Abstract 105: Free Cholesterol Determines the Phospholipid Domain Size and Stability of rHDL

Abstract

Background: Microsolubilization of dimyristoyl phosphatidylcholine (DMPC) by human apolipoprotein A-I is a frequently used model system for the identification of determinants of macrophage cholesterol efflux, the first step in reverse cholesterol transport (RCT) to hepatic disposal. Although many studies have focused on microsolubilization of DMPC, most have been conducted under stoichiometric conditions resulting in ∼9 nm rHDL, and few have included free cholesterol (FC), a key lipid in RCT. Methods: Various rHDL species (excess DMPC + FC), were prepared and analyzed by size exclusion chromatography (SEC), differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy as a function of temperature. Results: At each mol% of FC/DMPC, multiple rHDL species differing in size from ∼10 to ∼25 nm were formed. In SEC, rHDL size increased with the mol% FC in the microsolubilization reaction and in a given reaction mixture the large rHDL were always more FC-rich than the small rHDL. In DSC, the DMPC transition range of rHDL was broader than that of pure DMPC and although the transition temperature moderately increases with increasing mol% FC, the apparent enthalpy of the transition is diminished. The thermal dependence of the CD spectra showed that increasing mol% FC and rHDL size moderately increases the stability of apo A-I. Conclusion: Although large FC-rich and small FC-poor domains coexist on DMPC surfaces; the size of these domains increases with mol% FC. This effect of FC on rHDL size decreases the overall cooperativity of the DMPC phase transition and reduces the number of acyl chain interactions. Physiologically, these data suggest that increased FC-loading of macrophages should increase the size of nascent HDL produced by the interaction of apo A-I with cellular ABCA1. The lower stability of FC-poor HDL would likely make it release more lipid-free apo A-I in response to important plasma proteins including LCAT, PLTP, and CETP and in response to serum opacity factor.