Clustering of cardiovascular risk factors is common

Transcription

1 AJH 2004; 17: Low-Density Lipoprotein Particle Size and Coronary Atherosclerosis in Subjects Belonging to Hypertensive Sibships Iftikhar J. Kullo, Kent R. Bailey, Joseph P. McConnell, Patricia A. Peyser, Lawrence F. Bielak, Sharon L.R. Kardia, Patrick F. Sheedy II, Eric Boerwinkle, and Stephen T. Turner Background: Dyslipidemia in hypertensive sibships may be characterized by atherogenic small low-density lipoprotein (LDL) particles. Whether LDL particle size is associated with the extent of coronary atherosclerosis in hypertensive sibships is unknown. Methods: Subjects (n 792, mean age 62 years, 60% women) were ascertained through sibships containing at least two individuals with essential hypertension diagnosed before age 60 years. The LDL particle size was measured by polyacrylamide gel electrophoresis. Coronary artery calcium (CAC) was measured noninvasively by electron beam computed tomography, and CAC score was calculated using the method of Agatston et al. Sexspecific multiple regression models were used to assess independent predictors of LDL particle size and the association of LDL particle size with CAC. Results: In all, 76% of women and 77% of men were hypertensive. In each sex, independent predictors of smaller LDL particle size were total cholesterol, triglycerides, and lower HDL cholesterol. In women, greater age was an additional predictor of smaller LDL particle size. After adjustment for age and statin use, LDL particle size was significantly associated with the amount of CAC in women but not in men. After further adjustment for HDL cholesterol, triglycerides, diabetes, smoking, and hypertension, LDL particle size was not independently associated with CAC in either sex. Conclusions: After adjustment for age and statin use, LDL particle size was found to be significantly related to CAC quantity in women but not in men belonging to hypertensive sibships. In women, LDL particle size may mediate some of the atherogenic effects of low-hdl cholesterol-high-triglyceride dyslipidemia, but does not appear to be independently associated with the extent of coronary atherosclerosis in either sex. Am J Hypertens 2004;17: American Journal of Hypertension, Ltd. Key Words: Atherosclerosis, low-density lipoprotein particle size, coronary artery calcification, risk factors, electron beam computed tomography. Clustering of cardiovascular risk factors is common in subjects with essential hypertension. 1 In particular, dyslipidemia is frequent in hypertensive subjects, and up to 27 million American adults may have concomitant hypertension and dyslipidemia. 2 Heritable abnormalities in pathways common to both blood pressure (BP) regulation and lipoprotein metabolism may account in part for the clustering of hypertension and dyslipidemia. Williams et al were the first to describe the syndrome of familial dyslipidemic hypertension. 3 They noted that low high-density lipoprotein (HDL) cholesterol-high-triglyceride dyslipidemia was frequent in hypertensive sibships. Individuals with so-called dyslipidemic hypertension are at increased risk for coronary heart disease (CHD), and a Received March 4, First decision April 27, Accepted June 7, From the Divisions of Cardiovascular Diseases (IJK) and Biostatistics (KRB) and the Departments of Laboratory Medicine and Pathology (JPM), Diagnostic Radiology (PFS), and Hypertension (STT), Mayo Clinic and Foundation, Rochester, Minnesota; Department of Epidemiology (PAP, LFB, SLRK), University of Michigan, Ann Arbor, Michigan; and Human Genetics Center and Institute of Molecular Medicine (EB), University of Texas-Houston Health Science Center, Houston, Texas. This work was supported by a Grant-in-Aid ( Z) from the American Heart Association (Northland affiliate); a tored Patient- Oriented Research Career Development Award (K-23, RR17720) (to IJK), by grant U10-HL54464, and the General Clinical Research Center Grant M01 RR00585, from the National Institutes of Health (Bethesda, Maryland). Address correspondence and reprint requests to Iftikhar J. Kullo, Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, MN 55905; kullo.iftikhar@mayo.edu 2004 by the American Journal of Hypertension, Ltd. Downloaded from Published by Elsevier Inc /04/$30.00 doi: /j.amjhyper

