4.11. Erythrocyte Hemolysis Test

The RBC membrane is altered by free radical-induced oxidative stress, and the reduced antioxidant activity in erythrocytes may increase their sensitivity to hydrogen peroxide-induced hemolysis. Hydrogen peroxide-induced hemolysis was measured by the method from Greenberg et al. [59], with some modifications. A 2 mL erythrocyte sample was incubated with 100 μM of CA for 1 h at 37 °C under aerobic conditions. The cells were then centrifuged, washed and resuspended in PBS. Hydrogen peroxide (H₂O₂) was added to a final concentration of 100 μM, and the erythrocytes thus treated were incubated at 37 °C. Samples of 0.2 mL were taken at 30-min intervals starting from 0 min (immediately after adding H₂O₂) to 240 min. Then, they were diluted in 2 mL of PBS and centrifuged. We measured the hemolysis scales spectrophotometrically at 540 nm wavelength. The samples thus obtained were compared with controls prepared in an identical manner, except that the H₂O₂ solution was replaced with distilled water, and the extracellular hemoglobin contained in the samples was completely hemolyzed. The erythrocyte samples without preincubation with CA were also tested. We expressed the percentage of hemolysis according to Equation (11):

where A is the absorbance of the sample at 540 nm, and B is the absorbance of the fully hemolyzed reference sample at 540 nm.