Ascorbate- and hemoglobin-dependent brain chemiluminescence.
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 microM) 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-H2O2 complex) increased chemiluminescence in a time-dependent manner, as catalase reacted with accumulated H2O2, suggesting that ascorbic acid has a dual action being involved in the production of H2O2 and also maintaining Fe in the reduced state to catalyze a Fenton-like reaction. The excited species responsible for the chemiluminescence were partially 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).(ABSTRACT TRUNCATED AT 250 WORDS)
Prat
A G
AG
Department of Biochemistry, College of Medicine, University of South Alabama, Mobile 36688.
Turrens
J F
JF
D000818:Animals; D001205:Ascorbic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D001921:Brain / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D005609:Free Radicals; D006454:Hemoglobins / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D015227:Lipid Peroxidation; D008163:Luminescent Measurements; D008297:Male; D010101:Oxygen Consumption; D051381:Rats; D011919:Rats, Inbred Strains
D005456:Fluorescent Dyes; D005609:Free Radicals; D006454:Hemoglobins; D006861:Hydrogen Peroxide; D001205:Ascorbic Acid