2 846 LDL PARTICLE SIZE AND CORONARY ATHEROSCLEROSIS AJH September 2004 VOL. 17, NO. 9 high prevalence of familial dyslipidemic hypertension has been observed in CHD-prone pedigrees. 4 Small, dense low-density lipoprotein (LDL) particle phenotype is a heritable trait that has been found to coexist with low-hdl cholesterol high-triglyceride dyslipidemia. 5 Particles of LDL are heterogeneous in terms of size and density, and small LDL particles are considered to be more atherogenic than larger LDL particles. 5 Several prospective and retrospective studies have shown that LDL particle size is associated with an increased risk of CHD events However, other studies have failed to confirm LDL particle size as an independent predictor of CHD events. 11 We aimed to explore further the cardiovascular risk from smaller LDL particle size in hypertensive sibships by studying its relation to coronary artery calcium (CAC), a quantitative measure of coronary atherosclerotic burden. The presence and quantity of CAC as measured by electron beam computed tomography (EBCT) is a reliable surrogate for the presence and severity of coronary atherosclerosis and correlates with the extent of disease by angiography. 12 We hypothesized that LDL particle size would add to the prediction of CAC in subjects with hypertension or family history of hypertension, beyond conventional risk factors. Methods Study Population The study was approved by the Institutional Review Board of the Mayo Clinic, and subjects gave informed consent. Subjects were participating in the Genetic Epidemiology Network of Arteriopathy (GENOA) study, a communitybased multicenter study of hypertensive sibships that aims to identify genes influencing BP. 9 Sibships that had at least two full siblings with essential hypertension diagnosed before the age of 60 years were recruited into the GENOA study. In Rochester, Minnesota, all participants are of white, non-hispanic ethnicity. Probands were identified through the Rochester Epidemiology Project and the Mayo Medical Center diagnostic index for residents of Olmsted County, Minnesota. When a full sibling with hypertension was identified, the entire sibship was invited to participate. Enrollment in phase I of GENOA ended June In phase II of GENOA, participants are being assessed for measures of target organ disease. Subjects with history of coronary artery bypass surgery or percutaneous coronary intervention are not eligible for EBCT. Subjects who gave a previous history of myocardial infarction or stroke (22 women and 17 men) were included. Through November 2001, a total of 815 participants had completed the study protocol, including measurement of CAC by EBCT. We excluded 23 subjects with missing data. Height was measured by stadiometer, weight by electronic balance, and body mass index was calculated (kg/ m 2 ). Waist circumference was measured at the umbilicus. The ratio of waist circumference to hip circumference (waist-to-hip ratio) was calculated as a measure of body fat distribution. Resting systolic BP and diastolic BP levels were measured in the right arm with a random-zero sphygmomanometer (Hawksley and Sons, West Sussex, UK). Three measures at least 2 min apart were taken, and the average of the second and third measurements was used in the analysis. Blood samples were obtained by venipuncture after an overnight fast. Plasma glucose was measured by the glucose oxidase method. Standard enzymatic methods were used to measure total cholesterol, HDL cholesterol, and triglycerides. 13 Friedewald s equation 14 was used to calculate LDL cholesterol. Diabetes was considered to be present if an individual was being treated with insulin or oral agents or had a fasting glucose level 126 mg/dl. Ever smoking was defined as having smoked 100 cigarettes in the past. The diagnosis of hypertension was confirmed based on BP levels measured at the study visit (systolic BP 140 or diastolic BP 90 mm Hg) or report of a prior diagnosis of hypertension and current treatment with antihypertensive medications. Information about the use of statins was obtained from a questionnaire administered to the participants. Measurement of LDL Particle Size The LDL particle size was measured by polyacrylamide gel electrophoresis. 15 Plasma (25 L) was mixed with 200 L of loading gel (containing Sudan black B dye and riboflavin) and added to the top of a precast 3% polyacrylamide gel tube (Quantimetrix Corp., Redondo Beach, CA). After photopolymerization for 30 min, specimens were electrophoresed for 1 h. The dye preferentially binds to lipoprotein particles and remains with them during electrophoresis. Separation is based primarily on particle size, due to the sieving action of the polyacrylamide gel. The separated lipoprotein particles were scanned with an ArtixScan 1100 (Microtek, Carson, CA). The electrophoretograms were quantitatively analyzed using the public domain National Institutes of Health Image program (developed at the United States National Institutes of Health and available on the Internet at The program divides the electrophoretogram at designated electrophoretic mobility values and calculates the area under the curve for each mobility fraction. Particle diameter for each fraction was calculated as previously described 16 and the mean LDL particle size determined by averaging diameters for each fraction adjusted for area under the curve. Electron Beam Computed Tomography of the Heart The quantity of CAC was measured with an Imatron C-150 EBT scanner (Imatron Inc., South San Francisco, CA). 17,18 Each scan consisted of 40 continuous, 3-mm-

