Detection Of Superoxide Radicals Research Articles

Amperometric detection of superoxide dismutase at cytochrome c-immobilized electrodes: xanthine oxidase and ascorbate oxidase incorporated biopolymer membrane for in-vivo analysis.

Amperometric measurement of superoxide dismutase (SOD) was carried out at cytochrome c-immobilized monolayers and ascorbate oxidase (AOD)/xanthine oxidase (XOD)/cytochrome c- and (AOD, XOD)/cytochrome c-multilayers. Cytochrome c was covalently immobilized on mercaptopropionic acid-containing self-assembled monolayers on gold. A biopolymer membrane of poly-L-lysine confining XOD and AOD was cast on the monolayer of cytochrome c. While both the cytochrome c-immobilized monolayer and multilayer electrodes show anodic current responses to the generation of superoxide radical, the sensitivity of the multilayer system for the detection of superoxide radical was high relative to that of the monolayer system. In the case of the cytochrome c-multilayer electrodes, the generation of superoxide radical near the sensing element, cytochrome c, resulted in high sensitivity for the detection of superoxide. The use of a XOD and AOD-incorporated poly-L-lysine membrane enabled the detection of the generation of superoxide radical in the presence of L-ascorbic acid. Though L-ascorbic acid could scavenge superoxide radical, the biopolymer membrane confined with AOD will oxidize any L-ascorbic acid that permeated into the membrane. By using the multilayer electrodes, one could measure the activity of SOD in the presence of L-ascorbic acid.

Superoxide sensor based on hemin modified electrode

A hemin modified pyrolytic graphite (PG) electrode was prepared and applied for the electrochemical determination of superoxide. The rate constant of heterogeneous electron transfer of adsorbed hemin was determined with cyclic voltammetry (CV) to be 15 s −1. The hemin modified electrode was applied to detect superoxide radicals produced by xanthine oxidase (XOD) catalyzed hypoxanthine oxidation. Sensitivity was higher than compared to the established cytochrome c (cyt c)-based sensor. The antioxidative activity of superoxide dismutase (SOD) and uric acid was also investigated.

Detection of Superoxide Radicals and Peroxynitrite by 1-Hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine: Quantification of Extracellular Superoxide Radicals Formation

The reactions of the new sterically hindered hydroxylamine 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) with superoxide radical and peroxynitrite have been studied. These reactions produce the nitroxide 4-phosphonooxy-2,2,6,6-tetramethyl-piperidinyloxy. The rate constant for reaction of superoxide with PP-H is determined as (8.4±0.6)·102M−1s−1. It was found that PP-H provides almost the same spin trapping efficacy as 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background oxidation of PP-H in blood is much less than for CP-H. The extremely slow PP-H penetration into the cells makes possible the study of extracellular formation of superoxide radical. The acute treatment of blood with nitroglycerin is shown to induce an extracellular superoxide radical formation. PP-H is more sensitive for detection of reactive oxygen species as compared with CP-H. PP-H is an effective scavenger of superoxide radical and of peroxynitrite, and can be used to quantify the extracellular formation of these reactive oxygen species.

Oxidative stress in the Dahl hypertensive rat.

Enhanced production of oxygen free radicals may play a role in hypertension by affecting vascular smooth muscle contraction, resistance to blood flow, and organ damage. The aim of this study was to determine whether oxygen free radicals are involved in the development of salt-induced hypertension. Dahl salt-sensitive (Dahl-S) and salt-resistant (Dahl-R) rats were fed either a high salt (6.0% NaCl) or low salt (0.3% NaCl) diet for 4 weeks. The high salt diet caused the development of severe hypertension in Dahl-S animals and had no effect on blood pressure in Dahl-R animals. A tetranitroblue tetrazolium dye was used to detect superoxide radicals in microvessels of the mesentery. Light absorption measurements revealed enhanced staining along the endothelium of arterioles and venules in hypertensive Dahl-S animals, with significantly lower values in normotensive animals. In addition, a Clark electrochemical electrode was used to measure hydrogen peroxide levels in fresh plasma. Hypertensive Dahl-S animals had a higher plasma hydrogen peroxide concentration compared with their normotensive counterparts (2.81+/-0.43 versus 2.10+/-0.41 micromol/L), while no difference was detected between high- and low salt-treated Dahl-R animals (1.70+/-0.35 versus 1.56+/-0.51 micromol/L). The plasma hydrogen peroxide levels of all groups correlated with mean arterial pressure (r=.77). These findings demonstrate an enhanced production of oxygen free radicals in the microvasculature of hypertensive Dahl-S rats.

