1.5.4.2. Continuous monitoring of formation of conjugated dienes in LDL
After isolation, LDL was adjusted to 150 μg/mL of LDL protein with 10 mM PBS, pH = 7.4. 1ml of aliquots extracts/fractions at various concentration (0.25, 0.5 and 1mg/ml) were added to 1ml of LDL solution (150µg/ml). The oxidative modification of LDL was initiated by addition of 0.1ml of freshly prepared 10 µM CuSO4 solution at 37°C (Ahmadvandet al .,2011). Then the sample containing LDL and copper sulfate without extract/fractions (negative control sample) and samples containing copper sulfate and extracts/fractions (test samples) were prepared. Quercetin was used as the positive control sample and prepared in the same manner as the test samples. The appearance of conjugated diene was monitored at 234 nanometers once every 10 min for a period of 3 h and after 24 h at 37 °C in a UV/Visible spectrophotometer. The oxidation-time curve (lag time) was plotted.