High-Density Lipoprotein-Specific Phospholipid Efflux Assay

Plasma levels of high-density lipoprotein (HDL)-cholesterol (HDL-C) is currently a key metric for the clinical assessment of cardiovascular disease (CVD) risk. HDL-mediated removal of plaque lipids from cells and extracellular deposits in the arterial wall is one of several anti-atherogenic functions of HDL that may account for its inverse association with CVD risk. The HDL-mediated removal of cellular cholesterol in vitro has recently proven to be an even better predictor of CVD risk than HDL-C. HDL is composed of a heterogeneous population of particles, which perform different functions. HDL particles involved in the removal of atherosclerotic plaque lipids contain exchangeable apolipoproteins, mostly apolipoprotein A-I, that dissociate from the particle and then remove plaque lipids. Nascent HDL is formed by the solubilization and removal of both phospholipids and cholesterol from ABCA1-generated cellular plasma membrane domains by HDL-derived exchangeable apolipoproteins. This process plays a critical role in both the prevention and regression of atherosclerotic plaque. Herein, we describe the protocol for the cell-free, HDL-specific phospholipid efflux (HDL-SPE) assay, which we have previously shown can predict incident CVD risk. This assay specifically measures HDL apolipoprotein-mediated removal of a non-exchangeable fluorescent phospholipid from a lipid donor particle. Our case-control clinical studies have established that the HDL-SPE assay performs as well as and potentially even better than the "gold-standard" cell-based cholesterol efflux capacity assay, a laborious and technically complex assay that often utilizes radioactive cholesterol. The goal of this protocol is to enable basic and clinical researchers alike to assess the functionality of HDL in lipid mobilization, using a simple, standardized, cell-free, high-throughput assay. This assay can be used by basic researchers to gain insights into mechanisms underlying HDL-mediated lipid efflux and for screening new therapeutic agents that enhance HDL functionality. HDL-SPE can also serve as a clinical laboratory diagnostic assay for CVD risk assessment.