Simultaneous superoxide and hydrogen peroxide detection in peroxidase/NADH oscillator

A dual enzyme-based amperometric sensor was applied to simultaneous detection of superoxide radical and hydrogen peroxide in the peroxidase/NADH oscillator. Both species were detected at nanomolar concentrations. Concentration–time profiles for superoxide and hydrogen peroxide closely resembled qualitative and quantitative model predictions except during periods of large-d[O 2]/d t. Introduction of the sensor did not cause perturbation of the chemical behavior of the system within the reproducibility of the standard oscillator. Trace oxygen reduction on the sensors' glassy carbon surfaces was minimized by setting biasing potentials of both sensors to E=+170 mV (vs. WO 3 reference electrode). Characterization of hydrogen peroxide and superoxide sensors' membranes was performed by Fourier Transform-Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy, and electrochemistry.

A modified amperometric electrode for the determination of free radicals

In this paper we describe a new electrochemical system able to detect superoxide radicals produced by an enzymatic reaction: the system is based on the reduction of cytochrome c due to the superoxide radical produced in the oxidation reaction of xanthine catalysed by the xanthine oxidase; then the reduced form of the cytochrome is electrochemically detected. Measurements are carried out using first cyclic voltammetry, and then a true carbon paste electrode having cytochrome c as mediator and Fe(III)–protoporphyrin as promoter for continuous superoxide radical determination working at constant applied potential is also proposed.

Amperometric Sensors for Simultaneous Superoxide and Hydrogen Peroxide Detection

A two-channel sensor capable of almost instantaneous simultaneous detection of superoxide radical and hydrogen peroxide in the concentration range 10(-)(7)-10(-)(4) M is very important for understanding of a number of rapid kinetics processes. A glassy carbon working microelectrode covered by an electrodeposited polypyrrole/horseradish peroxidase (PPy/HRP) membrane was employed as a H(2)O(2) sensor. Another glassy carbon microelectrode covered by a composite membrane of an inside layer of PPy/HRP and an outside layer of superoxide dismutase was employed as a working electrode for superoxide detection. These two working electrodes with Pt counter and tungsten oxide (WO(3)) reference electrodes were contained in one 6 mm diameter Teflon cylinder. Simultaneous measurements were performed at a potential of -60 mV (vs WO(3) reference, pH 5.1). Additional sensor characterization was performed for pH 5.1-9.0. Superoxide sensor behavior as a function of membrane deposition conditions and coating time is reported. Sensors' mutual influence, selectivity, response times, linearity, stability, and sensitivity for hydrogen peroxide and superoxide are presented and discussed. A mathematical model of sensors' responses is proposed, with model calculation corresponding to experiment within 10%.

Continuous detection of superoxide in situ during ischemia and reperfusion in the rabbit heart.

