomized to gemfibrozil (1,200 mg/day) or placebo for 12 weeks. Lipids and lipoproteins were determined by enzymatic methods. The mass concentrations of lipoproteins in plasma were determined by analytic ultracentrifugation and included the S f intervals: 20 to 400 (very LDL), 12 to 20 (intermediatedensity lipoprotein), 0 to 12 (LDL), and HDL 2 mass (F to 9.0) and HDL 3 mass (F to 3.5). Postprandial measurements of triglycerides and lipoprotein(a) were taken after the patients consumed a 500 kcal/m 2 test meal. Treatment with gemfibrozil, compared with placebo, significantly reduced fasting plasma triglycerides (difference from placebo SE; mg/dl, p 0.02), total cholesterol ( mg/dl, p 0.04), apolipoprotein B ( mg/dl, p 0.006), very LDL mass of S f 20 to 400 ( mg/dl, p 0.02), S f 20 to 60 ( mg/dl, p 0.05), S f 60 to 100 ( mg/dl, p 0.05), and increased peak S F ( Svedberg, p 0.08). Gemfibrozil reduced the postprandial triglyceride level significantly at 3 (p 0.04) and 4 (p 0.05) hours after the test meal. A significantly different subclass response to gemfibrozil was observed in those with LDL pattern A versus B. Those with LDL pattern B had a significantly greater reduction in the small LDL mass S f 0to7(p 0.04), specifically regions S f 0to3 (p 0.009) and S f 3to5(p 0.009). In conclusion, normolipemic subjects with either predominantly dense or buoyant LDL respond differently to gemfibrozil as determined by the changes in LDL subclass distribution. Thus, treatment with gemfibrozil may have additional antiatherogenic effects in those with LDL pattern B by decreasing small dense LDL that is not apparent in those with pattern A Elsevier Inc. All rights reserved. (Am J Cardiol 2005;96: ) Although subjects classified as having low-density lipoprotein (LDL) subclass pattern A and B have been shown to respond differently to diet and nicotinic acid therapy, 1,2 a differential lipoprotein subclass response to fibrate in normolipemic patients has not been reported. Specifically, we hypothesized that those with LDL pattern B would Methods Subjects and trial design: In a double-blind, randomized, placebo-controlled trial design, 43 normolipemic suba Fuqua Heart Center/Piedmont Hospital, Atlanta, Georgia; b Cholesterol, Genetics, and Heart Disease Institute, Portola Valley, California; c University Hospital Bruderholz, Bruderholz, Switzerland; d Lawrence Orlando Berkeley National Laboratory, University of California, Berkeley, California; e Department of Clinical Medicine and Emerging Diseases, University of Palermo, Palermo, Italy; and f Stanford University School of Medicine, Palo Alto, California. Manuscript received January 31, 2005; revised manuscript received and accepted June 24, This study was supported by Parke Davis Pharmaceuticals, Warner- Lambert Co., Morris Plains, New Jersey. * Corresponding author: Tel: ; fax: address: robert.superko@piedmont.org (H.R. Superko). exhibit significantly greater reductions in fasting plasma concentrations of small, dense LDL and intermediate-density lipoprotein, postprandial lipemia, and a significantly greater increase in high-density lipoprotein (HDL) 2 concentrations compared with identically treated subjects with LDL pattern A. We therefore tested, in a randomized, controlled clinical trial, whether subjects with normolipemic LDL patterns A or B responded differently to gemfibrozil treatment. After a meal, a significant change can occur in postprandial lipemia that may affect coronary artery disease risk. 3 Therefore, we also examined whether the postprandial lipemia response to gemfibrozil treatment was related to the LDL subclass pattern /05/$ see front matter 2005 Elsevier Inc. All rights reserved. doi: /j.amjcard

2 Preventive Cardiology/Gemfibrozil Effect on LDL Subclasses 1267 jects (38 men and 5 women; aged 21 to 65 years) were randomized to gemfibrozil (1,200 mg/day) or placebo for 12 weeks after a 6-week American Heart Association phase I diet wash-out period. Subjects were excluded if they had a medical condition or used medications that could influence plasma lipoproteins. Subjects were required to have total cholesterol values of 250 mg/dl and fasting triglyceride values of 300 mg/dl. All participants completed 4-day diet records at baseline and at the end of treatment. These were analyzed for total calories; percentage of calories from fat, carbohydrate, protein, and alcohol; cholesterol content; polyunsaturated/saturated fat ratio; and micronutrients. The body mass index (BMI) was calculated as kilograms of weight divided by height in meters squared. The institutional human use committee approved the study, and all subjects signed an approved informed consent form. A modified definition of the metabolic syndrome was used to explore the potential difference in patients with the metabolic syndrome. 