Systematic Review: Association of Low-Density Lipoprotein Subfractions With Cardiovascular Outcomes

A critical component of lowering the cardiovascular disease burden across the population is identification and aggressive treatment of high-risk individuals. The Adult Treatment Panel III of the Expert Panel of the National Cholesterol Education Program (1) has identified a group of risk factors associated with cardiovascular disease, including elevated low-density lipoprotein (LDL) cholesterol concentrations, cigarette smoking, hypertension, reduced high-density lipoprotein (HDL) cholesterol concentrations, family history of premature coronary heart disease, and older age. Current efforts have focused on determining whether additional diagnostic criteria could improve the accuracy of cardiovascular disease risk estimation (25). Measures of LDL subfractions have been suggested as a potential risk factor. Many terms are used to describe the characteristics and distribution of LDL particles; these include LDL subclasses, particles, particle concentration, particle numbers, and various patterns. These terms describe separate, but sometimes overlapping, features of the LDL particle. To simplify matters, we use the generic term subfractions except when describing specific measurements. Despite this simplification, we are not suggesting that the disparate methods for analyzing LDL can be fully subsumed in a single concept. Numerous methods are used to measure or define LDL subfractions. Table 1 lists the principal methods used and the most commonly reported subfraction measures. Only a few of these disparate systems to estimate LDL subfractions are routinely available, and only from selected clinical laboratories. Table 1. Commonly Used LDL Subfraction Tests and Terms If LDL subfractions are predictive of cardiovascular risk and are of incremental value when added to established cardiovascular risk factors, it remains to be determined whether the different characteristics of the LDL subfractions assessed by various methods would result in similar predictive abilities for estimating cardiovascular risk. Lipid researchers have proposed that small, dense LDL particles confer greater atherogenic risk than larger, less dense LDL particles (6, 7). In vitro, small, dense LDL particles are more avidly taken up by macrophages than larger, less dense LDL particles; are more susceptible to oxidative modification, have a greater propensity for transport into the arterial subendothelial space; and have a greater binding potential to arterial wall proteoglycans (8, 9). The American Diabetes Association and the American College of Cardiology Foundation convened a panel of experts to develop a consensus position for patients with cardiometabolic risk (10). They noted that limited data from cross-sectional and prospective studies suggest that LDL particle number may be a better discriminator of cardiometabolic risk than LDL cholesterol concentrations. They pointed out several limitations, including availability and accuracy of the method and consistency of the predictive power across ethnic groups, ages, and conditions that affect lipid metabolism. They concluded that it is yet to be determined whether treatment decisions would be improved if LDL subfraction measurements were added to the current risk factors used to estimate cardiovascular risk. We sought to evaluate the association between LDL subfractions and incidence and progression of clinical cardiovascular disease. We focus primarily on the LDL subfraction tests that are available for routine use by clinical laboratories and are thus available to all U.S. clinicians and their patients. We also summarize the potential value of LDL subfraction tests used only in research laboratories. An earlier version of this systematic review was conducted as part of a Technology Assessment for the Centers for Medicare & Medicaid Services (11). Methods Data Sources and Searches We conducted a comprehensive search of the scientific literature to identify relevant studies in MEDLINE (1950 to 5 January 2009), CAB Abstracts (1973 to 30 June 2008), and the Cochrane Central Register of Controlled Trials (second quarter of 2008). Appendix Table 1 lists search terms for LDL, particle size or subfractions, and test methodologies. We limited the literature searches to humans and English-language publications. The searches were supplemented by screening reference lists of included studies and selected reviews and requesting more information from domain experts. Appendix Table 1. Search Strategy Study Selection Three investigators screened all citations and retrieved articles for eligibility. We included studies of any prospective, longitudinal design that reported an association between any measure of LDL subfractions and either incident cardiovascular disease (cardiac, cerebrovascular, or peripheral vascular disease) or progression of disease severity (for example, coronary atherosclerosis) and had at least 10 adults per study group. Serum (or