cytochrome-c-t and 10-10--dimethyl-9-9--biacridinium

The radical anion of dioxygen superoxide (O2.-) is a physiological free radical formed in various enzymatic processes. On the one hand superoxide is a precursor of reactive oxygen and nitrogen species (hydroxyl radicals, peroxy radicals, hydrogen peroxide, peroxynitrite, etc.), -the initiators of cellular damage; on the other hand it is a signaling molecule regulating numerous physiological processes including apoptosis, aging, and senescence. Therefore, the detection and measurement of superoxide in cells, tissues, and whole organisms is of a vital importance for in vitro and in vivo studies of many physiological and pathophysiological processes. At present different efficient methods were developed, which allow to identificate and measure superoxide in biological systems. In present review the credibility and efficiency of principal mostly applied methods of superoxide detection based on one-electron transfer and nucleophilic reactions are discussed, and spectrophotometrical, chemiluminescent, fluorescent, and ESR spin trapping methods are compared.

Topics: Acridines; Cytochromes c; Imidazoles; Luminescent Measurements; Luminol; Oxygen; Pyrazines; Spectrophotometry; Spin Trapping; Superoxides

Allopurinol is a prodrug converted to oxypurinol by xanthine oxidase, a process followed by an efficient enzyme inhibition. Using a lucigenin-enhanced chemiluminescence method, we found that, under alkaline conditions, superoxide radicals are produced in large amounts in the first step of the interaction between the enzyme and the inhibitor. A comparison between lucigenin and cytochrome c as final detectors revealed that only the chemiluminescence technique is able to detect the superoxide anions from allopurinol oxidation. The allopurinol-xanthine oxidase-lucigenin system can be used for the quantification of various free-radical scavengers, in particular superoxide dismutase mimics. Three manganese compounds from different structural classes [manganese(II) chloride, manganese N,N'-bis(salicylidiene)ethylenediamine chloride, and manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin] were compared at five concentrations (0.01, 0.1, 1, 10, and 100 μM). The method is fast, 16 times more sensitive than the cytochrome c assay at pH 10.1 and could be used for in vivo investigations avoiding the lucigenin redox cycle. If the concentrations of the reagents are increased and Tween 20 is added, the method is also operative at pH 7.4.

Topics: Acridines; Allopurinol; Animals; Cattle; Cytochromes c; Enzyme Inhibitors; Hydrogen-Ion Concentration; Luminescent Agents; Luminescent Measurements; Prodrugs; Superoxide Dismutase; Superoxides; Xanthine Oxidase

The ability of dipyridamole (DIP) to scavenge oxygen metabolites generated by either activated human neutrophils (PMNs) or cell-free systems using luminol(s)- and lucigenin-enhanced chemiluminescence was investigated. In the presence of DIP (15-50 microM) a dose-dependent inhibition period was seen in phorbol myristate acetate (PMA)-stimulated PMNs as assayed by isoluminol-enhanced chemiluminescence (ILCL) with horseradish peroxidase (HRP). Although such a lag period was not observed in the absence of HRP, 50 microM DIP inhibited extracellular ILCL by more than 50%. Intracellular luminol-enhanced chemiluminescence (LCL) as assayed in either PMA- or in ionomycin-activated PMNs was not affected by dipyridamole (15-50 microM). In cell-free systems, DIP produced concentration-dependent inhibition in H2O2-(45% at 50 microM), OH- (40%, at 0.1 microM) and HOCl-(20% at 10 microM). Both absorbance and fluorescence scans revealed that DIP is able to react with equimolar quantities of either H202 or HOCl. These results suggest that DIP scavenges reactive oxygen species (ROS) presumably secreted by activated human PMNs in the following decreasing order: *OH > HOCl > H2O2 >> O2-.

Topics: Acridines; Antioxidants; Cytochromes c; Dipyridamole; Extracellular Space; Humans; Hydrogen Peroxide; Hypochlorous Acid; In Vitro Techniques; Luminescent Measurements; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxidants; Oxidation-Reduction; Reactive Oxygen Species