4 Because uncontrolled hypertension and medications that affect plasma lipoproteins were exclusion criteria, hypertension was not present in this population. For the purposes of analysis, the metabolic syndrome was defined as fasting triglycerides of 150 mg/dl and a BMI 25 kg/m 2. Baseline fasting blood samples for lipid and lipoprotein analysis were obtained from the patients after a 12- to 16-hour fast, avoidance of vigorous physical activity, and before the initiation of any therapy. Venous blood samples were drawn with the subject in a sitting position. Postprandial measurements of triglycerides and lipoprotein(a) (Lp(a)) were made after the patients consumed a 500 kcal/m 2 test meal of cream, sugar, and milk that contained 45% of its calories from fat. The test meal was consumed after the fasting blood draw. Additional blood samples were taken at 3, 4, 5, and 8 hours after the test meal while the subjects remained in the clinic area and consumed nothing except water. Laboratory methods: Plasma was prepared from the blood samples within 30 minutes and the blood and plasma samples were kept at 4 C. Plasma lipid and lipoprotein cholesterol concentrations were determined using the methods of the Lipid Research Clinics. 5 Triglycerides, total cholesterol, and lipoprotein cholesterol values were measured by enzymatic procedures (Abbott ABA 200 instrument, Abbott Park, Illinois). HDL cholesterol was determined by the dextran sulfate-magnesium precipitation procedure. 6 LDL cholesterol was calculated from the following equation: LDL cholesterol total cholesterol [HDL cholesterol (triglycerides/5)]. During the study, the laboratory remained standardized for lipid measurements through the Centers for Disease Control National Heart, Lung, and Blood Institute Lipoprotein Standardization Program. 7 The concentrations of lipoproteins in plasma (as total mass) were determined at the Donner Laboratory, University of California at Berkeley, using computer analysis of the results of analytic ultracentrifugation. 8 This technique generates a Schlieren curve, which describes the distribution of lipoproteins according to their high-density flotation (F) and low- to very-low-density flotation (S f ) rates. The intervals include the total lipoprotein mass concentrations of the following S f intervals: 20 to 400 (very-low-density lipoprotein [VLDL]), 12 to 20 (intermediate-density lipoprotein), and 0 to 12 (LDL). The HDL 2 mass and HDL 3 mass were determined as the sum of the flotation intervals F to 9.0 and F to 3.5, respectively. Classification of LDL pattern A versus B was based on whether the LDL peak (S F ) was 5.2 (pattern A) or 5.2 (pattern B). 9 Apolipoprotein A-I and B assays were performed by a competitive enzyme-linked immunoassay procedure using well-characterized and specific monoclonal antibodies. 10,11 Lp(a) concentrations were measured with an enzyme-linked immunosorbent assay kit (Macra Lp(a) Terumo Diagnostics Division, Tokyo, Japan). Internal quality assurance for apolipoproteins was monitored at 2 levels for each analyte on an on-going basis using specifically prepared frozen pools. Throughout the period in which all apolipoprotein measurements were performed, the laboratory participated in the Centers for Disease Control International Union of Immunology Societies apolipoprotein standardization program. 12 Apolipoprotein E isoforms were determined by isoelectric focusing of VLDL apolipoproteins and phenotypes designated according to recommended nomenclature. 13 Statistical analysis: Mean differences between placebo and gemfibrozil were compared using an unpaired t test. Two-way analysis of variance was used to test whether treatment differences in the lipoprotein change between baseline and follow-up were affected by the LDL pattern at baseline. Correction for the change in BMI was performed using analysis of covariance. Results Baseline values: No significant differences were found between the treatment arms for baseline BMI, lipids, apolipoproteins, Lp(a), VLDL mass distribution, intermediatedensity lipoprotein mass distribution, LDL mass distribution, HDL mass distribution, postprandial triglycerides, or Lp(a) (Table 1). The distribution of the apolipoprotein E isoform was not different between groups. Of the patients in the gemfibrozil and placebo groups, 15 and 17 had apolipoprotein E 3/3, 4 and 4 had apolipoprotein E 4/3 or 4/4, and 2 and 1 had apolipoprotein E 2/3, respectively. The treatment arms also did not differ in their nutrient intake at baseline, including dietary cholesterol, total calories, percentage of calories from saturated, unsaturated, and monounsaturated fats, and percentage of calories from carbohydrates and alcohol (analyses not shown). As expected, subjects with pattern B had significantly higher mean triglycerides, VLDL (S F 20 to 400), intermediate-density lipoprotein (S F 12 to 20), and apolipoprotein B and significantly lower HDL cholesterol, HDL 2 mass, HDL 3 mass, and apolipoprotein A-I concen-

3 1268 The American Journal of Cardiology ( Table 1 Baseline lipid, lipoprotein subclasses, and apolipoproteins in the placebo and gemfibrozil groups Variable Gemfibrozil Placebo p Value BMI (kg/m 2 ) Triglycerides (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Apolipoprotein A I (mg/dl) Apolipoprotein B (mg/dl) Lp(a) (mg/dl) LDL mass (mg/dl) S f S f S f S f S f IDL mass S f (mg/dl) VLDL mass (mg/dl) S f S f S f S f LDL peak flotation rate (S f ) HDL mass (mg/dl) F F Postprandial triglycerides (mg/dl) Average h h h h Postprandial Lp(a) (mg/dl) 4 h h Data are presented as means SDs. IDL intermediate-density lipoprotein. trations compared with those with pattern A (Table 2). Patients with pattern B had denser LDL, as reflected by a significantly higher mass for the patient flotation intervals between S f 0 to 5 and significantly lower mass concentrations for patient flotation intervals within S f 7 to 12. They also had higher postprandial triglycerides (p ) than those with pattern A. Fasting and postprandial Lp(a) did not differ by LDL pattern. In the patient group with metabolic syndrome (n 12), 81.8% had LDL pattern B compared with 35.5% in the group without metabolic syndrome (n 31, chi-square p 0.008). At baseline, the metabolic syndrome group had significantly higher fasting triglycerides (p ), apolipoprotein B (p 0.004), dense LDL S f 3to5(p 0.03), VLDL S f 20 to 60 (p 0.003), S f 60 to 100 (p ), and S f 100 to 400 (p ) and significantly lower HDL cholesterol (p 0.02), peak S f (p 0.007), and buoyant LDL S f 7to12(p 0.02). Treatment response: Unexpectedly, a significantly greater reduction occurred in BMI in the gemfibrozil than in the Table 2 Differences between low-density lipoprotein (LDL) patterns A and B at baseline Variable Pattern A (n 22) Pattern B (n 21) P Value BMI (kg/m 2 ) Triglycerides (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Apolipoprotein A I (mg/dl) Apolipoprotein B (mg/dl) Lp(a) (mg/dl) LDL mass (mg/dl) S f S f S f S f S f IDL mass S f 2 20 (mg/dl) VLDL mass (mg/dl) S f S f S f S f LDL peak flotation rate (S f ) HDL mass (mg/dl) F F Postprandial triglycerides (mg/dl) Average h h h h Postprandial Lp(a) (mg/dl) 4 h h Data are presented as means SDs. Abbreviation as in Table 1. placebo group ( kg/m 2,p 0.001). When adjusted for changes in BMI (Table 3), gemfibrozil-treated subjects had significantly greater (mean SE) reductions in plasma concentrations of triglycerides ( mg/ dl), total cholesterol ( mg/dl), apolipoprotein B ( mg/dl), VLDL mass of S f 20 to 400 ( mg/dl), S f 20 to 60 ( mg/dl), and S f 60 to 100 ( mg/dl), and borderline significantly greater increases in peak S F ( Svedberg, p 0.08). In addition, adjustment for BMI also increased the significance of the marginal treatment effects to p 0.05 for LDL mass of S f 5to7( mg/dl) and apolipoprotein A-I ( mg/dl). Gemfibrozil had no significant effect on fasting and postprandial Lp(a). Treatment response by LDL patterns A and B: Table 3 also lists the changes in the gemfibrozil and placebo groups separated by LDL patterns A and B. Analysis for an interaction between phenotype and treatment group re-

