High-Density Lipoprotein Particle Size and Concentration and Coronary Risk

Context High-density lipoprotein (HDL) particles are heterogeneous in size and composition. Contribution This nested casecontrol study found that both HDL size and HDL particle concentration were associated with risk for coronary artery disease (CAD). High-density lipoprotein size was strongly associated with risk factors characteristic of the metabolic syndrome, such as triglyceride and apolipoprotein B levels. Adjustment for those factors explained the association between HDL size and CAD risk, but not the association between HDL particle concentration and CAD risk. Implication High-density lipoprotein size and HDL particle concentration are differentially associated with cardiovascular risk factors and CAD risk. The association between HDL size and CAD risk may be explained by metabolic parameters. The Editors The strong inverse relationship between high-density lipoprotein (HDL) (see Glossary) cholesterol levels and risk for coronary artery disease (CAD) is well established (1). However, at any given level of HDL cholesterol (see Glossary), HDL particle concentration (see Glossary) and HDL size (see Glossary) distribution may differ substantially between individuals. The atheroprotective role of HDL cholesterol is believed to be mediated mainly by its role in reverse cholesterol transport, the biological pathway that facilitates removal of cholesterol from macrophages in the arterial wall back to the liver (2). Substantial evidence suggests that HDL particles are heterogeneous in their efficacy to facilitate adenosine triphosphatebinding cassette A1 (see Glossary)mediated cholesterol efflux from macrophages and scavenger receptor class B1 (see Glossary)mediated hepatic uptake of cholesterol from HDL particles. In particular, this heterogeneity has been associated with HDL particle size (35). Accumulating evidence suggests that, in addition to their role in reverse cholesterol transport, HDL particles have anticoagulant, antioxidative, and anti-inflammatory properties that contribute to the antiatherogenic capacity of HDL cholesterol. In fact, HDL particles were recently shown to carry a wide array of proteins that mediate these properties (6). The binding affinity of these proteins to the surface of HDL particles may depend on their size (7, 8). Under certain conditions, HDL particles may lose their antiatherogenic capacity and become dysfunctional (9, 10). As a consequence, various HDL subpopulations may differ substantially in their capacity to play an atheroprotective role. Despite this functional heterogeneity, HDL has thus far been regarded as a single entity in epidemiologic studies, and the amount of cholesterol transported by HDL particles is traditionally being used for this purpose. However, we have recently shown that higher HDL cholesterol levels may not necessarily be associated with lower cardiovascular risk (11), and the HDL cholesterol levelincreasing drug torcetrapib has been shown to have detrimental effects (12). These findings emphasize that a broader perspective on HDL metabolism is warranted. We hypothesized that HDL particle concentration and HDL size distribution are differentially associated with cardiovascular risk factors and with cardiovascular risk. We tested these hypotheses in a casecontrol study nested in the EPIC (European Prospective Investigation into Cancer and Nutrition)-Norfolk cohort. Methods We performed a nested casecontrol study among participants of the EPIC-Norfolk study, a prospective population-based study of 25663 men and women age 45 to 79 years living in Norfolk, United Kingdom, who completed a baseline questionnaire survey and attended a clinic visit (Figure). Participants were recruited from agesex registers of general practices in Norfolk as part of the 10-country collaborative EPIC study, which was designed to investigate dietary and other determinants of cancer. Additional data were obtained in EPIC-Norfolk to enable the assessment of determinants of other diseases. Figure. Study flow diagram. ApoAI = apolipoprotein A-I; ApoB = apolipoprotein B; CRP = C-reactive protein; EPIC = European Prospective Investigation into Cancer and Nutrition; HDL = high-density lipoprotein; LDL = low-density lipoprotein; NMR = nuclear magnetic resonance. *Individual case patients and control participants may have several missing variables. The design and methods of the study have been described in detail (13). Eligible participants were recruited by mail. At the baseline survey between 1993 and 1997, participants completed a detailed health and lifestyle questionnaire. Nonfasting blood samples were obtained by venipuncture into plain and citrate bottles. Blood samples were processed for assay at the Department of Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom, or were stored at 80C. All individuals have been monitored for death certification at the United Kingdom Office of National Statistics, with vital status