Metal Ion Complexes of EDTA:  A Solute System for Density Gradient Ultracentrifugation Analysis of Lipoproteins

Abstract

In the study reported here, we apply some of the features of coordination chemistry to solve a long-standing problem in the separation and characterization of lipoprotein particles. Lipoproteins are circulating micelle-like particles responsible for lipid transport. They exist in three major classes: very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein in well-defined density ranges using the density gradient ultracentrifugation (DGU) method. The analytical instrumentation of DGU has improved over the years in response to clinical evidence that certain lipoprotein species are linked to a high risk for developing cardiovascular disease. A long-standing problem has been a lack of appropriate gradient-forming solutes that can generate a useful gradient from a homogeneous solution. We have found that a new class of solutes based on metal ion complexes has the potential of providing a wide selection of compounds where the features can be modulated by choice of ligand, complexing metal ion, and counterion. In this study, we have chosen the cesium salt of BiEDTA (CsBiEDTA) and have investigated the dynamics of density gradient formation in the ultracentrifuge. We show that a useful density gradient can be formed within a few hours beginning with a homogeneous solution. We also present data on the migration behavior of lipoproteins under gradient-forming conditions and show that high-resolution density profiles can be obtained with good precision. The resolution of the CsBiEDTA profile is compared with those obtained using high molecular weight organic solutes.

Effect of sterol carrier protein-2 gene ablation on HDL-mediated cholesterol efflux from cultured primary mouse hepatocytes

“…NBD-cholesterol was obtained from Molecular Probes (Eugene, OR); alkaline phosphatase-conjugated rabbit anti-goat IgG and goat anti-rabbit IgG from Sigma-Aldrich (St. Louis, MO); and purified human HDL, LDL, VLDL, and apoliprotein A1 (apoA1) from CalBiochem, EMD Biosciences (San Diego, CA). HDL was subfractionated to obtain HDL2 and HDL3 (14,17,18). [1,2,6, H(N)]cholesterol and methyl-␤-cyclodextrin (M␤CD) were obtained from NEN/PerkinElmer (Waltham, MA) and Sigma (St. Louis, MO), respectively.…”

Electronegative LDLs from familial hypercholesterolemic patients are physicochemically heterogeneous but uniformly proapoptotic

“…After centrifugation, tubes were placed in a custom fluorescence imaging station comprising a Plexiglas tube holder, a digital Optronics Microfire Camera (Goleta, CA), and a DolanJenner (Lawrence, MA) MH-100 metal halide light source fitted with a liquid light guide (22). The tube holder, camera, and light source were mounted orthogonal to each other on an optical breadboard in a dark room.…”

“…Here, we report the density distribution of LDL À isolated from hypercholesterolemic (FH) and normolipidemic (NL) subjects as assessed by equilibrium density gradient ultracentrifugation (EDGUC) (22) and an assay of biological effects, EC apoptosis.…”

Centrifuge microextraction coupled with sweeping–MEKC to analyze trace steroid hormones in urine samples

“…It is also used to separate the extraction solvent/solid extractant from the sample after extraction [27]. Another important application is density gradient ultracentrifugation, which is used to separate compounds with slightly different densities [28][29][30][31] or determine the density of biological macromolecules, such as proteins [32][33][34].…”