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).