1.5.4. In vitro CuSO4-induced LDL Oxidation inhibition study
1.5.4.1. Isolation of LDL
The LDL fraction was isolated from fresh plasma by single vertical discontinuous density gradient ultracentrifugation (Richard et al .,1999). The density of the plasma was adjusted to 1.21 g/ml by the addition of solid potassium bromide (KBr) (0.365 g/ml). Centrifuge tubes were loaded by layering 1.5 ml of density-adjusted plasma under 3.5 ml of 0.154 mol/L NaCl, and centrifuged in a Beckman L7-55 ultracentrifuge at 40000 rpm at 10 °C for 150 min. The yellow LDL band, located in the upper middle portion of the tube, was collected into a syringe by puncturing the tube. The isolated LDL was dialyzed for 48h at 4°C against three changes of deoxygenated-PBS (0.01 mol/L Na₂HPO4, 0.16 mol/L NaCl, pH 7.4) containing 0.01% NaN3 and 0.01% EDTA. After isolation of total LDL, the protein content of LDL was measured (Bradford et al .,1979).