ascertained for the entire cohort. In addition, hospitalized participants were identified by their unique National Health Service number and data linkage with the East Norfolk Health Authority database, which identifies all hospital contacts throughout England and Wales for Norfolk residents. Coronary artery disease was defined as codes 410 to 414 according to the International Classification of Diseases, Ninth Revision. Participants were identified as having CAD during follow-up if they had had a hospital admission or had died with CAD as the underlying cause. Previous validation studies in our cohort indicate high specificity of such case ascertainment (14). We report results with follow-up to November 2003, an average of about 6 years. The study was approved by the Norwich District Health Authority Ethics Committee, and all participants gave signed, informed consent. Participants We have previously described other analyses within this prospective, nested casecontrol study (14, 15). We excluded all individuals who reported a history of heart attack or stroke or use of lipid-lowering drugs at the baseline clinic visit. Case patients were individuals who developed fatal or nonfatal CAD during follow-up until November 2003. Control participants were study participants who remained free of cardiovascular disease during follow-up. We matched 2 control participants to each case patient by age (within 5 years), sex, and time of enrollment (within 3 months). We performed the current analysis on all participants who had a complete data set available for baseline characteristics, apolipoproteins A-I and B, gradient gel electrophoresis (GGE)measured HDL size, and lipoprotein nuclear magnetic resonance (NMR) spectroscopy. We selected 1138 persons who were apparently healthy at baseline but did develop CAD during follow-up. For every case patient, we aimed to select 2 control participants who were healthy at baseline and remained free of cardiovascular disease during follow-up. From the original data set of 1138 case patients and 2237 control participants, 270 case patients and 430 control participants were excluded because at least 1 value was missing for any of the parameters mentioned. We excluded 46 case patients because they had no matching control participants and 406 control participants because they had no matching case patients. The analyses are therefore based on a data set of 822 case patients and 1401 control participants (243 case patients were matched with 1 control participant each, and 579 case patients were matched with 2 control participants each). Measurements We obtained data on smoking and alcohol consumption by health questionnaires at the baseline clinic visit. We obtained information on physical activity with a physical activity questionnaire (13). In the original cohort, we classified smoking into current cigarette smokers, former smokers, and those who never smoked. In the present study, we recorded smoking as yes or no. We divided physical activity into 4 categories: inactive, moderately inactive, moderately active, and active. We defined use of large amounts of alcohol as more than 21 units of alcohol per week. Diabetes mellitus and use of hormone replacement therapy was self-reported. We asked participants about medical history with the question, Has a doctor ever told you that you have any of the following? , followed by a number of choices, including diabetes. We recorded blood pressure by taking 2 measurements of diastolic and systolic blood pressure by using the Accutorr sphygmomanometer (Datascope, Huntingdon, United Kingdom) after 3 minutes of resting. We measured serum levels of total cholesterol, HDL cholesterol, and triglycerides on fresh samples with the RA 1000 (Bayer Diagnostics, Basingstoke, United Kingdom). We calculated low-density lipoprotein (LDL) cholesterol levels with the Friedewald formula to closely approach current clinical procedures. We measured plasma concentrations of C-reactive protein with a sandwich-type enzyme-linked immunosorbent assay as previously described (16). We measured serum levels of apolipoprotein A-I and apolipoprotein B by using rate immunonephelometry (Behring Nephelometer BNII, Dade Behring, Marburg, Germany) with calibration traceable to the International Federation of Clinical Chemistry primary standards (17). We measured serum concentration of myeloperoxidase (see Glossary) by using a commercially available enzyme-linked immunosorbent assay (CardioMPO Test, Prognostix, Cleveland, Ohio) (15). We measured paraoxonase 1 (see Glossary) activity as previously described (18). We measured HDL size by 4% to 30% nondenaturing polyacrylamide GGE as previously described (19). We measured lipoprotein subclass particle concentrations and average size of particles by proton NMR spectroscopy (LipoScience, Raleigh, North Carolina) as previously described (20). In brief, we obtained particle concentrations of lipoprotein subclasses of different size directly from the measured amplitudes of t