4 Preventive Cardiology/Gemfibrozil Effect on LDL Subclasses 1269 Table 3 Effects of gemfibrozil treatment on plasma lipids, lipoprotein subclasses, and apolipoproteins Variable LDL Pattern A LDL Pattern B Significance* Gemfibrozil Placebo Gemfibrozil Placebo Treatment Interaction Triglycerides (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Apo A I (mg/dl) Apo B (mg/dl) Lp(a) (mg/dl) LDL peak flotation rate (S f ) LDL mass (mg/dl) S f S f S f S f S f IDL mass S f mg/dl VLDL mass (mg/dl) S f S f S f S f HDL 2 mass F mg/dl HDL 3 mass F mg/dl Postprandial triglycerides (mg/dl) Average h h h h Postprandial Lp(a) (mg/dl) 4h h Data are presented as means SEs. * Treatment significance is the difference between combined (pattern A and B) gemfibrozil group compared with placebo (drug effect) adjusted for change in BMI; interaction is interaction between phenotype and treatment group. Abbreviation as in Table 1. vealed a significantly different response to gemfibrozil in those with LDL pattern A versus pattern B. Total small, dense LDL mass S f 0 to 7 and, in particular, the dense regions S f 0to3andS f 3 to 5 were reduced significantly more in those with LDL pattern B compared with those with pattern A. A borderline significant difference was observed for VLDL mass S f 100 to 400 (p 0.07) and HDL 2 mass F to 9.0 (p 0.07). Figure 1 shows that compared with those with pattern A at baseline, gemfibrozil in those with pattern B produced significantly greater decreases in small LDL mass concentrations of S f 0 to 7 (pattern B vs A difference SE vs mg/dl) and, more specifically, LDL of S f 0to3( vs mg/dl) and S f 3to5( vs mg/dl). Treatment response by metabolic syndrome classification: In the gemfibrozil treatment group, no significant difference was found between those classified with or without the metabolic syndrome for changes in BMI and all lipoprotein measurements, with the exception of significantly greater LDL cholesterol (p 0.04) and HDL 2 (F3.5 to 9.0, p 0.08) increases in patients with the metabolic syndrome. Postprandial triglyceride response: Thirty-seven subjects (19 placebo and 18 gemfibrozil) provided postprandial lipoprotein measurements at baseline and the end of treatment. Figure 2 shows the significant differences in fasting and postprandial triglycerides in those with LDL pattern A versus pattern B at baseline. Gemfibrozil had a greater effect on average triglyceride reduction compared with placebo (Table 3). When adjusted for changes in BMI, the reduction in postprandial triglycerides by gemfibrozil was significant for triglycerides at 3 and 4 hours and marginally significant when averaged over the 4 time points. Lp(a) values did not change significantly in the postprandial period. Those with LDL pattern B, compared with pattern A, had significantly greater postprandial triglyceride reduction at 3 and 8 hours. Averaged over the 8-hour postprandial period, those with LDL pattern B had significantly greater triglyceride reduction.

5 1270 The American Journal of Cardiology ( Figure 1. Net effect of 12 weeks of gemfibrozil treatment on LDL mass concentrations by LDL flotation interval in those with LDL patterns A or B. Plotted values are mean differences between gemfibrozil and placebo, and bars represent SE. Figure 2. Baseline difference in postprandial triglyceride (TG) values in those with LDL subclass patterns A or B. Discussion Fibrate therapy has been shown to improve the lipoprotein profile in patients with combined hyperlipidemia and type 2 diabetes mellitus with mixed hyperlipoproteinemia by reducing levels of triglyceride-rich lipoproteins and small dense LDLs and increasing HDL. 14,15 Consistent with other reports, 16,17 we have shown that 1,200 mg/day of gemfibrozil reduced fasting triglycerides by 40% (p 0.02), LDL cholesterol by 5% (p NS), and increased HDL cholesterol by 6% (p NS). At the end of treatment, BMI had decreased significantly more in the gemfibrozil group than in the placebo group. However, after statistical adjustment for the change in BMI, the differences in triglycerides, total cholesterol, small LDL and apolipoprotein B remained significantly different between the gemfibrozil and placebo groups. The greatest percentage of reduction in lipoproteins was in the very largest VLDL particles (S f 100 to 400, 73% reduction) and intermediate-size VLDL particles (S f 60 to 100, 58% reduction). The percentage of reduction in smaller VLDL particles (S f 20 to 60, 16% reduction) was only 1/2 the magnitude (29% reduction) as the reduction in larger VLDL particles. This suggests a preferential reduction of the largest VLDL particles. This may