Abstract Body (Do not enter title and authors here): Introduction/Background
Cholesterol efflux capacity (CEC) is one of the atheroprotective functions exerted by cholesterol acceptors such as high-density lipoproteins (HDL) or apolipoprotein A-I (apoA-I), and it is a promising indicator for cardiovascular disease (CVD) risk assessment. While CEC is ordinally evaluated using cell-based methods, these are not suitable for clinical laboratories due to their complexity. In this context, we established a novel, clinically applicable CEC assay, the immobilized liposome-bound magnetic beads (ILM) method, which has shown good correlation with traditional cell-based methods. However, whether this cell-free method can reflect variations in CEC based on the inherent features of cholesterol acceptors remained unclear.

Research Questions/Hypothesis
Can the ILM method reflect the differences in CEC arising from variations in the lipid-to-protein composition of cholesterol acceptors?

Goals/Aim
This study aims to clarify the relationship between the protein-to-lipid composition of cholesterol acceptors and the CEC values measured by the ILM method, and to demonstrate the utility for assessing the functional quality of cholesterol acceptor.

Methods/Approach
CECs of HDL isolated by ultracentrifugation and apoA-I purified from HDL were measured using the ILM method at several protein concentrations. Additionally, reconstituted HDL (rHDL) was prepared by mixing apoA-I, free-cholesterol and lecithin at various ratios. The resulting rHDL particles were analyzed for size distribution, and their protein, cholesterol, phospholipid, and particle concentrations were determined. CECs of these rHDL samples were then measurementd using the ILM method.

Results/Data
The CEC of both HDL and apoA-I increased in a concentration-dependent manner, however, HDL exhibited extremely higher CEC than apoA-I. The prepared rHDLs exhibited a single, uniform size distribution respectively, and their particle diameters were positively correlated with molar ratio of cholesterol or phospholipid to apoA-I (chol/apoA-I, PL/apoA-I). Moreover, CEC per particle of rHDL was positively correlated with the chol/apoA-I and PL/apoA-I.

Conclusion
Cholesterol acceptors with higher lipid content, such as HDL or larger rHDL particles, presented higher CEC values in ILM the method. These results indicate that our assay can detect variations in CEC based on the degree of apoA-I lipidation, implying its potential utility for HDL quality evaluation and CVD risk assessment.