3 AJH September 2004 VOL. 17, NO. 9 LDL PARTICLE SIZE AND CORONARY ATHEROSCLEROSIS 847 Table 1. Subject characteristics (n 470) (n 322) P Age (y) LDL particle size (Å) Small LDL ( 266 Å), n (%) 119 (25%) 111 (34%).005 Waist circumference (cm) Body mass index Waist-to-hip ratio Total cholesterol (mg/dl) Triglycerides (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Systolic bloodpressure mm Hg Diastolic bloodpressure mm Hg Hypertension, n (%) 360 (77%) 246 (76%).95 Fasting glucose mg/dl Diabetes, n (%) 65 (14%) 60 (19%).07 Statin use, n (%) 119 (25%) 109 (34%).009 Ever smoked, n (%) 187 (40%) 198 (61%).001 Detectable CAC, n (%) 276 (59%) 280 (87%).001 CAC score Å angstrom; CAC, coronary artery calcium; HDL high-density lipoprotein; LDL low-density lipoprotein. Continuous variables are presentedas mean SD, andcategorical variables as counts andpercentages. P value is for difference between women andmen using Wilcoxon rank-sum test for continuous variables and 2 test for dichotomous variables. LDL cholesterol could not be calculated in 17 individuals with triglycerides 400 mg/dl. thick computed tomograms (transverse two-dimensional images) commencing at the root of the aorta cephalad to the coronary sinuses and proceeding caudally through the entire coronary arterial tree (12 cm). An electrocardiograph triggering at 600 msec past the peak of the R wave was used so that all images were obtained during diastole when cardiac motion was minimal. Exposure time for each tomogram was 100 msec. Tomograms were scored by a technologist using an automated scoring system. 19 Calcification was defined as a hyperattenuating focus of four or more adjacent pixels in size (1.38 mm 2 under a field of view of 30 cm) with a CT number 130 Hounsfield Units within 5 mm of the arterial midline. An experienced radiologist interpreted the findings of each tomogram. According to the method of Agatston et al, 20 a score for each focus of CAC was determined and the total calcium score was obtained by summing individual lesion scores from each of the four anatomic sites (left main, left anterior descending, circumflex, and right coronary arteries). Statistical Analysis All analyses were performed separately for women and men. To understand better the relationship between LDL particle size and CAC, we first assessed for potential confounding due to possible association of LDL particle size with CHD risk factors. Using linear regression models, we assessed the association of LDL particle size with each of the following: 1) risk factors for CHD such as age, smoking, diabetes, hypertension, total cholesterol, HDL cholesterol, and triglycerides; 2) measures of adiposity such as body mass index, waist circumference, and waistto-hip ratio; and 3) statin use. Stepwise multiple linear regression models were then fit to identify independent significant predictors of LDL particle size in each sex. In the second set of analyses we studied the relationship of LDL particle size to CAC. Because the distribution of CAC scores was positively skewed and because not all subjects had detectable CAC, the scores were log-transformed after adding 1. Using linear regression analyses, we assessed the association of transformed CAC (referred to hereafter as CAC) with each of the following lipid measures: total cholesterol, HDL cholesterol, LDL cholesterol, log triglycerides, and LDL particle size, and each of the other CHD risk factors such as ever smoking, diabetes, and hypertension, after adjustment for age and statin use. Multivariable regression models were then constructed to assess independent predictors of CAC, including LDL particle size and the other lipid and nonlipid measures that were identified in the preceding analyses to be significantly related to CAC in either sex. Statistical significance was inferred at P values.05. Statistical analyses were performed with SAS version 8.2 software (SAS Institute, Cary, NC). Because of the presence of sibships in the sample, population-averaged generalized estimating equations (GEE) were used to assess the possible impact of familial correlations on the relationships between predictor and outcome variables. Because inferences were the same, only regression results are reported.

