Abstract 556: Lipoprotein Remodeling and Cholesterol Exchange Monitored Using Fluorescent Lipids and Proteins

We developed a sensitive and robust in vitro method to monitor lipoprotein cholesterol and protein exchange and lipoprotein remodeling, using non-exchangeable fluorescent phosphatidylethanolamine (PE) as a lipoprotein marker. We applied this method to monitor the exchange of unesterified cholesterol (FC) and apoA-I among isolated human lipoproteins and synthetic lipoprotein-X (LpX). Fluorescent FC, but not PE, rapidly equilibrated between VLDL and HDL, and transferred almost entirely from VLDL or HDL to LDL. Fluorescent apoA-I bound specifically to HDL and remodeled fluorescent PE and FC-labeled LpX into a new lipoprotein particle that contained both fluorescent lipids and apoA-I. LpX-derived fluorescent PE incorporated into plasma HDL only. The incorporation of LpX-derived fluorescent FC into plasma lipoproteins was similar to fluorescent FC alone, consistent with remodeling of LpX to HDL with concomitant exchange of FC between lipoproteins. LPL remodeled fluorescent PE and FC-tagged VLDL into a new particle containing both fluorescent lipids and apoA-I. We also developed a model system to study lipid transfer in vitro and in vivo by depositing lipids on calcium silicate hydrate crystals to form dense lipid coated donor particles that are readily separated from acceptor membranes and can be used as a surrogate for cell-dependent cholesterol efflux. These methodologies can readily be applied to study the other members of the vast lipoprotein proteome and the wide variety of remodeling events involved in lipoprotein-mediated lipid homeostasis in health and disease.