The detection of superoxide anion from the reaction of oxyhemoglobin and phenylhydrazine using EPR spectroscopy

Summary The low temperature EPR spectrum of a quickly reacted mixture of oxyhemoglobin and phenylhydrazine was studied. With the use of a computer, the spectral contribution of methemoglobin in the region of g=2 was subtracted. The remaining spectrum was that of an axial free radical (g=2.00, g=2.06) having the magnetic parameters of superoxide anion. In the presence of superoxide dismutase, this axial radical was not seen, confirming that superoxide anion is indeed generated by the reaction.The portion of this investigation carried out at the Albert Einstein College of Medicine was supported in part by US Public Health Service Research Grant HL-93399 from the Heart and Lung Institute and by National Institute Contract Nol-CP-55606 to J.P. This is communication No. 378 from the Joan and Lester Avnet Institute of Molecular Biology.Predoctoral fellow in the Medical Scientist Training Program (United States Public Health Service Grant 5-TO-5-GM 01669-12) at the New York University School of Medicine.Recipient of a grant-in-aid from the New York Heart Association.     

The autooxidation of 2,3,5,6-tetrahydroxy-2,5-cyclohexadiene-1,4-dione under physiological conditions

Summary A radical anion of 2,3,5,6-tetrahydroxy-2,5-cyclohexadiene-1,4-dione was detected using the EPR technique by complexing with Zn ions. Hydrogen peroxide, superoxide radical anion and hydroxyl radical were also detected in the reaction mixture. Kinetic study and product distribution indicated a probable mixed type one- and two-electron transfer mechanism. A possible relationship between the autooxidation process and the biological activity of substituted quinones was suggested.     

Vanadium induced hemolysis of vitamin E deficient erythrocytes in Hepes buffer

Several vanadium compounds were tested for their ability to induce in vitro hemolysis of vitamin E-deficient hamster erythrocytes. Free vanadyl caused hemolysis in Hepes buffer but not in Tris or phosphate buffer, while hemolysis was inhibited by catalase, chelators such as deferoxamine mesylate and EDTA, and hydroxyl radical scavengers such as ethanol andd-mannitol. Although metavanadate itself could not induce hemolysis, metavanadate with NAD(P)H caused hemolysis in Hepes buffer only, and superoxide dismutase prevented it. Hydrogen peroxide, hydroxyl radical and Hepes radical were involved in vanadyl-induced hemolysis; superoxide anion was further involved in metavanadate plus NAD(P)H-induced hemolysis. Vitamin E prevented hemolysis under both conditions.     

Superoxide anion scavenging effect and superoxide dismutase activity of Ginkgo biloba extract

Ginkgo biloba extract is known to be efficient in diseases associated with free radical generation. The purpose of this work was to study, under in vitro conditions, the action of Ginkgo biloba extract (Gbe) against superoxide anion (O2-.), which is directly or indirectly implicated in cell damage. Gbe appears to have both an O2-. scavenging effect and also a superoxide dismutase activity. Its antiradical effect was demonstrated by low temperature electron spin resonance and in a non-enzymatic system (phenazine methosulfate-NADH), and its enzymatic activity was shown by polarographic determination.     

Vanadium induced hemolysis of vitamin E deficient erythrocytes in Hepes buffer

Several vanadium compounds were tested for their ability to induce in vitro hemolysis of vitamin E-deficient hamster erythrocytes. Free vanadyl caused hemolysis in Hepes buffer but not in Tris or phosphate buffer, while hemolysis was inhibited by catalase, chelators such as deferoxamine mesylate and EDTA, and hydroxyl radical scavengers such as ethanol andd-mannitol. Although metavanadate itself could not induce hemolysis, metavanadate with NAD(P)H caused hemolysis in Hepes buffer only, and superoxide dismutase prevented it. Hydrogen peroxide, hydroxyl radical and Hepes radical were involved in vanadyl-induced hemolysis; superoxide anion was further involved in metavanadate plus NAD(P)H-induced hemolysis. Vitamin E prevented hemolysis under both conditions.     

Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin

Depending on the availability of suitable reducing agents, alloxan can be either a prooxidant or an antioxidant. Alloxan and its reduced derivative, dialuric acid, act as a redox couple, driven by reduced glutathione (GSH) or L-cysteine, generating in vitro in the presence of oxygen, both superoxide radical and hydrogen peroxide. The production of superoxide radicals was shown by the appearance of lucigenin chemiluminescence (CL) as well as by the generation of formazan from nitroblue tetrazolium (NBT). The lucigenin CL as well as the NBT reduction was inhibited by superoxide dismutase and partially by catalase. Melatonin inhibited alloxan-mediated CL. In contrast, in the absence of reducing agents, alloxan is a scavenger of superoxide radicals formed by other reactions. Because of the high content of reducing compounds in the cell (e.g. glutathione), it is suggested that alloxan acts in vivo mainly as a generator of reactive oxygen species. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999     

Superoxide anion scavenging effect and superoxide dismutase activity ofGinkgo biloba extract

Summary Ginkgo biloba extract is known to be efficient in diseases associated with free radical generation. The purpose of this work was to study, under in vitro conditions, the action ofGinkgo biloba extract (Gbe) against superoxide anion ( ), which is directly or indirectly implicated in cell damage.Gbe appears to have both an scavenging effect and also a superoxide dismutase activity. Its antiradical effect was demonstrated by low temperature electron spin resonance and in a non-enzymatic system (phenazine methosulfate-NADH), and its enzymatic activity was shown by polarographic determination.     

Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes

A Ginkgo biloba extract (Gbe) containing flavonoids, among other compounds, was tested for the release of activated oxygen species (O-2, H2O2, OH.) during the stimulation of human neutrophils (PMNs) by a soluble agonist. The extract slows down O2 consumption (respiratory burst) of stimulated cells by its inhibitory action on NADPH-oxidase, the enzyme responsible for the reduction of O2 to O-2. Consequently, superoxide anion (O-.2) and hydrogen peroxide (H2O2) production is significantly decreased when the PMNs stimulation is done in the presence of the extract at concentrations of 500, 250 and 125 micrograms/ml. Moreover, the hydroxyl radical generation (OH.) is very much decreased at concentrations as low as 15.6 micrograms Gbe/ml, which indicates that the extract also has free radical scavenging activity. Gbe is able at least to reduce very severely the activity of myeloperoxidase contained in neutrophils. This enzyme, secreted into the intra and extracellular medium, catalyzes the oxidation of chloride (Cl-) by H2O2 to yield strong oxidants (HOCl, chloramines) which are implicated in inflammatory processes.     

A method for detecing superoxide anion in presence of peroxidases

Summary Cytochrome c, used as a scavenger for superoxide anion in some oxidase-like reactions of horseradish peroxidase, diminishes the conversion of the starting products to the oxidized forms. This behavior can be used as a tool for determining superoxide in the presence of peroxidases and hydrogen peroxide.     

Superoxide dismutase activity in the Spanish population

Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen. This superoxide radical is produced by all aerobic cells as a normal metabolic intermediate of molecular oxygen, and is dangerous for the cell because it induces the inactivation of various enzymes, lipid peroxidation and mutations. Superoxide dismutase can therefore be considered as a protective enzyme. The purpose of this work was to determine the level of superoxide dismutase activity in the Spanish population, and to study the factors that influence this activity. The superoxide dismutase activity of 2397 individuals was determined using the method described by Minami and Yoshikawa. The superoxide dismutase activity level in the adult Spanish population was found to be 4.16 +/- 0.89 Units/ml of blood. No significant variations with respect to sex were detected. But it was observed that the superoxide dismutase activity level was 9% higher in the young urban Spanish population.     

Superoxide dismutase activity in the Spanish population

Summary Superoxide dismutase is an enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen. This superoxide radical is produced by all aerobic cells as a normal metabolic intermediate of molecular oxygen, and is dangerous for the cell because it induces the inactivation of various enzymes, lipid peroxidation and mutations. Superoxide dismutase can therefore be considered as a protective enzyme. The purpose of this work was to determine the level of superoxide dismutase activity in the Spanish population, and to study the factors that influence this activity. The superoxide dismutase activity of 2397 individuals was determined using the method described by Minami and Yoshikawa. The superoxide dismutase activity level in the adult Spanish population was found to be 4.16±0.89 Units/ml of blood. No significant variations with respect to sex were detected. But it was observed that the superoxide dismutase activity level was 9% higher in the young urban Spanish population.     

Evidence that superoxide radicals are involved in the hemolytic mechanism of phenylhydrazine

Summary Phenylhydrazine produces in the red blood cell the same effect as the enzymic system xanthine oxidase-xanthine, a superoxide radical generator system. Both effects are inhibited by the enzyme superoxide dismutase.This work was supported in part by grants Nos. 1045 and 3243 of Oficina Técnica de Desarrollo Científico y Creación Artística de la Universidad de Chile.     