4 848 LDL PARTICLE SIZE AND CORONARY ATHEROSCLEROSIS AJH September 2004 VOL. 17, NO. 9 Table 2. Predictors of low-density lipoprotein (LDL) particle size in women and men (from a stepwise multiple regression model) (n 470) (n 322) SE P SE P Age Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Log triglycerides Model R R HDL high-density lipoprotein; SE standard error. regression coefficient for a 1-unit change for quantitative variables. Results Of the subjects, 60% were women; the mean age of both women and men was 62 years (Table 1). Mean LDL particle size was significantly greater in women than in men, and small dense LDL phenotype (defined as LDL particle size 266 angstroms, [Å]) was significantly less common in women than in men. The prevalence of detectable CAC and the quantity of CAC was significantly lower in women than in men. In separate linear regression models, body mass index, total cholesterol, HDL cholesterol, and log triglycerides were each significantly associated with LDL particle size in women and in men. Age, waist circumference, waistto-hip ratio, fasting blood sugar, and statin use were also predictors of LDL particle size in women (analyses not shown). In multivariable regression models, independent predictors of LDL particle size included total cholesterol, HDL cholesterol, and log triglycerides in men and in women (Table 2). Age was also an independent predictor of LDL particle size in women but not in men. Measures of adiposity including body mass index, waist circumference, and waist-to-hip ratio were not independently associated with LDL particle size in either sex. After adjustment for age and statin use, LDL particle size was found to be a significant predictor of CAC in women but not in men (Table 3). Likewise, other lipid measures including total cholesterol, LDL cholesterol, and triglycerides were also significantly related to CAC in women but not in men. No statistically significant interactions were noted between LDL particle size and conventional risk factors (diabetes, ever smoking, elevated total cholesterol, and low-hdl cholesterol) in the prediction of CAC (analyses not shown). In a multivariable model that considered LDL particle size, HDL cholesterol, triglycerides, and other conventional risk factors, we observed that only age, HDL cholesterol, statin use, total cholesterol, smoking history, diabetes, and hypertension remained significant predictors of CAC in women, and only age, smoking, and diabetes remained significant predictors of CAC in men (Table 4). Discussion The main finding of our study is that in subjects belonging to hypertensive sibships, LDL particle size is significantly (inversely) associated with the quantity of CAC in women Table 3. Sex-specific prediction of coronary artery calcium (CAC) by various lipid measures and other conventional risk factors, considered one-at-a-time, after adjusting for age and statin-use SE P SE P Age* Statin use Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Log triglycerides LDL particle size (Å) Ever smoking Diabetes Hypertension Abbreviations as in Table 2. LDL cholesterol could not be calculated in 17 individuals with triglycerides 400 mg/dl. * Age estimates are adjusted for statin use; statin use estimates are adjusted for age.

5 AJH September 2004 VOL. 17, NO. 9 LDL PARTICLE SIZE AND CORONARY ATHEROSCLEROSIS 849 Table 4. Predictors of coronary artery calcium (CAC) in multiple regression models in women and men SE P SE P Age Statin use Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Log triglycerides LDL particle size (Å) Ever smoked Diabetes Hypertension Abbreviations as in Table 2. but not in men, after adjusting for age and statin use. The association of LDL particle size with CAC in women, however, was not independent of HDL cholesterol and other conventional risk factors. The LDL particle size, a component of low-hdl cholesterol high-triglyceride dyslipidemia in hypertensive families, may mediate some of the atherogenic effects of this syndrome, but does not independently predict the extent of coronary atherosclerosis. Several prospective studies have demonstrated that small LDL particle size may be a predictor of future cardiovascular events. 10,21,22 In the Quebec Heart Study, 22 men in the first tertile of the LDL particle diameter had a 3.6-fold increase in the risk of CHD compared with those in the third tertile. The relationship remained significant after adjustment for variations in LDL cholesterol, triglycerides, HDL cholesterol, and apolipoprotein B concentrations. In the other two prospective studies, however, an independent association of LDL particle size to the risk of future cardiovascular events was not demonstrated. 10,21 In none of the studies was an interaction noted between sex and LDL particle size in the prediction of CHD events. The relationship of LDL particle size to the extent of coronary atherosclerosis has been less well studied. After adjusting for age and statin use, we noted LDL particle size to be significantly related to CAC in women but not in men. However, mirroring the results of studies looking at cardiovascular events as an endpoint, this relationship is not independent of other risk factors. Mackey et al, 23 in a study of 286 postmenopausal women, found smaller LDL particle size to be significantly associated with increased quantity of CAC after adjustment for LDL cholesterol and triglycerides but not after further adjustment for age, total cholesterol, smoking, and systolic BP. Both HDL cholesterol and triglycerides were significant independent predictors of LDL particle size. Significant intercorrelation was noted between these three lipid variables (Fig. 1), and the resulting multicollinearity makes it difficult to determine the independent contribution of LDL particle size to the extent of coronary atherosclerosis. It is possible that in women, smaller LDL particle size may be a mediator of the atherogenicity of low-hdl cholesterol high-triglyceride dyslipidemia. 5,24 A causal role for small LDL particles is suggested by studies showing that such particles may be more atherogenic compared to larger LDL particles. Small LDL particles have been shown to be more susceptible to oxidation 25 and to have increased affinity for intimal proteoglycans, 26 increased ability to cross into the subintimal space, 27 and reduced affinity for the LDL receptor. 28 In this study, we noted that other lipid variables (total cholesterol, HDL cholesterol, and triglycerides) were as- FIG. 1. A) Correlations between LDL particle size (angstroms [Å]) andhdl cholesterol (mg/dl) in women andmen. B) Correlations between LDL particle size andlog triglycerides in women andmen.

