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Journal of Lipid Research, Vol. 43, 1986-1993, November 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

The differential apoA-I enrichment of preß₁ and HDL is detectable by gel filtration separation

Maud Chétiveaux^*, Hassan Nazih^*,, Veronique Ferchaud-Roucher^*, Gilles Lambert^*, Yassine Zaïr^*,, Martial Masson^*, Khadija Ouguerram^*, Daniele Bouhours^* and Michel Krempf^1,*,

^* Inserm U539, Centre de Recherche en Nutrition Humaine, CHU Hôtel Dieu, Nantes, France
Laboratoire de Biochimie Fondamentale et Appliquée, UFR de Pharmacie, Nantes, France
Clinique d'Endocrinologie, Maladies Métaboliques. Nutrition, CHU Hôtel-Dieu, Nantes, France

¹ To whom correspondence should be addressed. e-mail : mkrempf{at}sante.univ-nantes.fr

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 [5.5.5-²H₃]leucine for 14 h in five subjects. Using FPLC, preß₁ HDL and HDL (HDL₂ and HDL₃) were separated from 200 µl of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in preß₁ 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 preß₁ HDL and HDL 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.

Supplementary key words fast protein liquid chromatography • apolipoprotein A-I • kinetic analysis

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