Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis

Oxidative modification of low density lipoproteins (LDLs) is an important p athogenetic factor in atherosclerosis. The various steps in oxidative modif ications of LDL can be monitored using different methodologies with varying degrees of complexity In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequenti al ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were char acterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP meth od to be highly sensitive, as changes in peak area of the separated LDL sub fractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhi bited an increased migration velocity of the fractions, in summary, we pres ent the first study investigating LDL-subfractions separated by CITP and th e alterations of these LDL-subfractions after gradual in vitro oxidation an d after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.