CHARACTERIZATION OF A MORE ELECTRONEGATIVELY CHARGED LDL SUBFRACTION BY ION-EXCHANGE HPLC

Low density lipoproteins (LDL), collected from 32 normal male subjects (aged 30-60), were subfractionated by high resolution ion exchange chromatography (IE-HPLC). By this procedure two LDL subfractions were eluted. The first corresponds to normal LDL (nLDL); while the second one corresponds to a more electronegative subfraction, called LDL-. The mean percentage contribution of LDL- to native plasma LDL was of 3.9% (range 0.5-9.8%). The percentage concentration of LDL- in total native LDL did not correlate with plasma total cholesterol, triglycerides, and LDL cholesterol, whereas a significant negative correlation with high density lipoprotein cholesterol was found (r = -.38; p < .05). LDL- was negatively correlated with LDL phospholipids (r = -.59; p < .001), and with the LDL vitamin E content (r = -.63; p < .001), and positively correlated with LDL proteins (r = -.35; p < .05) and the content of thiobarbituric acid reactive substances (TBARS) in total LDL (r = .43; p < .05). The TBARS molar content of LDL- was three times higher than in nLDL, with a mean concentration in LDL- of 7.3 mol/mol lipoprotein. By preparative IE-HPLC significant differences of the LDL- chemical composition were observed. The percentage content of cholesterol esters and of phospholipids was decreased, whereas proteins and free cholesterol were increased. Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis reveled that besides apolipoprotein B-100 there was evidence of peptides with a higher molecular weight in LDL-. LDL oxidation with cupric ions induced a significant increase of a peak having the same retention time of LDL-, coupled with a significant increase of TBARS, a dramatic reduction of vitamin E and an apolipoprotein B fragmentation. The relationship of this LDL- component to oxidatively modified LDL is discussed.