Application of Fluorescence Correlation Spectroscopy to Measure High-Density Lipoprotein (HDL) Metabolism

Abstract

High-density lipoprotein (HDL) protects vascular system from atherosclerosis by several mechanisms, including reverse cholesterol and therefore understanding its metabolism has important implications for public health. A major factor in HDL metabolism is endothelial lipase (EL). The goal of this study was to elucidate the kinetics of EL-HDL metabolism using fluorescence correlation spectroscopy (FCS). FCS is an advanced microscopy technique in which fluctuations in the fluorescence of a dye or dye-labeled molecule is recorded as the particles freely diffuse through a small focal volume. In this case, we measured the fluorescence of dye-labeled (Nile Red) HDL in the presence of wild type and mutant EL. The data can be analyzed mathematically using the cross-correlation function, from which the diffusion coefficient of the molecule is obtained. The lipase activity of EL changes HDL size, in turn affecting the diffusion coefficient, and can be calculated using the Stokes-Einstein relation. Our preliminary results suggest the hydrolysis of HDL occurs rapidly and proportionately to the concentration of EL. Several mutations in EL have been identified in human population studies. Our future goal is to compare the rate of hydrolysis between wild type and mutant EL and with normal and oxidized HDL. In addition, fluorescence measurements were used to investigate the composition of HDL. The fluorescence spectrum of Nile Red is dependent upon the local lipid environment. By monitoring the change in fluorescence emission as a function of EL metabolism, we investigate the content of HDL while being remodeled by EL. These fluorescence techniques allow us to answer some of the key questions regarding the HDL lipid collection and distribution function.