Characterization of a novel high-density lipoprotein antioxidant capacity assay and its application to high-density lipoprotein fractions

Abstract

ObjectivesHigh-density lipoprotein (HDL) inhibits low-density lipoprotein (LDL) oxidation therefore it is involved in the prevention of atherogenesis. HDL particles originating from different persons possess different antioxidant activities. Our aim was to establish a method for the measurement of HDL antioxidant capacity, which is suitable for testing the antioxidant activity of HDL samples in a wide range and produces data relevant to in vivo HDL–LDL interactions. Hemin was used as pro-oxidant since its role in the course of LDL oxidation and atherosclerosis is proven. MethodsHemin-induced and hydrogen peroxide catalyzed lipid peroxidation of LDL was performed in the presence and absence of HDL. The time interval required for reaching the maximum reaction velocity (ΔTVmax) was determined and HDL antioxidant capacity was expressed as the ratio of the ΔTVmax with and without HDL. HDL fractions (n=8) isolated by ultracentrifugation from healthy donors were analyzed and their antioxidant capacities were compared. ResultsIn parallel with their increasing density, HDL fractions expressed increasing antioxidant capacity (106.12–194.12%). Within-run and within-laboratory CVs of the method were 1.72–1.87% and 4.09–4.93%, respectively. Alterations of hydrogen peroxide concentration in the range of 50–125μmol/L did not influence the assay results, while the elevation of hemin concentration (between 3 and 9μmol/L) resulted in decreased antioxidant capacity. The values for hemin degradation correlated well with conjugated diene formation. ConclusionsHemin-induced LDL oxidation is a reliable assay system to test the antioxidant capacity of HDL and its subpopulations.