Electrophoretic analysis of oxidative modification of apolipoprotein E in very low density lipoprotein from fresh human plasma

Ferrous ion-induced oxidative modification of apoE in lipid peroxidation of human very low density lipoprotein (VLDL) and the role of the cysteinyl gr oup, present in apoE3 but absent in apoE4, were examined. Fresh human VLDL was obtained from healthy volunteers with different apoE phenotypes as dete rmined by isoelectric focusing (IEF). The VLDL was oxidized by incubating w ith FeSD4. The time course of the lipid peroxidation was determined by thio barbituric acid (TBA) assay. The sequential oxidative modification of the a poE was demonstrated by sodium dodecyl sulfate - polyacrylamide gel electro phoresis (SDS-PAGE) and immunoblotting with an anti-apoE antibody. To modif y the cysteinyl group of apoE3, the VLDL from the apoE3/3 subject was pretr eated with cysteamine before the TEA assay and electrophoresis. The ferrous ion-induced peroxidation of the VLDL was pH-dependent, with increased reac tivity at acidic pH ranges. In the initial stage of the lipid peroxidation of the VLDL, aggregates were formed involving apoE dimers and apoB via disu lfide bonds, followed by the intra- and intermolecular cross-linkings of ap oE monomers. The VLDL with apoE3/4 showed a shorter lag time than the VLDL with apoE3/3 in the TEA assay, suggesting a lower antioxidative effect with apoE3/4. The apoE3/3 pretreated with cysteamine showed the patterns of apo E3/4 in IEF and SDS-PAGE with immunoblotting, but it did not show the short ened lag time in the TEA assay. This suggests that factors other than cyste inyl groups contribute to the reduced antioxidative activity of apoE4.