Improved Method to Capture a Broader Array of High Density Lipoprotein Particles Including Those of Intestinal Origin

Abstract

The isolation of high density lipoprotein (HDL) particles by density‐based methods using a density range of 1.063–1.21 g/mL is considered to be the gold standard. However, it is known that several HDL subclasses, including discoidal/nascent HDL particles, as well as ApoE‐containing, and intestinally derived ApoA‐IV‐containing HDL particles, are found at a higher density of up to 1.25 g/mL. In addition, using density‐based approaches alone it is not possible to purify HDL particles from a range of other particles such as extracellular vesicles, which overlap in density but not size. In this study, a novel method was optimized to isolate a broader array of HDL particles in a purified fraction that minimizes contamination by extracellular vesicles. The method utilizes density‐based sequential flotation ultracentrifugation followed by size exclusion chromatography with an FPLC system to isolate HDL fractions of different sizes, which were then analyzed for their cholesterol efflux capacity and protein content. The results demonstrate a higher yield of HDL and an increased level of HDL from earlier fractions using the 1.25 density cut‐off compared to the 1.21 density cut‐off. The cholesterol efflux capacity of early and late eluting fractions from the 1.25 density cut‐off method is increased, suggesting that both smaller and larger higher‐density particles, both with increased efflux capacity, are being isolated with the new method. Gel electrophoresis and Western Blot results from these fractions indicate enrichment of ApoE as well as ApoA‐IV in HDL fractions collected using the 1.25 density cut‐off method, particularly from plasma samples collected in the postprandial vs. the fasted state. In conclusion, we demonstrate that the use of the optimized HDL isolation method yields a broader array of HDL particles, which may be lost when the 1.21 density cut‐off is used.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.