6 850 LDL PARTICLE SIZE AND CORONARY ATHEROSCLEROSIS AJH September 2004 VOL. 17, NO. 9 sociated with CAC in women but not in men. When we assessed the relationship of lipid variables to CAC in the entire sample in age-adjusted multiple regression models, a significant interaction was noted between log triglycerides and sex, and between LDL particle size and sex in the prediction of CAC (analyses not shown). A recent crosssectional study of risk factors and CAC demonstrated total cholesterol to be independently associated with CAC in women but not in men. 29 These findings suggest that the atherogenicity of certain lipid variables may be modified by sex. An alternative explanation could be that the association of lipid variables with coronary atherosclerosis is weakened in the setting of the relatively advanced CAC noted in the men in this study. 30 A limitation of our study is the use of cross-sectional data, which does not allow us to infer causal pathways underlying the observations. The findings may be applicable only to individuals from hypertensive sibships. When we limited the analyses to hypertensive subjects (n 606), the inferences did not change. It is possible that the relationship of LDL particle size to CAC differs in younger persons, in normotensive persons, and in individuals of other ethnicities. We did not assess the relationship of individual subclasses of LDL particle size to CAC. Williams et al recently showed that only the smallest fraction of LDL particle size was significantly related to progression of coronary atherosclerosis. 31 In conclusion, among subjects belonging to hypertensive sibships, LDL particle size was significantly related to CAC in women but not in men, after adjustment for age and statin use. In addition, LDL particle size was significantly correlated with HDL cholesterol and triglycerides. In women, LDL particle size may mediate some of the atherogenic effects of low-hdl cholesterol high-triglyceride dyslipidemia, but does not appear to be independently associated with the extent of coronary atherosclerosis in either sex. Acknowledgment The authors thank Brandon Grossardt, MS, for assistance with data analyses. References 1. Kannel WB: Risk stratification in hypertension: new insights from the Framingham Study. Am J Hypertens 2000;13:3S 10S. 2. American Heart Association: Heart Disease and Stroke Statistics 2004 Update. American Heart Association, Dallas, Williams RR, Hunt SC, Hopkins PN, Stults BM, Wu LL, Hasstedt SJ, Barlow GK, Stephenson SH, Lalouel JM, Kuida H: Familial dyslipidemic hypertension. Evidence from 58 Utah families for a syndrome present in approximately 12% of patients with essential hypertension. JAMA 1988;259: Williams RR, Hopkins PN, Hunt SC, Wu LL, Hasstedt SJ, Lalouel JM, Ash KO, Stults BM, Kuida H: Population-based frequency of dyslipidemia syndromes in coronary-prone families in Utah. Arch Intern Med 1990;150: Austin MA, Brunzell JD, Fitch WL, Krauss RM: Inheritance of low density lipoprotein subclass patterns in familial combined hyperlipidemia. Arteriosclerosis 1990;10: Campos H, Blijlevens E, McNamara JR, Ordovas JM, Posner BM, Wilson PW, Castelli WP, Schaefer EJ: LDL particle size distribution. Results from the Framingham Offspring Study. Arterioscler Thromb 1992;12: Coresh J, Kwiterovich PO Jr, Smith HH, Bachorik PS: Association of plasma triglyceride concentration and LDL particle diameter, density, and chemical composition with premature coronary artery disease in men and women. J Lipid Res 1993;34: Austin MA, Breslow JL, Hennekens CH, Buring JE, Willett WC, Krauss RM: Low-density lipoprotein subclass patterns and risk of myocardial infarction. JAMA 1988;260: Griffin BA, Freeman DJ, Tait GW, Thomson J, Caslake MJ, Packard CJ, Shepherd J: Role of plasma triglyceride in the regulation of plasma