account for the smaller triglyceride reduction in normolipidemic subjects who have less VLDL between S f 60 to 400 than do dyslipidemic subjects. The atherogenic lipoprotein profile is characterized by elevated triglycerides, VLDL, intermediate-density lipoprotein, low HDL, insulin resistance, and an abundance of small, dense LDL particles, termed pattern B. 18 Our primary hypothesis was to test whether the response was significantly different from gemfibrozil treatment in patients without classic hyperlipidemia who were classified as having either LDL pattern B or A with regard to improvements in lipoprotein subclasses and fasting and postprandial lipoprotein concentrations. Our analyses indicated that gemfibrozil produced significantly greater reductions in small LDL in those subjects who were initially classified as having LDL pattern B compared with those with pattern A. The results were unlikely to be a result of regression to the mean because the differential response was revealed in a controlled study (i.e., regression to the mean would affect the gemfibrozil and placebo conditions equally). The plasma concentrations of baseline LDL S f 0to3andS f 3to5and the LDL peak flotation rate were similar in the gemfibrozil and placebo groups. No significant treatment by LDL pattern interaction was found for baseline S f 0to3andS f 3to 5. The decrease in small dense LDL in those with pattern B was confirmed by gradient gel electrophoresis (data not shown). These observations all argue against the differential response being a result of regression to the mean. The lipoprotein subclass distribution characteristics of LDL patterns B and A in this normolipemic sample exhibited the same characteristic lipoprotein subclass profile as has been reported for hyperlipidemic populations. At baseline, fasting apolipoprotein B and fasting and postprandial triglycerides were significantly higher in subjects with LDL pattern B versus A and HDL cholesterol and apolipoprotein A to I were lower in subjects with pattern B versus A. No differences were found in total cholesterol, LDL cholesterol, or Lp(a) by LDL pattern. The LDL and HDL subclass distributions revealed a predominance of dense LDL, greater intermediate-density lipoprotein (S F 12 to 20), greater postprandial lipemia, and less HDL 2 (F to

6 Preventive Cardiology/Gemfibrozil Effect on LDL Subclasses ) in those with pattern B compared with those with pattern A. The metabolic syndrome, like the LDL pattern B, is associated with elevated triglycerides and VLDL. 4,19 This study revealed that the atherogenic lipoprotein profile, characterized by an abundance of VLDL, small LDL, and reduced HDL 2, exists in 36% of patients without the metabolic syndrome. In this study, the lipoprotein subclass distribution changes induced by gemfibrozil were not significantly different in patients with or without the metabolic syndrome. The postprandial triglyceride response revealed a significant reduction in the average postprandial triglycerides of 30% in response to gemfibrozil compared with placebo. This reduction was significant at 3 and 4 hours after the test meal. Those with LDL pattern B exhibited significantly greater postprandial triglyceride reduction in response to gemfibrozil compared with those with LDL pattern A. No effect was found of the fat load on Lp(a) and no effect of gemfibrozil on Lp(a). Conflicting studies have reported that Lp(a) may, or may not, increase after a meal Our study results extend this observation to healthy patients with LDL pattern B or A. The clinical significance of this investigation is linked to the importance of lipoprotein subclass distribution and postprandial lipemia to clinical events and arteriographic outcomes. The plasma lipoprotein profile accompanying a predominance of small dense LDL particles is associated with approximately a 3-fold increased risk of coronary artery disease. This has been demonstrated in case-control studies of myocardial infarction and angiographically documented coronary disease In several of these studies, the disease risk associated with small dense LDL was no longer significant after adjusting for covariates, including triglycerides or other risk factors. However, statistical analysis of these trials has indicated that the LDL peak particle diameter is a coronary artery disease risk factor independent of traditional lipid risk factors. 