In order to clarify the time course of superoxide generation in situ during ischemia and reperfusion in the rabbit heart, we used a method of enhanced chemiluminescence (CL) with 2-methyl-6-[p-methoxyphenyl]-3, 7-dihydroimidazo [1, 2-alpha]pyrazin-3-one (MCLA) as a specific probe for detecting superoxide radicals. The surface of the rabbit heart was exposed to a photomultiplier tube in a light-proof box. We introduced a reversible snare occluder into the box to continuously observe the light emission. An ischemia-reperfusion group (I/R, n = 7) was subjected to 30 mins of coronary occlusion, followed by 90 mins of reperfusion. We performed the same procedure (except for coronary occlusion) in the sham-operated group (n = 4). Another group of rabbits (n = 4) subjected to I/R received superoxide dismutase (SOD: 20 mg/kg, i.v.) during reperfusion to observe the CL response. In the I/R-group, the increase in CL began at 13 +/- 2 (mean +/- SEM) mins and peaked at 52 +/- 12 mins of reperfusion. CL in the I/R-group gradually increased from 818 +/- 350 counts/10 secs in the preischemic period to 1077 +/- 401 counts/10 secs during reperfusion (p < 0.01). In contrast, there was no increase in CL in the sham-operated group. The administration of SOD briefly attenuated CL by 24.1 +/- 6.8% for a period of 24.3 +/- 6.8 mins. The superoxide generation in situ in the ischemic rabbit heart appears to increase gradually and persists for a period following reperfusion.

A chemiluminescent detection of superoxide radical produced by adherent leucocytes to the subendothelium following thrombolysis: studies with a photochemically induced thrombosis model in the guinea pig femoral artery

Reocclusion following thrombolysis is a major limitation of thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) because denuded vessel wall exposed to blood following thrombolysis is a favourable surface for platelet and leucocyte deposition. We have applied a chemiluminescence technique to detect superoxide radical (O 2 −) produced by leucocytes adherent to the femoral artery 24 h after photochemically induced thrombogenesis in the guinea pig in vivo and subsequent thrombolysis by rt-PA. Intravenous administration of MCLA, a specific chemiluminescence reagent for detecting O 2 −, markedly increased photon emission. The photon emission was markedly potentiated by phorbol myristate acetate and was suppressed by superoxide dismutase. Reocclusion 24 h after rt-PA induced thrombolysis was observed in 10 of 16 animals. Histological observations revealed extensive polymorphonuclear leucocytes adherent to the vessel wall at the site of thrombogenesis and thrombolysis. A higher level of O 2 − could be detected from the arteries in which thrombolysis was induced compared with those without thrombolysis. Further, the level of O 2 − detected was greater in reoccluded arteries compared with those in which reflow was established. These observations suggest that O 2 − is produced by adherent leucocytes at the site of thrombolysis and that leucocytes are involved in reocclusion after thrombolysis.

A method to detect superoxide radicals using Teflon membrane and superoxide dismutase

A method to detect superoxide radicals using Teflon membrane and superoxide dismutase

Bioluminescence in scale-worm photosomes: the photoprotein polynoidin is specific for the detection of superoxide radicals.

Photosomes are the characteristic organelles of the luminous epithelium in the elytral appendages of polynoïd annelids. They are paracrystals of endoplasmic reticulum and emit a flash of bioluminescence in response to stimulation. The series of flashes in response to repetitive stimulation begins with a period of facilitation because the number of reacting photosomes increases in each photogenic cell. Reacting photosomes are coupled to the plasma membrane by dyad junctions which are established under stimulation and dedifferentiate in the resting system. The calcium influx of an action potential propagated through the conducting elytral epithelium triggers the luminous reaction. This reaction is based on a membrane photoprotein, polynoidin, which is specifically triggered by superoxide radicals. These oxy radicals result from the oxydation of riboflavin, which is present in a compartment of the photosomes. Polynoidin proved to be an interesting probe in the detection of superoxide radicals produced by activated white blood cells. Its potential applications are discussed.

A simple method for evaluation of superoxide radical production in neural cells under various culture conditions: application to hypoxia.

