Standardization of a Method to Evaluate the Antioxidant Capacity of High-Density Lipoproteins

Background: A method to evaluate the antioxidant capacity of high-density lipoprotein (HDL) was developed and standardized. Methods: This method measure conjugated diene (CD) formation and electrophoretic mobility of low-density lipoprotein (LDL) in agarose gels in the presence and absence of HDL. HDL was isolated from 1 mL of plasma within 24 hours and oxidation assays were performed within 6 hours. Oxidation was induced by adding CuSO(4). The lag phase increase in CD kinetics and the inhibition of electrophoretic mobility were defined as the HDL antioxidant capacity. Results: The optimal conditions for the CD assay were 2.5 μM CuSO(4), LDL at 0.1 g apoB/L, HDL at 0.1 g apoA-I/L, at 37°C and for 3h 50 min. Agarose electrophoresis at 100 V, at 4°C for 50 min was then performed immediately. CD formation variability was 21.1% for inter-assay CV and 12.7% for intra-assay CV. Electrophoretic mobility was 26.5% for inter-assay CV and 2.4% for intra-assay CV. Correlation analysis showed a significant association between the antioxidant capacity of HDL and its neutral/polar lipid ratio. Conclusions: The method herein described measures of the HDL antioxidant capacity in a reproducible and rapid manner that can be applied to a relatively high number of samples.