Increase in the survival time of mice exposed to ionizing radiation by a new class of free radical scavengers

N-acyl dehydroalanines react with and scavenge mainly superoxide radical (O-2.) and hydroxyl radical (HO.). The ortho-methoxyphenylacetyl dehydroalanine derivative, indexed as AD-20, protects mice against damage resulting from total body X-irradiation, as measured by the increase in their survival time. AD-20 increases the LD50 at 30 days from 6.1 to 7.3 Gy in animals exposed to a wide range of X-rays (6 to 10 Gy). The dose reduction factor (D R F) of AD-20 is 1.20. We postulate that such radioprotective effect may result from its free radical scavenging activity.     

Role of active oxygen species in diethyldithiocarbamate-induced gastric ulcer in the rat

Diethyldithiocarbamate, an inhibitor of Cu,Zn-superoxide dismutase, was recently found to be ulcerogenic in the rat stomach, and active oxygen species were found to be responsible for its ulcerogenicity. To clarify which active oxygen species play a role in ulcerogenesis, the effects of various scavengers and iron-chelators were studied. As superoxide dismutase and catalase reduced the ulcerogenesis induced by diethyldithiocarbamate, the superoxide radical and hydrogen peroxide were considered to play a pathogenic role in this ulcer model.     

A simple,sensitive, non-stimulated photon counting system for detection of superoxide anion in whole blood

A simple, sensitive, non-stimulated assay was developed to measure the superoxide anion concentration in whole blood, using an ultra-sensitive chemiluminescence (CL) analyzer, and lucignin amplification. The assay system can be performed without leukocyte isolation or stimulant administration. The blood CL levels of healthy males (362.8±337.7 counts/10 sec) were not different from those of females (335±308.7 counts/10 sec) (p=0.64), whereas the CL levels in whole blood in patients with acute pancreatitis (2522±2014 counts/10 sec) were significantly higher than those of healthy controls (p<0.001). This assay system may be valuable in the future for quantitative measurement of reactive oxygen species in various disorders.     

Role of active oxygen species in diethyldithiocarbamate-induced gastric ulcer in the rat

Summary Diethyldithiocarbamate, an inhibitor of Cu,Zn-superoxide dismutase, was recently found to be ulcerogenic in the rat stomach, and active oxygen species were found to be responsible for its ulcerogenicity. To clarify which active oxygen species play a role in ulcerogenesis, the effects of various scavengers and iron-chelators were studied. As superoxide dismutase and catalase reduced the ulcerogenesis induced by diethyldithiocarbamate, the superoxide radical and hydrogen peroxide were considered to play a pathogenic role in this ulcer model.     

Proton-induced dismutation of superoxide in aprotic media by bile pigments

Cyclic voltammetry of molecular oxygen in aprotic media (dimethylformamide) and in the presence of bilirubin and other bile pigments shows that superoxide anion (O2-.) undergoes proton-induced dismutation. Lactam hydrogens of bile pigments are sufficiently acid to induce (O2-.) disproportionation to O2 and H2O2. Because of its characteristic lipophilic behavior, a biological role for natural bilirubin similar to that of other non-enzymatic lipophilic scavengers of (O2-.) is suggested.     

A working hypothesis for negative ion generators

Summary The effects of negative ion generators may be explained by reactions of carbonate radical anion, or alternatively, by physical factors such as the electric field of the ions generated by these apparatuses.     

Ribonucleotide reductases and radical reactions

Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to deoxyribonucleotides. They play a pivotal role in the regulation of DNA synthesis and are targets for antiproliferative drugs. Ribonucleotide reductases are unique enzymes in that they all require a protein radical for activity. Class I nonheme iron RNRs (mammals, plants, Escherichia coli) use a tyrosyl/cysteinyl radical pair, class II adenosylcobalamin RNRs (prokaryotes, archaea) a cysteinyl radical, class III iron-sulphur RNRs (facultative anaerobes) a glycyl radical. Here we describe the reactivity of these radicals with respect to the natural ribonucleotide substrates as well as to a variety of enzyme inhibitors, radical scavengers, nitric oxide, superoxide radicals and substrate analogues. Received 3 December 1997; received after revision 26 February 1998; accepted 27 February 1998