Measurement of antioxidant activity in lipoproteins using enhanced chemiluminescence

We describe a new assay for antioxidant activity (AOA) in lipoprotein solutions based upon their potential to quench light emission from a glowing horseradish peroxidase-catalyzed enhanced chemiluminescent reaction. By comparison with the quenching activity of the tocopherol-analogue trolox the AOA can be quantified. These measurements suggest that all lipoprotein fractions have significant AOA and that this has a non-linear relationship with lipoprotein concentration increasing significantly on a per particle basis at higher concentrations. Mean AOA in very low density, low density and high density lipoprotein fractions were 39.9 +/- 5.3, 20.3 +/- 4.0 and 5.3 +/- 1.0 mumol of trolox equivalents per litre, respectively, when measured at 1 mg protein/ml. Using known values for the protein content of the lipoprotein fractions, these values correspond to 79.8 +/- 10.7, 10.3 +/- 2.0 and 0.84 +/- 0.15 equivalents per particle. Parallel measurements of light emission and conjugated diene formation suggest that the oxidative stress imposed by the peroxidase-catalyzed reaction leads to lipid peroxidation but only after all AOA has been exhausted. AOA was significantly correlated with the alpha-tocopherol content in 30 lipoprotein samples (r = 0.764). This assay offers a rapid and simple method for investigating the effects of diseases, drugs or dietary manipulation on lipoprotein AOA.