To evaluate the potential deleterious influence of oxygen-derived free radicals following hypoxia in a model of primary culture of neurons obtained from the fetal rat brain, superoxide radicals were measured as a function of time in the extracellular medium. Neuronal cells were grown for 8 days in the presence or absence of serum, then incubated in a buffered Krebs-Ringer solution containing 60 microM acetyl-cytochrome c. The rate of superoxide radical formation was quantified spectrophotometrically by measuring the specific reduction of acetyl-cytochrome c. Under normoxic conditions (95% air-5% CO2), basal production of superoxide that increased with time was recorded. It was significantly more pronounced in cells grown in serum-free medium. Under both culture conditions, acute hypoxia (95% N2-5% CO2) for 6 h increased superoxide radical amounts in the extracellular medium, and they were still enhanced 3 h after reoxygenation. The addition of superoxide dismutase to the incubating medium abolished the detection of superoxide radicals. The present study describes a new reliable method for superoxide radical measurement in cells in vitro and demonstrates hypoxia/reoxygenation-induced overproduction of superoxide in cultured neurons that may account for cell injury.

32] In vivo determination of superoxide and vitamin C radicals using cytochrome c and superoxide dismutase derivatives

Publisher Summary Most of the reactive oxygen species rapidly react with various molecules and interfere with cellular functions. Based on experiments using antioxidant enzymes and scavengers for reactive oxygen species and free radicals, such as Cu/Zn-type superoxide dismutase (SOD), glutathione, ascorbic acid, and dimethyl sulfoxide (DMSO), critical roles of these reactive species in the pathogenesis of various diseases are presented in this chapter. However, these antioxidants react with a wide variety of compounds and are involved in various metabolic pathways. Thus, even if these scavengers inhibited tissue injury, the corresponding reactive oxygen species may not always be involved in their pathogenesis. To get direct evidence for the involvement of reactive oxygen species in the pathogenesis of various diseases, these species should be determined quantitatively in vivo. Cytochrome c (Cyt c) is used for determining superoxide radicals in vitro. Because Cyt c reacts not only with superoxide radicals but also with other compounds with reducing activity and serves as a substrate for cytochrome c-reductase and cytochrome c-oxidase, acetylated Cyt c (AC) is used for the detection of superoxide radicals, particularly in complex biological systems.

Photochemical oxygen activation: Superoxide radical detection and production rates in the eastern Caribbean

Superoxide ion‐radical, O2‐, the one‐electron reduction product of molecular oxygen, is a long‐suspected first intermediate in chemical reactions using oxygen as the ultimate electron acceptor. We have detected photochemical production of O2‐ in a variety of eastern Caribbean waters studied in Spring and Fall by using a chemically direct O2‐ trapping reaction combined with 15N and/or 18O isotopic labelling of reactants to provide sensitivity and specificity. Photochemical superoxide production rates ranged from 0.1–6 nmol L−1 min−1 of full‐sun irradiation in Spring and from 0.2–8 nmol L−1 min−1 in Fall. These superoxide fluxes correlate well with independently measured total (NO‐scavengeable) radical fluxes in both seasons and account for ∼ 1/3 of the total radical production. Hence we confirm that O2‐ is a quantitatively important, perhaps dominant, reactive transient species in the photochemical radical array. Semiquantitative estimates suggest that O2‐ dismutation to HOOH and O2 can account for most or all of the photochemical HOOH production in surface waters. O2‐ production rates also correlated negatively with salinity.

Photosensitization by anticancer agents 11. Mechanisms of photosensitization of human leukemic cells by diaminoanthraquinones: Singlet oxygen and radical reactions

