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

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-H-2(3)]leucine for 14 h in five subjects. Using FPLC, prep, HDL and alphaHDL (HDL2 and HDL3) were separated from 200 mul of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in prebeta(1) HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to alphaHDL 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 alphaHDL, 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 prebeta(1) HDL and alphaHDL from total HDL previously used for kinetic measurements. This original and new method should help to understand the kinetics of HDL in humans and the reverse cholesterol transport dynamics.