Open tubular CE for in vitro oxidation studies of human very-low-density lipoprotein particles

Human very-low-density lipoprotein (VLDL) particles were immobilised on the inner wall of electrochromatographic fused-silica capillaries, and the applicability of these capillary columns in oxidation studies was investigated. Capillaries coated with radiolabelled VLDL particles showed a coating efficiency of 97%, and allowed estimation of the amount of VLDL present in a capillary. Radioactivity measurements and atomic force microscopy with tapping mode confirmed the presence of VLDL particles as a monolayer. The pI determined for the VLDL was 4.7-4.8 varying with the human source. The effects of VLDL concentration, coating time and pH on the coating stability were clarified, and the stability was examined in terms of the repeatability of EOF and retention factors of selected steroids. The repeatability of run-to-run and the coating-to-coating reproducibility ranged from 2.6 to 4.9% and 3.2 to 6.6%, respectively. The lifetime of a coating was at least 7 days or 84 consecutive runs. The in situ copper-mediated VLDL oxidation carried out in the capillary with optimised VLDL coating showed that, during the oxidation of VLDL particles, the negative charges of the particles are increased, leading to enhanced EOF mobilities. Several oxidation parameters, including copper sulfate concentration, amount of EDTA needed to stop the reaction, pH and the oxidation procedure, were examined. Effect of the oxidation process on the stability of the coating in one capillary, and in five different capillaries ranged between 0.4-4.1% and 0.8-6.6%, respectively. The in situ oxidation of VLDL particles was compared with that of low-density lipoproteins.