Exogenous glutathione is essential in the testing of antioxidant capacity using radical-induced haemolysis

Radical-induced haemolysis has been employed by many investigators to determine the antioxidant capacity of novel compounds. However the free radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) results in the complete depletion of intracellular reduced glutathione (GSH) in cells that can no longer synthesise macromolecules. As GSH is essential in recycling certain antioxidants back to their active form, the current study examined the effects of exogenous GSH on the antioxidant capacity of quercetin, phenol, ebselen and nitroxide detected using AAPH-induced haemolysis. Here we report a modification that increases the likelihood of detecting antioxidant activity in a radical-induced haemolysis assay.

C57Bl/6 mouse erythrocyte suspensions were pre-incubated with 1, 3 or 10 μM of phenol, ebselen, nitroxide or 10, 20 or 30 μM of quercetin for 30 min in the presence or absence of 1mM of glutathione. AAPH (150 mM) was added to each well to induce haemolysis. Absorbance of erythrocytes was measured spectrophotometrically at 690 nm over 5h. Haemolysis in the presence of different pre-treatments was quantified by calculating the time to 50% lysis.

AAPH in the presence and absence of GSH resulted in a decrease in absorbance over time as cells lysed. Pre-incubating cells with ebselen or phenol (10 μM) delayed AAPH-induced haemolysis by 37 and 74 min only in the presence of exogenous GSH. Nitroxide accelerated radical-induced haemolysis by 40 min in the absence of exogenous GSH, however delayed haemolysis by 38 min in the presence of exogenous GSH. The antioxidant actions of quercetin were unaffected by the presence of exogenous GSH.

The results demonstrate that exogenous GSH is required to detect the antioxidant capacity of certain antioxidant moieties using the radical-induced haemolysis assay. This is particularly important as numerous groups use this technique as a high throughput screening assay of antioxidant activity.