Electronegativity-based subfractionation of intermediate-density lipoproteins: Comparison with main lipoprotein classes.

Studying the chemical characteristics of intermediate-density lipoprotein (IDL), a transient metabolite and direct precursor to LDL, is crucial for understanding LDL heterogeneity and atherogenic potential. This study aimed to characterize the physicochemical properties and lipidomic composition of distinct IDL subfractions and evaluate their proatherogenic effects. Using ultracentrifugation and anion-exchange chromatography, we fractionated IDL into five increasingly electronegative subpopulations (I1-I5), analogous to LDL, VLDL, and HDL subclasses with high resolution. We then comprehensively characterized these overlooked remnants using TEM, electrophoresis, and exploratory LC-MS lipidomics. Our analysis showed I1 (49.6%) was the least and I5 (6.3%) the most electronegative subfraction. TEM revealed a significant size difference, with I5 particles (averaged 37.9 ± 11.2 nm) being larger than I1 particles (averaged 25.1 ± 4.6 nm; p < 0.05). Furthermore, electrophoretic protein fingerprinting suggested complex remodeling of the putative surface apolipoprotein cargo in highly electronegative particles. Exploratory lipidomic profiling indicated that I5 was enriched in lipid species associated with proatherogenic pathways, including triacylglycerides, lysophosphatidylcholines, and lysophosphatidylethanolamines, whereas I1 particles were enriched in structural phospholipids. These patterns highlight IDL's profound lipidomic heterogeneity. Similarities among V5, I5, and L5 strongly support a shared metabolic continuum, with I5 potentially representing an intermediate transition stage. Crucially, functional assays revealed that the electronegative I5 subfraction possessed potent cytotoxicity in endothelial and monocytic cell models compared to other lipoprotein subfractions and oxLDL, significantly reducing viability and increasing cell death. Given IDL's limited circulation time, the presence of the highly electronegative and lipotoxic I5 subfraction may substantially enhance the overall proatherogenic potential of electronegative lipoproteins.