Free radical generation in hydroperoxide-treated erythrocytes monitored continuously by luminol-amplified chemiluminescence.

Abstract

Organic hydroperoxides induce oxidative damage to mammalian cells. We describe how luminol-amplified chemiluminescence can be used to monitor free radical generation (following treatment of erythrocytes in vitro with organic hydroperoxides) throughout the entire time-course of oxidative stress. Enrichment of erythrocyte alpha-tocopherol levels increased the induction time by 25% and led peak chemiluminescence fall of 30%. Furthermore, ascorbate loading reduced the signal four-fold during the induction period. The catalytic role of haemoglobin was shown by the abolition of chemiluminescence by azide and a low (but detectable) signal in haemoglobin-depleted erythrocyte ghosts. Luminol-amplified chemiluminescence enables the kinetics of free radical generation to be monitored continuously. Furthermore, it may enable features of the mechanism of interaction between cellular antioxidants and antioxidant enzymes to be elucidated.