27 Also, a 13-year follow-up study of the Quebec Cardiovascular Study has reported a strong and independent association between small LDL levels and coronary artery disease risk in men. 28 The presence of an abundance of dense, small LDL particles identifies a high-risk group (LDL pattern B) that may benefit from a differential response to treatment with regard to LDL and HDL subclass distribution, as we have demonstrated for the first time in this investigation in a normolipidemic population. The Helsinki investigation hinted at such a possibility when they reported a 34% reduction in clinical events in all men treated with gemfibrozil, but in men with a baseline triglycerides 204 mg/dl and a LDL cholesterol/hdl cholesterol ratio 5, a 71% reduction in clinical events was reported. 29 The elevated triglycerides and high total cholesterol/hdl cholesterol ratio in this Helsinki subgroup suggest the effect would be enriched in those with LDL pattern B. Our investigation suggests that part of this enhanced benefit attributed to gemfibrozil in this Helsinki subset may have been due to the greater effect of gemfibrozil on small, dense LDL in those with pattern B compared with those with pattern A. Investigations using arteriographic change as an outcome variable have reported that arteriographic progression in the control group is significantly greater in patients with a predominance of small, dense LDL and that arteriographic benefit is concentrated in patients with a predominance of small, dense LDL who receive treatment that tends to lower small dense LDL Gemfibrozil improves the abnormal lipoprotein subclass distribution in normolipidemic patients with a predominance of dense, small LDL (LDL pattern B). The differential effect of gemfibrozil on lipoprotein subclasses in those with pattern B may help explain the greater reduction in clinical end points reported in subsets of patients in clinical trials with triglyceride and HDL cholesterol parameters likely to reflect a predominance of subjects with the LDL subclass pattern B trait. 1. Dreon DM, Fernstrom H, Miller B, Krauss RM. Low density lipoprotein subclass patterns and lipoprotein response to a reduced-fat diet in men. FASEB J 1994;8: Superko HR, McGovern ME, Raul E, Garrett B. Nicotinic acid has a differential effect on low density lipoprotein subclass distribution in patients classified as LDL pattern A, B, or I. Am J Cardiol 2004;94: Zilversmit DB. Atherogenesis: a postprandial phenomenon. Circulation 1979;60: Superko HR. The metabolic syndrome and coronary artery disease. In: Fuster V, Alexander RW, O Rourke RA, Roberts R, King SB III, Nash IS, eds. Hurst s The Heart. New York: McGraw-Hill, 2004:P2127 P Office USGP. Lipid Research Clinics Manual of Laboratory Operations: Lipid and Lipoprotein Analysis. Vol. I. HEW Publication No. NIH Washington, DC: Government Printing Office, Warnick GR, Benderson J, Albers JJ. Dextran sulfate-mg2 precipitation procedure for quantitation of high-density lipoprotein cholesterol. Clin Chem 1982;28: Myers G, Cooper G, Winn C, Smith S. The Center for Disease Control-National Heart, Lung and Blood Institute Lipid Standardization Program: an approach to accurate and precise lipid measurements. Clin Lab Med 1989;9: Lindgren FT, LCJ, Hatach FT. The isolation and quantitative analysis of serum lipoproteins. In: Nelson GJ, ed. Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. New York: Wiley- Interscience, 1972: Miller BD, Alderman EL, Haskell WL, Fair JM, Krauss RM. Predominance of dense low-density lipoprotein particles predicts angiographic benefit of therapy in the Stanford Coronary Risk Intervention Project. Circulation 1996;94: Hogle DM, Smith RS, Curtiss LK. Quantitation of plasma apolipoprotein A-I using two monoclonal antibodies in an enzyme-linked immunosorbent assay. J Lipid Res 1988;29: Young SG, Smith RS, Hogle DM, Curtiss LK, Witztum JL. Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B. Clin Chem 1986;32: Cooper GR, Smith SJ, Wiebe DA, Kuchmak M, Hannon WH. International survey of apolipoproteins A1 and B measurements ( ). Clin Chem 1985;31: Weisgraber KH, Rall SC Jr, Mahley RW. Human E apoprotein heterogeneity: cysteine-arginine interchanges in the amino acid sequence of the apo-e isoforms. J Biol Chem 1981;256:

7 1272 The American Journal of Cardiology ( 14. Guerin ME, Bruckert PJ, Dolphin G, Turpin MJ, Chapman MJ. Fenofibrate reduces plasma cholesteryl ester transfer from HDL to VLDL and normalizes the atherogenic, dense LDL profile in combined hyperlipidemia. Arterioscler Thromb Vasc Biol 1996;16: Frost RJ, Otto C, Geiss HC, Schwandt P, Parhofer KG. Effects of atorvastatin versus fenofibrate on lipoprotein profiles, low-density lipoprotein subfraction distribution, and hemorheologic parameters in type 2 diabetes mellitus with mixed hyperlipoproteinemia. Am J Cardiol 2001;87: Robins SJ, Collins D, Wittes JT, Papademetriou V, Deedwania PC, Schaefer EJ, McNamara JR, Kashyap ML, Hershman JM, Wexler LF, Rubins HB. Relation of gemfibrozil treatment and lipid levels with major coronary events: VA-HIT a randomized controlled trial. JAMA 2001;285: Gotto AM Jr, Breen WJ, Corder CN, Dunn JK, Goldberg A, Knopp RH, Schrott H, Sprecher D, for the Lopid SR Work Group I. Oncedaily, extended-release gemfibrozil in patients with dyslipidemia. Am J Cardiol 1993;71: Superko HR, Chronos NA. Hypercholesterolemia and dyslipidemia. issues for clinicians. Curr Treatment Options CV Med 2004;5: Isomaa B, Henricsson M, Almgren P, Tuomi T, Taskinen MR, Groop L. The metabolic syndrome influences the risk of chronic complications in patients with type II diabetes. Diabetologia 2001;44: Pfaffinger D, Schuelke J, Kim C, Fless GM, Scanu AM. Relationship between Apo[a] isoforms and Lp[a] density in subjects with different Apo[a] phenotype: a study before and after a fatty meal. J Lipid Res 1991;32: Cohn JS, Lam CW, Sullivan DR, Hensley WJ. Plasma lipoprotein distribution of apolipoprotein(a) in the fed and fasted states. Atherosclerosis 1991;90: Hoppichler F, Kraft HG, Sandholzer C, Lechleitner M, Patsch JR, Utermann G. Lipoprotein(a) is increased in triglyceride-rich lipoproteins in men with coronary heart disease, but does not change acutely following oral fat ingestion. Atherosclerosis 1996;122: Miller BD, Alderman EL, Haskell WL, Fair JM, Krauss RM. Predominance of dense low-density lipoprotein particles predicts angiographic benefit or therapy in the Stanford Coronary Risk Intervention Project. Circulation 1996;94: Stampfer MJ, Krauss RM, Blanche PJ, Holl LG, Sacks FM, Hennekens CH. A prospective study of triglyceride level, low density lipoprotein particle diameter, and risk of myocardial infarction JAMA 1996; 276: Gardner CD, Fortmann SP, Krauss RM. Association of small lowdensity lipoprotein particles with the incidence of coronary artery disease in men and women. JAMA 1996;276: Lamarche B, Tchernof A, Moorjani S, Cantin B, Dagenais GR, Lupien PJ, Despres JP. Small, dense low-density lipoprotein particles as a predictor of the risk of ischemic heart disease in men: prospective results from the Quebec cardiovascular study. Circulation 1997;95: Austin MA. Triglyceride, small, dense low-density lipoprotein, and the atherogenic lipoprotein phenotype. Curr Atheroscler Rep 2000;2: St. Pierre AC, Cantin B, Dagenais GR, Mauriege P, Bernard PM, Despres JP, Lamarche B. Low-density lipoprotein subfractions and the long-term risk of ischemic heart disease in men. Arterioscler Thromb Vasc Biol 2005;25: Manninen V, Tenkanen L, Koskinen P, Huttunen JK, Manttari M, Heinonen OP, Frick MH. Joint effects of serum triglyceride and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study: implications for treatment. Circulation 1992;85: Krauss RM. Relationship of intermediate and low-density lipoprotein subspecies to risk of coronary artery disease. Am Heart J 1987;113(2 Pt 2): Watts GF, Mandalia S, Brunt JN, Slavin BM, Coltart DJ, Lewis B. Independent associations between plasma lipoprotein subfraction levels and the course of coronary artery disease in the St. Thomas Atherosclerosis Regression Study (STARS). Metabolism 1993;42: Haskell WL, Alderman EL, Fair JM, Superko HR, Maron DJ, Champagne MA, Mackey SF, Williams PT, Krauss RM, Farquhar JW. Beneficial angiographic and clinical response to multifactor modification in the Stanford Coronary Risk Intervention Project (SCRIP). Circulation 1994;89: Zambon A, Hokanson JE, Brown BG, Brunzell JD. Evidence for a new pathophysiological mechanism for coronary artery disease regression: hepatic lipase-mediated changes in LDL density. Circulation 1999;99: Williams PT, Superko HR, Haskell WL, Alderman EA, Blanche PJ, Holl LG, Krauss RM. Smallest LDL particles are most strongly related to coronary disease progression in men. Arterioscler Thromb Vasc Biol 2003;23: