Objective-We measured efflux from macrophages to apolipoprotein B-depleted serum from 263 specimens and found instances in which serum having similar high-density lipoprotein cholesterol (HDL-C) differed in their efflux capacity. Thus, we wanted to elucidate why efflux capacity could be independent of total HDL-C or apolipoprotein A-I (apoA-I). Methods and Results-To understand why sera with similar HDL-C or apoA-I could differ in total efflux capacity, we assessed their ability to promote efflux via the pathways expressed in cAMP-treated J774 macrophages. Briefly, macrophages were preincubated with probucol to block ABCA1, with BLT-1 to block SR-BI, and with both inhibitors to measure residual efflux. ABCG1 efflux was measured with transfected BHK-1 cells. We used apolipoprotein B-depleted serum from specimens with similar HDL-C values at the 25 th and 75 th percentiles. Specimens in each group were classified as having high or low efflux based on total efflux being above or below the group average. We found that independently of HDL-C, sera with higher efflux capacity had a significant increase in ABCA1-mediated efflux, which was significantly correlated to the concentration of pre␤-1 HDL. The same result was obtained when these sera were similarly analyzed based on similar apoA-I. Key Words: ABCA1 Ⅲ apolipoprotein A-I Ⅲ cholesterol efflux Ⅲ high-density lipoprotein cholesterol Ⅲ macrophages E pidemiological and interventional studies 1-4 demonstrate an inverse relationship between high-density lipoprotein cholesterol (HDL-C) levels and coronary heart disease, which is an observation also supported by animal studies. 5 Thus, high HDL-C levels are thought to independently reduce coronary heart disease risk. Although HDL has been shown to have both antioxidant and antiinflammatory properties, 6 its beneficial antiatherogenic effect is likely attributable to its central role in reverse cholesterol transport, ie, the transport of cholesterol from peripheral tissues to the liver for excretion to reduce its accumulation in tissue cells such as vessel wall macrophages. 7 Because both HDL metabolism and cholesterol transport are complex processes, it has been difficult to obtain in vivo evidence that modulating HDL levels can affect removal of cholesterol from macrophage foam cells in the vessel wall and reduce atherosclerotic lesions. Overexpression of apolipoprotein (apo) A-I in mice can reduce progression of atherosclerotic lesions, 8 and infusion of apoA-I/phospholipid complexes in humans promotes lesion regression and increases fecal excretion of bile acids. 9 However, results of studies in subjects with monogenic disorders of HDL metabolism 10,11 and post hoc analyses of epidemiological studies raise questions regarding the mechanism underlying the association between HDL-C levels and coronary heart disease. 12 We recently demonstrated that in healthy individuals having a wide range of HDL-C and apoA-I levels, the capacity of serum HDL to promote cholesterol efflux from macrophages in vitro is negatively correla...

Objective-High-density lipoprotein (HDL) is a heterogeneous lipoprotein class and there is no consensus on the value of HDL subspecies in coronary heart disease (CHD) risk assessment. We tested the hypothesis whether specific HDL subpopulations are significantly associated with CHD-prevalence. Methods and Results-ApoA-I concentrations (mg/dL) in HDL subpopulations were quantitatively determined by native 2d gel electrophoresis, immunoblotting, and image analysis in male participants in the Framingham Offspring Study (FOS). CHD cases (nϭ169) had higher pre␤-1 and ␣-3 particle and lower ␣-1, pre␣-3, and pre␣-1 particle levels than either all (nϭ1277) or HDL cholesterol-matched (nϭ358) controls. ␣-1 and pre␣-3 levels had an inverse association, whereas ␣-3 and pre␣-1 particle levels had a positive association with CHD prevalence after adjusting the data for established CHD risk factors. Standardized logit coefficients indicated that ␣-1 HDL was most significantly associated with CHD prevalence. Moreover, each mg/dL increase in ␣-1 particle level decreased odds of CHD by 26% (PϽ0.0001), whereas each mg/dL increase in HDL cholesterol decreased odds of CHD by 2% in a model including all established CHD risk factors. Conclusions-Specific HDL subpopulations were positively correlated, whereas others were inversely correlated with CHD prevalence in male subject in the FOS, indicating that the various HDL particles might have different roles in the cause of CHD. [1][2][3][4] Traditionally, HDL has been separated into major subclasses by polyanion precipitation, ultracentrifugation (HDL2 and HDL3), or by the apolipoprotein content, distinguishing particles containing only apoA-I (LpA-I), the major apolipoprotein of HDL, from particles containing both apoA-I and apoA-II (LpA-I:A-II). None of these techniques has provided any convincing evidence that 1 kind of HDL subfraction has any greater cardioprotective function than another. [5][6][7][8][9][10] The lack of agreement among these studies is probably related to the fact that all of these HDL subfractions are themselves heterogeneous, containing a variety of different HDL subspecies with possibly different physiological functions.Our laboratory uses native 2-dimensional gel electrophoresis, immunoblotting, and image analysis to separate HDL subpopulations quantitatively from plasma with highresolution based on electrophoretic charge and particle size. 11 We determine apoA-I content, not cholesterol, in these particles. This method has been useful in studies of HDL metabolism and cholesterol transport from cells because it separates intermediates in these processes. 12 A small casecontrol study indicated that coronary heart disease (CHD) patients not only had HDL deficiency but also had a major rearrangement in the apoA-I-containing HDL subpopulations with significantly lower levels of the large ␣-1 and pre␣-1 (Ϸ11 nm), and higher levels of the small ␣-3 (Ϸ8.4 nm) and pre␤-1 (Ϸ5.6 nm) HDL particles than controls. 13 Among these particles, ␣-3 contains both apoA-I and apoA-...