The differential apoA-I enrichment of preβ1 and αHDL is detectable by gel filtration separation

Abstract

The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-2 H 3 ]leucine for 14 h in five subjects. Using FPLC, pre ␤ 1 HDL and ␣ HDL (HDL 2 and HDL 3 ) were separated from 200 l of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in pre ␤ 1 HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to ␣ HDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be ␣ HDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in pre ␤ 1 HDL and ␣ HDL from total HDL previously used for kinetic measurements. Plasma HDL and apolipoprotein A-I (apoA-I) concentrations are inversely correlated with the risk of coronary heart disease (1, 2). Kinetics studies are a good tool for a better understanding of the role that HDL play in the reverse cholesterol transport. Pre ␤ 1 HDL are considered as the initial acceptor of cellular cholesterol and are transformed into spherical HDL 3 as a result of the LCAT activity. Nascent discoidal pre ␤ 1 HDL are either directly synthesized by the liver and the intestine or generated when other classes of lipoproteins including ␣ HDL are remodeled by hepatic lipase (3). In plasma, pre ␤ 1 HDL represented 5.5 Ϯ 3.3% and 7.2 Ϯ 4% of total apoA-I in women and men, respectively (4).ApoA-I, a major protein component of HDL, could be endogenously labeled using stable isotopes to study HDL metabolism in humans (5). Plasma lipoprotein and HDL are usually separated by ultracentrifugation based on differential flotation using either sequential (6) or one-step gradient methods (7). It appears that the combination of high salt concentrations and high centrifugal forces may cause structural damages to the lipoproteins (8) that could change results of kinetics studies (9). Alternatively, plasma lipoproteins may also be separated by size-exclusion chromatography on agarose. Fast Protein Liquid Chromatography (FPLC), based on the gel filtration property, is a rapid, quantitative, and non-destructive method used to separate lipoproteins. Nascent pre ␤ 1 HDL can also be separated from ␣ HDL by FPLC (10). The aim of the present study is to validate the separation of HDL subclasses by FPLC in metabolic study after an endogenous labeling with stable isotopes. In this study, we have compared the leucine enrichment of apoA-I-HDL separated by FPLC to that of HDL separated by the traditional ultracentrifugation of plasma samples.

Single session exercise stimulates formation of preβ1-HDL in leg muscle

“…It was suggested that pre␤ 1 -HDL may be an initial acceptor of cellular cholesterol during cholesterol efflux (14,30) and/or a first product of lipidation of lipid-free apoA-I by ABCA1-dependent formation of HDL (16). The role of pre␤ 1 -HDL as a precursor of mature HDL has been strengthened by a recent finding of a greater rate of incorporation of newly synthesized apoA-I into pre␤-HDL (29). Its concentration increases with dyslipidemia (31) and with overweight (22).…”

“…Contributing to this high flux of pre␤ 1 -HDL may be a possible defect of removal of this particle from plasma. Chetiveaux et al (29) have also demonstrated the existence of a separate pool of pre␤-HDL with kinetic parameters different from ␣-HDL. If formation of pre␤ 1 -HDL and remodeling of pre␤ 1 -HDL to ␣-HDL is accompanied by cholesterol efflux (16), that may represent a significant contribution to reverse cholesterol transport.…”

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

“…SDS-PAGE, silver staining, and Western blotting of the appropriate lipoprotein was used to assay the purity of each lipoprotein fraction. In addition, lipoproteins were fractionated by size exclusion chromatography by using an Akta purifier UPC (GE Healthcare Bio-sciences, Uppsala, Sweden) (10,14). Approximately 175 l of the sample was injected into a 500-l sample loop.…”

Age-related impairment of HDL-mediated cholesterol efflux

“…Under our conditions, HDL 2 and HDL 3 were isolated by ultracentrifugation, which excluded the presence of preb 1 -HDL in our preparations, thus eliminating its possible role in the difference of the RCT capacity between young and elderly (32,33).…”