The synthesis of several aminoanthraquinone derivatives (AAQs), designed to suppress the dark toxicity and to promote more efficient cancer cell photosensitization for potential use in photodynamic therapy (PDT), is described. The following AAQs were synthesized: 1-NH 2-4,5-(MeO) 2-AQ ( 1), 1,5-(NH 2) 2-4,8-(MeO) 2-AQ ( 2), 1,8-(NH 2) 2-4,5-(MeO) 2-AQ ( 3), and 1,5-(NHPhMe) 2-4,8-(MeO) 2-AQ ( 8). The agents exhibit strong absorption in the region 480–620 nm. Possible mechanisms of photosensitization were studied by measuring 1O 2 phosphorescence at 1270 nm, detecting superoxide radicals employing an electron paramagnetic resonance (EPR)-spin trapping technique, and measuring oxygen consumption during the photo-oxidation of a representative biological electron donor, NADH. Strong phosphorescence from 1O 2 was observed upon illumination of 2 and 3 in C 6H 6 (quantum yield of 0.25 and 0.5 respectively), and in EtOH (quantum yield of 0.23 and 0.34). The 1-amino-AQ ( 1) was the weakest 1O 2 sensitizer, with quantum yield of 0.13 in benzene. No phosphorescence was observed in EtOH. A superoxide radical was detected as a spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in irradiated benzene solutions of 1, 2 or 3 and DMPO. AAQs 2 and 3 sensitized photo-oxidation of NADH in H 2O/EtOH mixture with the intermediacy of singlet oxygen as judged by the effect of sodium azide on the photostimulated oxygen consumption. Evolution of O 2 upon addition of catalase to the illuminated solution confirmed the ultimate formation of hydrogen peroxide. These findings suggested that the (di)amino-dimethoxyanthraquinones might exert photosensitization via both Type I and Type II mechanisms. The AAQs were tested for their ability to photosensitize K562 human chronic myeloid leukemic cells in culture. Viability was measured using the 3,4,5-diethylthiazol-2,5-diphenyl tetrazolium blue assay, and DNA and possible membrane damage were assessed. The results from illuminating cells with light > 475 nm show that for the 1,5-compounds, the presence of methoxy substituents at 4,8 positions reduces the dark toxicity from ID 50 of 23 to 250 μM and for the 1,8-compounds correspondingly from ID 50 of 53 to > 300 μM. In the 1,5-series,this decrease of the dark toxicity is accompanied by an increase in light-induced dose modification from 8.85 to 14.4.Differences exist in the mechanisms of cytotoxicity between the prototype phenolic AAQs and their methoxy counterparts. It appears that the cytotoxic action of the latter causes cell damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks. No evidence for membrane damage, as determined by transport of the model amino acid cycloleucine, could be observed even at supralethal doses.

Synthesis of a cytochrome c derivative with prolonged in vivo half-life and determination of ascorbyl radicals in the circulation of the rat.

Since cytochrome c and acetylated cytochrome c disappear from the circulation with a half-life of 4 min, these proteins cannot be used for in vivo detection of superoxide radicals and related metabolites. To determine superoxide and other radicals in vivo, a cytochrome c derivative (SMAC) was synthesized by linking 1 mol of poly(styrene-co-maleic acid) butyl ester (SM) to cytochrome c, followed by acetylation of its lysyl amino groups. SMAC retained 8 and 80% of cytochrome c activity to react with ascorbyl and superoxide radicals, respectively. However, SMAC did not serve as a substrate for cytochrome c reductase and cytochrome c oxidase. When injected intravenously to the rat, SMAC circulated bound to albumin with a half-life of 130 min. SMAC was rapidly reduced in the circulation of intact animals. Treatment of animals with paraquat markedly enhanced the reduction of the circulating SMAC. We have synthesized an SM-conjugated superoxide dismutase (SOD) derivative (SM-SOD) that circulates bound to albumin with a half-life of 6 h. Kinetic analysis revealed that SM-SOD effectively inhibited the superoxide-dependent reduction of SMAC either in the presence or absence of 0.5 mM albumin. However, the reduction of the circulating SMAC was not inhibited by SM-SOD both in normal and paraquat-treated animals. Plasma samples from both animal groups also reduced cytochrome c and SMAC by an SOD-insensitive mechanism. However, after treatment with ascorbate oxidase, both plasma samples lost their activity to reduce cytochrome c and SMAC. These and other results suggest that ascorbyl radical might principally be responsible for the reduction of circulating SMAC and that plasma levels of ascorbyl radical might increase in paraquat-treated animals.

