Ascorbate- and hemoglobin-dependent brain chemiluminescence

Abstract

It has been indicated recently that ascorbic acid is responsible for the hemoglobin-mediated oxidative damage to the central nervous system (Sadrzadeh & Eaton, J. Clin. Invest. 82: 1510–1515, 1988). In this paper we describe the changes in chemiluminescence accompanying hemoglobin- and ascorbate-dependent oxidative injury to brain tissue. Addition of either hemoglobin (15 μM) or ascorbate (1 or 2 mM) to rat brain homogenates stimulated spontaneous chemiluminescence in a synergistic manner. This increase in chemiluminescence was inhibited by desferrioxamine indicating that free iron was involved in the reactions leading to lipid peroxidation. Preincubation with ascorbate oxidase inhibited both spontaneous and hemoglobin-dependent chemiluminescence, suggesting that ascorbate was required for the reactions leading to lipid peroxidation. Supplementation with aminotriazole (an irreversible inhibitor of the catalase-H 2O 2 complex) increased chemiluminescence in a time- dependent manner, as catalase reacted with accumulated H 2O 2, suggesting that ascorbic acid has a dual action being involved in the production of H 2O 2 and also maintaining Fe in the reduced state to catalyze a Fenton-like reaction. The excited species responsible for the chemiluminescence were partly characterized by adding specific fluorescent energy acceptors: dibromoanthracene (DBA) and diphenylanthracene (DPA). Both DBA and DPA stimulated chemiluminescence several-fold indicating that triplet and singlet species are responsible for the observed chemiluminescence. Excited singlet carbonyls (identified with DPA) may be produced during the collision of two ROO·. Singlet oxygen may also be generated during the same reaction. It decays to the triplet state (emitting chemiluminescence at 634 nm) and reacts with double bonds producing dioxetanes, which may breakdown generating triplet carbonyls (identified with DBA). These results support the hypothesis that during stroke, the release of hemoglobin into the central nervous system (with high concentration of ascorbic acid) leads to the formation of oxidative species responsible for lipid peroxidation.