An improved method for the sensitive monitoring of low density lipoprotein modification by myeloperoxidase

Abstract

The aim of this investigation was to compare an improved fluorometric method with an UV absorbance assay for their ability to monitor low density lipoprotein (LDL) modification by myeloperoxidase (MPO) and to evaluate determining factors influencing the modification of LDL. Using absorbance at 234 nm to study the kinetics of LDL aggregation, and a native fluorescence assay for protein oxidation, we found that all components of the MPO/H₂O₂/Cl^- system may have rate determining effects on LDL modification. While the lipoprotein modification rate correlated positively with enzyme concentration, variation of the concentration of H₂O₂ had a biphasic effect on the maximal rate of LDL modification with both methods. Furthermore, a positive association was found between the maximal rate of LDL modification and the acidity of the medium, with a pathophysiologically relevant optimal rate at a slightly acidic pH of 5–6, but hardly any modification above pH 6.8. In summary, both methods provide simple and useful tools for the continuous monitoring of LDL modification by the MPO/H₂O₂/Cl^- system, but the more sensitive fluorometric method is preferable, since it allows the application of experimental conditions which are much closer to the situation in vivo.