Oxygen-free radicals in traumatic brain oedema

Oxygen-free radicals have been implicated as causative factors in ischaemic and traumatic processes. Oxygen-free radical scavengers can be used potentially to treat brain oedema. We investigated the effects of superoxide dismutase and dimethylthiourea on brain oedema. Vasogenic brain oedema was produced in 44 cats by a cortical freezing lesion. Animals were separated into three groups: (1) cold-induced oedema with sacrifice at 6, 24 and 48 h; (2) cold-induced oedema with sacrifice at 6, 24 and 48 h: subgroup A was pretreated with 10,000 u./kg polyethylene glycol and superoxide dismutase; subgroup B received a bolus injection of free superoxide dismutase (4 mg/kg) and then 1 mg/kg/min for 20 min; (3) cold-induced oedema with sacrifice at 6 and 24 h: this group was pretreated with 500 mg/kg dimethylthiourea. Brain water content was measured by the specific gravity method. Detection of superoxide radicals was carried out by the direct cortical application of nitroblue tetrazolium. Free and polyethylene glycol-superoxide dismutase did not prevent the development of brain oedema, but superoxide radicals were detected in the cold lesion. Dimethylthiourea prevented the development of brain oedema in the white matter adjacent to the lesion at 6 h but not at 24 h. These findings indicate that oxygen-free radicals are generated by the brain following cold injury and the demonstration of these radicals offers an important clue in the genesis of traumatic brain oedema.

Oxygen Free Radicals in the Genesis of Traumatic and Peritumoral Brain Edema

This study evaluates the effect of superoxide dismutase, a scavenger of oxygen free radicals, on peritumoral and cold injury edema. Vasogenic brain edema was produced in 39 cats by a standardized cortical freezing lesion. Peritumoral edema was produced by the transplantation of VX2 carcinoma cells into the brain of New Zealand White rabbits. Detection of superoxide radicals was studied by topical application of nitroblue tetrazolium to the cortical injury and by incubation of the VX2 carcinoma cells. The animals were treated with free superoxide dismutase (SOD) and polyethylene glycol superoxide dismutase (PEG-SOD). The following groups were studied: 1) control, 2) untreated tumor group, 3) tumor group treated with PEG-SOD, 4) a group treated with PEG-SOD before injury, and 5) a group treated with free SOD after injury. Brain edema was evaluated by measurement of specific gravity and planimetry study of the spread of Evans blue dye. Preliminary data indicate that superoxide radicals are present in the brain after cold injury and in the cytoplasm and nucleus of VX2 carcinoma cells. Free SOD and PEG-SOD had no beneficial effect upon the vasogenic brain edema produced in either model. It is concluded that intracellular uptake of SOD may be one of the factors necessary for an effect upon either form of edema.

Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems

In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems. This nitroso spin trap recently has been reported to yield very persistent spin adducts with O 2 −· as well as with various carbon-centered radicals. In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied. no significant toxicity was found. In cellular systems, however, DBNBS did not produce detectable adducts with O 2 −·; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system. Both DBNBS and a DBNBS adduct preformed extracellularly and then added to cell suspensions were rapidly metabolized by cells. Intracellular spin adducts were not detected under any condition. Evidently, in spiute of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.

Measurement of Serum Superoxide Dismutase Activity by Electron Spin Resonance

We measured serum superoxide dismutase activity by electron spin resonance, using 100mM 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a trap. The addition of superoxide dismutase decreased the height of the DMPO-OOH signal produced by the hypoxanthine-xanthine oxidase system. Serum superoxide dismutase activity was determined from a quantitative line for superoxide dismutase. When bovine Cu, Zn-superoxide dismutase (50, 000U/kg) was injected intravenously into rats, the superoxide dismutase activity decreased almost linearly with a half life of about 6min. This method allowed direct detection of superoxide radicals and new measurement of superoxide dismutase activity.