Fractionation of charge-modified low density lipoproteins by fast protein liquid chromatography

We describe a methodology developed to separate different forms of charge-modified low density lipoproteins (LDL) using the fast protein liquid chromatography (FPLC) system from Pharmacia. Lipoproteins were isolated by sequential ultracentrifugation and introduced onto an anion-exchange column (Mono Q HR 5/5). The multistep NaCl gradient elution was optimized and the analytical variables were determined on copper-oxidized LDL. After oxidation by copper for various times (up to 48 h), five forms were obtained (fractions A, B, C, D, and E). Within-run and day-to-day reproducibility were better than 8.6% and 10%, respectively. Protein and cholesterol recovery after the chromatographic separation was good (greater than 82%) and the detection limit was about 1 microgram. The more negative forms of collected LDL were mainly characterized by an increase in the lipid peroxidation product content, a depletion of vitamin E, an alteration of apoB and increased degradation by macrophages. The proposed methodology was applied to the study of LDL modifications generated by human umbilical endothelial cells and the protective effect of antioxidants (vitamin E and probucol).