Figure 4

Oxidation of DCFH in the presence of six different samples of HDL [3 from patients with coronary artery disease (CAD) and 3 from healthy volunteers (Non CAD)] was assessed as described in Methods, using 2.5 μg (cholesterol) of added HDL. The slope of oxidation of each sample was normalized to the slope of oxidation of DCFH and the % relative oxidation slope and quenching is shown on the y axis. The data (means of quadruplicates) from three independent experiments are plotted. The experiments were repeated after DCFH (grey columns) was almost fully oxidized (air oxidation at room temperature for 5 hours). There was a ~40-fold increase in the slope of oxidation of DCFH after 5 hours of air oxidation. The oxidized DCF (DCF ox) was diluted 40-fold to give comparable fluorescence to the baseline DCFH and experiments after addition of HDL were repeated. Addition of HDL did not cause significant fluorescence quenching and there was a significant increase in the variability of oxidation of DCFH compared to baseline DCFH. These results indicate that most of DCFH had converted into DCF and did not react with HDL confirming the fact that fluorescence quenching is the result of DCFH-HDL interaction.