plasma) samples must have been obtained before determination of outcomes. We evaluated only clinical outcomes or measures of atherosclerosis on which clinical decisions are made (for example, minimum lumen diameter). We placed no further restrictions on study populations and included studies of people with and without cardiovascular disease at baseline. No minimum follow-up duration was required. Data Extraction One of the three authors extracted data from each study, and at least 1 additional author reviewed and verified the extractions. Full data extraction, including quality assessment, was performed for studies that used specific methods or kits that are currently available to clinical laboratories (as opposed to research laboratories). From the best information available to us from the Centers for Medicare & Medicaid Services, the U.S. Food and Drug Administration, domain experts, the reviewed studies, internet searches, invited reviewers, and conversations with several laboratories, we limited the full analysis to nuclear magnetic resonance (NMR), the LipoPrint kit (Quantimetrix, Redondo Beach, California) for linear polyacrylamide gel electrophoresis, gradient gel electrophoresis performed at Berkeley HeartLab (LDL-S3 GGE Test, Berkeley HeartLab, Burlingame, California), an ultracentrifugation technique performed at the University of Washington's Northwest Lipid Research Laboratory, and the Vertical Auto Profile (Atherotech, Birmingham, Alabama). For other laboratory methods, we extracted only limited results data: the type of LDL subfraction measurement (particle size, particle concentration or number, or pattern of LDL subfraction distribution [small, medium, or large LDL, or other subfractions]) and the direction and statistical significance of the association. These other laboratory methods included a range of gel electrophoresis and ultracentrifugation methods that are generally not standardized and are used only in the research setting, high-pressure liquid chromatography, capillary isotachophoresis, and other techniques. We analyzed both unadjusted and adjusted associations between LDL subfractions and clinical cardiovascular outcomes. For the purposes of this review, adjusted analyses were multivariable analyses in which the association between LDL subfraction and cardiovascular outcomes were adjusted for LDL cholesterol, HDL cholesterol, non-HDL cholesterol, or triglyceride concentrations; unadjusted analyses did not adjust for cholesterol concentrations but may have adjusted for other variables, such as other lipoprotein subfractions, clinical history, demographic characteristics, or blood pressure. Quality Assessment We assessed the methodological quality of each fully extracted study on the basis of predefined criteria (12). The primary data extractor determined the study quality, and at least 1 other extractor confirmed it. We used a 3-category grading system to denote the methodological quality of each study. Good-quality studies adhere most closely to the commonly held concepts of high quality, including clear descriptions of the population, setting, LDL subfraction measures, and analytic technique; appropriate measurement of outcomes; appropriate statistical analysis, including multivariable analysis adjusting for lipid measures; no obvious reporting omissions or errors; clear reporting of dropouts; and complete reporting of associations of interest for this systematic review. Fair-quality studies have some deficiencies, but these are unlikely to cause major bias. Poor-quality studies failed to adequately describe the measures, analyses, or results of interest or had substantial flaws in reporting or statistical analyses, such that major bias could not be excluded. The quality assessment was based specifically on the analysis of LDL subfractions and clinical cardiovascular outcomes, regardless of the primary analysis of interest to the original researchers. Role of the Funding Source The Agency for Healthcare Research and Quality participated in formulating the study questions but did not participate in the literature search; determination of study eligibility; data analysis or interpretation; or preparation, review, or approval of the manuscript for publication. Results The literature searches yielded 6724 citations (Figure), of which 476 were retrieved for further consideration for this and other research questions of interest. Of these, 24 met eligibility criteria. Ten studies (1322) used NMR to measure LDL subfractions. Although LipoPrint gel electrophoresis is among the methods more commonly used by clinical laboratories, we identified no study that used this kit to evaluate incidence or progression of cardiovascular disease (at least 6 LipoPrint studies all evaluated prevalent disease). Also, none of the eligible studies used the gradient gel electrophoresis performed at the Berkeley HeartLab or the ultracentrifugation method available at the Northwest Lipid Research Laboratory or the Vertical Auto Profile. An additional 14 studies