low density lipoprotein (LDL) subfractions: relative contribution of small, dense LDL to coronary heart disease risk. Atherosclerosis 1994;106: 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: Sacks FM, Campos H: Low-density lipoprotein size and cardiovascular disease: a reappraisal. J Clin Endocrinol Metab 2003;88: Bielak LF, Peyser PA, Sheedy PF II: Electron-beam computed tomography screening for asymptomatic coronary artery disease. Semin Roentgenol 2003;38: Kottke BA, Moll PP, Michels VV, Weidman WH: Levels of lipids, lipoproteins, and apolipoproteins in a defined population. Mayo Clin Proc 1991;66: Friedewald WT, Levy RI, Fredrickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18: Hoefner DM, Hodel SD, O Brien JF, Branum EL, Sun D, Meissner I, McConnell JP: Development of a rapid, quantitative method for LDL subfractionation with use of the Quantimetrix Lipoprint LDL System. Clin Chem 2001;47: Kazumi T, Kawaguchi A, Hozumi T, Nagao M, Iwahashi M, Hayakawa M, Ishihara K, Yoshino G: Low density lipoprotein particle diameter in young, nonobese, normolipidemic Japanese men. Atherosclerosis 1999;142: Turner ST, Bielak LF, Narayana AK, Sheedy PF, Schwartz GL, Peyser PA: Ambulatory blood pressure and coronary artery calcification in middle-aged and younger adults. Am J Hypertens 2002; 15: Lange LA, Lange EM, Bielak LF, Langefeld CD, Kardia SL, Royston P, Turner ST, Sheedy PF, Boerwinkle E, Peyser PA: Autosomal genome-wide scan for coronary artery calcification loci in sibships at high risk for hypertension. Arterioscler Thromb Vasc Biol 2002;22: Reed JE, Rumberger JA, Davitt PJ. Reed JE, Rumberger JA, Davitt PJ: System for quantitative analyses of coronary calcification via electron beam tomography, in Hoffman EA, Acharya RS (eds): Medical Imaging 1994: Physiology and Function From Multidimensional Images. Bellingham, WA, International Society for Optical Engineering, 1994, pp Agatston AS, Janowitz WR, Hildner FJ, Zusmer NR, Viamonte M Jr, Detrano R: Quantification of coronary artery calcium using ultrafast computed tomography. J Am Coll Cardiol 1990;15: Stampfer MJ, Krauss RM, Ma J, Blanche PJ, Holl LG, Sacks FM, Hennekens CH: A prospective study of triglyceride level, lowdensity lipoprotein particle diameter, and risk of myocardial infarction. JAMA 1996;276:

7 AJH September 2004 VOL. 17, NO. 9 LDL PARTICLE SIZE AND CORONARY ATHEROSCLEROSIS 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: Mackey RH, Kuller LH, Sutton-Tyrrell K, Evans RW, Holubkov R, Matthews KA: Lipoprotein subclasses and coronary artery calcium in postmenopausal women from the healthy women study. Am J Cardiol 2002;90: Austin MA, King MC, Vranizan KM, Newman B, Krauss RM: Inheritance of low-density lipoprotein subclass patterns: results of complex segregation analysis. Am J Hum Genet 1988;43: de Graaf J, Hak-Lemmers HL, Hectors MP, Demacker PN, Hendriks JC, Stalenhoef AF: Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects. Arterioscler Thromb 1991;11: Camejo G, Hurt-Camejo E, Bondjers G: Effect of proteoglycans on lipoprotein-cell interactions: possible contributions to atherogenesis. Curr Opin Lipidol 1990;1: Nordestgaard BG, Nielsen LB: Atherosclerosis and arterial influx of lipoproteins. Curr Opin Lipidol 1994;5: Nigon F, Lesnik P, Rouis M, Chapman MJ: Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor. J Lipid Res 1991;32: Reilly MP, Wolfe ML, Localio AR, Rader DJ: Coronary artery calcification and cardiovascular risk factors: impact of the analytic approach. Atherosclerosis 2004;173: Newman AB, Naydeck BL, Sutton-Tyrrell K, Feldman A, Edmundowicz D, Kuller LH: Coronary artery calcification in older adults to age 99: prevalence and risk factors. Circulation 2001;104: Williams PT, Superko HR, Haskell WL, Alderman EL, 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: