Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

What do we measure by a luminol-dependent chemiluminescence of phagocytes?

The review presents a survey of published findings concerning the mechanism of luminol-dependent chemiluminescence in biological systems. The potential of various oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) to react with luminol is discussed. The ability of commonly used enzymes (superoxide dismutase, catalase), inhibitors, and oxygen radical scavengers to discriminate between individual oxygen species is assessed together with the potential of a variety of substances encountered in biological systems to interfere in luminol-dependent chemiluminescence reactions. It is concluded that luminol-dependent chemiluminescence gives at present very little ability to discriminate between individual oxygen or radical species. Furthermore, luminol-dependent chemiluminescence used in biological systems is extremely prone to many interferences, which are very difficult to control.     

Electron paramagnetic resonance spin trapping investigation into the kinetics of glutathione oxidation by the superoxide radical: re-evaluation of the rate constant

The ability of glutathione to scavenge the superoxide radical is a matter of serious contention in the literature: reported values for the second-order rate constant range from 10(2) to greater than 10(5) M(-1) s(-1). The physiological implications of this discrepancy will determine, for example, whether or not glutathione can compete with Mn-superoxide dismutase for reaction with the radical in the mitochondrial matrix, leading to formation of the potentially harmful glutathionyl radical. Several authors have investigated the kinetics of glutathione oxidation by superoxide using spectrophotometric assays, based on competition between either ferricytochrome c or epinephrine for reaction with the radical. However, these approaches have received criticism because the contributions of various secondary reactions to the overall kinetics have been largely overlooked (e.g., the reduction of ferricytochrome c by glutathione). In the present investigation, we have used electron paramagnetic resonance spectroscopy to monitor competition between GSH and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide. This method has been used previously and a rate constant of 1.8 x 10(5) M(-1) s(-1) obtained (Dikalov, S.; Khramtsov, V.; Zimmer, G. Arch. Biochem. Biophys. 326:207-218; 1996). However, we demonstrate that this value is a gross overestimation because the spectrum of the hydroxyl radical adduct of the spin trap was incorrectly assigned to the glutathionyl radical adduct. The relatively high yield of the DMPO hydroxyl radical adduct is shown to be due to the two-electron reduction of the corresponding superoxide radical adduct by glutathione. Taking these factors into consideration, we estimate the second order rate constant for the oxidation of glutathione by superoxide to be approximately 200 M(-1) s(-1).     

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H₂O₂, hydroxyl radical, or peroxynitrite (ONOO⁻). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes.     

Carbonate anions; effects on the oxidation of luminol, oxidative hemolysis, gamma-irradiation and the reaction of activated oxygen species with enzymes containing various active centres

Presence of carbonate anions increases the oxidation of luminol in different chemical systems. Lysis of human erythrocytes due to the action of dihydroxyfumaric acid or of perborate is also stimulated by carbonate ions. These anions also change considerably the loss of activity of different enzymes treated with superoxide, hydroxyl or formate radicals and can increase or decrease the effect as a function of the nature of the active centre of the enzyme. The relative effects of superoxide, hydroxyl, formate and carbonate radicals for the inactivation of various enzymes (superoxide dismutases, catalase, ribonuclease, glucose oxidase and glutathione peroxidase) have been examined. Three systems were used: gamma-irradiation under different conditions, photoproduction of radicals and sonication. Inactivation of the enzymes is a function not only of the radical used but also of the nature of the active site. Thus glutathione peroxidase is remarkably resistant to hydroxyl radicals while the superoxide dismutases are rapidly inactivated by carbonate radicals. All of the results combine to show that the presence or absence of carbonate anions must be considered in all studies of oxygen containing free radicals whether chemical, biochemical or biological or high energy irradiation.     

Superoxide dismutase and free radical pathology

Different defense systems against oxidative damage leading to pathological conditions are described. The superoxide radical plays a primary role in initiating and sustaining biological damage and is responsible for the production of other free radicals and lipoperoxides. Certain pathologies are associated with these events such as post-irradiation necrosis, or the Spanish Toxic Oil Syndrome. Superoxide dismutase has been used clinically with considerable success, particularly the liposomal form, to treat various diseases in which it has been shown that the superoxide radical plays an important role. Although the mechanism of the enzymic reaction catalysed by superoxide dismutase is now well defined, a complete explanation of the anti-inflammatory properties in vivo of the enzyme has not yet been established.     

Detection of oxygen radicals during reperfusion of intestinal cells in vitro

The nitroxide OXANO. (2-Ethyl-2,5,5-trimethyl-3-oxazolidinoxyl) which in its reduced form, OXANOH (2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine), is capable of reacting with short-lived radicals, forming a secondary stable radical, was used for ESR-detection of radical production in isolated cells. The properties of OXANO. and OXANOH in terms of stability in cellular and subcellular systems, membrane permeability and effects on cellular viability were evaluated. Ischemia and reperfusion was simulated in vitro in a preparation of cells from rat intestinal mucosa by incubation at high density (4 X 10(8) cells/ml) under an atmosphere of nitrogen for 25 min and resuspended with fresh oxygenated buffer containing 5 mM OXANOH. A significant increase in radical formation during the 15 min reperfusion period studied was obtained in cells exposed to ischemia compared to control cells incubated at normal density under an atmosphere of oxygen. The addition of 5 microM of the scavenging enzyme superoxide dismutase reduced the radical formation by 50%. The time sequence of the superoxide formation was calculated as the difference in radical production in the presence and absence of superoxide dismutase.     

Reassignment of organic peroxyl radical adducts.

The study of the important role of peroxyl radicals in biological systems is limited by their difficult detection with direct electron spin resonance (ESR). Many ESR spectra were assigned to 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/peroxyl radical adducts based only on the close similarity of their ESR spectra to that of DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the radical adduct from DMPO/superoxide radical adduct. Later, the spin-trapping literature reported that DMPO/peroxyl radical adducts have virtually the same hyperfine coupling constants as synthesized alkoxyl radical adducts, raising the issue of the correct assignment of peroxyl radical adducts. However, using 17O-isotope labelling, the methylperoxyl and methoxyl radical adducts should be distinguishable. We have reinvestigated the spin trapping of the methylperoxyl radical. The methylperoxyl radical was generated in aerobic solution with 17O-molecular oxygen either in a Fenton system with dimethylsulfoxide or in a chloroperoxidase system with tert-butyl hydroperoxide. Two different spin traps, DMPO and 2,2,4-trimethyl-2H-imidazole-1-oxide (TMIO), were used to trap methylperoxyl radical. 17O-labelled methanol was used to synthesize methoxyl radical adducts by nucleophylic addition. It was shown that the 17O hyperfine coupling constants of radical adducts formed in methylperoxyl radical-generating systems are identical to that of the methoxyl radical adduct. Therefore, methylperoxyl radical-producing systems form detectable methoxyl radical adduct, but not detectable methylperoxyl radical adducts at room temperature. One of the possible mechanisms is the decomposition of peroxyl radical adduct with the formation of secondary alkoxyl radical adduct. These results allow us to reinterpret previously published data reporting detection of peroxyl radical adducts. We suggest that detection of 17O-alkoxyl radical adduct from 17O-labelled molecular oxygen can be used as indirect evidence for peroxyl radical generation.     

Intratracheal injection of nickel chloride and copper-zinc superoxide dismutase activity in lung of rats.

Superoxide radical (O2-) is a free radical that may be involved in various toxic processes. Cu--Zn superoxide dismutase catalyses the dismutation of the superoxide free radical and protects cells from oxidative damage, and it has been used clinically. The concentration of Ni2+ and Cu--Zn superoxide dismutase activity were measured in lungs of rats at time intervals of 5, 12, 19, 26, 33, and 40 days following an intratracheal injection of 127 nmol of NiCl2. Nickel chloride increased nickel content and resulted in a significant increase of Cu--Zn superoxide dismutase activity in lungs. This elevation of Cu--Zn superoxide dismutase activity was highest on the 12th day (approximately threefold) and is at levels comparable to controls rats on day 40 onwards. Since Cu--Zn superoxide dismutase activity was increased in lung throughout our experimental period without corresponding increases of Cu2+ and Zn2+, we speculate that the elevation of Cu--Zn superoxide dismutase activity might be due to an increased half-life of the enzyme, induced by nickel.     

Role of antioxidant enzymes in cell immortalization and transformation

Summary The role of antioxidant enzymes, particularly superoxide dismutase (SOD), in immortalization and malignant transformation is discussed. SOD (generally MnSOD) has been found to be lowered in a wide variety of tumor types when compared to an appropriate normal cell control. Levels of immunoreactive MnSOD protein and mRNA for MnSOD also appear to be lowered in tumor cells. Tumor cells have the capacity to produce superoxide radical, the substrate for SOD. This suggests that superoxide production coupled with diminished amounts of MnSOD may be a general characteristic of tumor cells. The levels of MnSOD in certain cells correlates with their degree of differentiation; non-differentiating cells, whether normal or malignant, appear to have lost the ability to undergo MnSOD induction. These observations are used to elucidate a two-step model of cancer. This model involves not only the antioxidant enzymes, but also organelle (particularly mitochondria and peroxisomes) function as a dominant theme in carcinogenesis.     

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.     

Selective detection of superoxide anion radicals generated from macrophages by using a novel fluorescent probe

Quantitation of superoxide radical (O (2)(-).) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O(2)(-). using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohexene), coupled with a spectra character-signaling increase event. A high-specificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O(2)(-). in living cells. Better selectivity for O(2)(-). over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 x 10(-9)-3.33 x 10(-6) M. The detection limit was 1.68 x 10(-9) M. Fluorescence images of probe-stained macrophages stimulated with 4beta-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope.     

Depolarization-Induced Superoxide Radical Formation in Rat Hippocampal Slices

Glutamate is the major excitatory neurotransmitter in the brain. Activation of glutamatergic receptors induces neuronal depolarization, and if this activation is excessive, it can lead to cellular damage. Evidence for the participation of glutamatergic receptor systems in the production of oxygen free radicals in neuronal cells is accumulating. In the present study, we have kept hippocampal slices under depolarization conditions induced by including 50 mM K⁺ in artificial cerebrospinal fluid (dACSF) and followed superoxide radical formation. Superoxide radical formation was increased in dACSF-incubated hippocampal slices. We have also attempted to determine the relative contribution of agonist- and voltage-sensitive channels to superoxide radical formation by using their selective blockers. Superoxide radical formation was suppressed by MK 801, memantine, APV, CNQX, and TTX application to dACSF-incubated hippocampal slices. Similar studies on different experimental systems may help to unravel the underlying critical events and active mechanisms that may lead to superoxide radical generation and subsequent neuronal cell death.     

Manganese complexes and the generation and scavenging of hydroxyl free radicals

In a wide variety of biological systems non-enzyme complexes of the metals copper (Cu) and iron (Fe) have been shown to enhance oxygen radical damage by increasing the production of an oxidative species generally believed to be the hydroxyl free radical (.OH) via "Fenton" and possibly "Haber-Weiss" type reactions. However, the behavior of the chemically and biologically similar transition metal manganese (Mn) with .OH is unknown. Unlike Fe and Cu, inorganic complexes of Mn are known to exist in high concentrations in certain cells. Three different oxygen free radical generating systems and four .OH detection methods were used to investigate the activity of biologically relevant inorganic Mn complexes. These complexes were compared to compounds reported to scavenge and generate .OH. The direct and indirect effects of Mn on the .OH flux were compared by attempting to distinguish the effects of hydrogen peroxide (H2O2), superoxide (O2-), and .OH through the use of selective scavengers and generators. Mn-EDTA and biologically relevant Mn-pyrophosphates and polyphosphates, in contrast to Fe-EDTA, do not generate .OH in these systems. The results suggest that Mn in various forms does, indeed, inhibit oxy-radical damage mediated by .OH, but only if the .OH production is dependent on the presence of O2- or H2O2. Thus, with .OH, as with O2- and H2O2, Mn complexes appear to behave in a fundamentally different fashion from Cu and Fe.     

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H₂O₂), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.     

The participation of coenzyme Q in free radical production and antioxidation

Published experimental data pertaining to the participation of coenzyme Q as a site of free radical formation in the mitochondrial electron transfer chain and the conditions required for free radical production have been reviewed critically. The evidence suggests that a component from each of the mitochondrial NADH-coenzyme Q, succinate-coenzyme Q, and coenzyme QH₂-cytochrome c reductases (complexes I, II, and III, most likely a nonheme iron-sulfur protein of each complex, is involved in free radical formation. Although the semiquinone form of coenzyme Q may be formed during electron transport, its unpaired electron most likely serves to aid in the dismutation of superoxide radicals instead of participating in free radical formation. Results of studies with electron transfer chain inhibitors make the conclusion dubious that coenzyme Q is a major free radical generator under normal physiological conditions but may be involved in superoxide radical formation during ischemia and subsequent reperfusion. Experiments at various levels of organization including subcellular systems, intact animals, and human subjects in theclinical setting, support the view that coenzyme Q, mainly in its reduced state, may act as an antioxidant protecting a number of cellular membranes from free radical damage.     

Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses

Scavenging or quenching of the reactive oxygen species (ROS) involved in oxidative stress has been the subject of many recent studies. Resveratrol, found in various natural food products, has been linked to decreased coronary artery disease and preventing cancer development. The present study measured the effect of resveratrol on several different systems involving the hydroxyl, superoxide, metal/enzymatic-induced, and cellular generated radicals. The rate constant for reaction of resveratrol with the hydroxyl radical was determined, and resveratrol was found to be an effective scavenger of hydroxyl, superoxide, and metal-induced radicals as well as showing antioxidant abilities in cells producing ROS. Resveratrol exhibits a protective effect against lipid peroxidation in cell membranes and DNA damage caused by ROS. Resveratrol was also found to have a significant inhibitory effect on the NF-kappaB signaling pathway after cellular exposure to metal-induced radicals. It was concluded that resveratrol in foods plays an important antioxidant role.     

Luminol-enhanced chemiluminescence after reaction of hydroperoxides with opsonized zymosan

Luminol-enhanced chemiluminescence (LEC) is very sensitive in detecting free radicals but relatively insensitive for hydroperoxides (hydrogen peroxide, tert-butyl hydroperoxide). However, in the presence of opsonized zymosan (often used for stimulation of phagocytic cells) hydroperoxides also induce LEC, suggesting that free radicals are produced under these conditions. Therefore careful interpretation with respect to the nature of the reactive species is necessary when LEC is used for characterization of zymosan-induced phagocytosis. We studied the properties of zymosan-induced LEC under different test conditions and with various inhibitors. Typical radical scavengers, e.g. nordihydroguaiaretic acid and superoxide dismutase, are strong inhibitors, indicating the importance of the superoxide anion. This system is useful for drug testing with respect to antioxidative or radical scavenging activity.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

黄芪有效成分对氧自由基清除作用的ESR研究

用电子自旋共振技术研究了黄芪总黄酮(TFA)、黄芪总皂甙(TSA)和黄芪总多糖(TPA)对次黄嘌呤／黄嘌呤氧化酶体系产生的超氧阴离子自由基和H₂O₂－Fe²⁺体系产生的羟自由基的清除作用．结果表明,这3种成分均有清除氧自由基的作用;对超氧阴离子自由基的清除效能大于对羟自由基的清除作用;其作用强度依次为TFA＞TSA＞TPA．结果提示清除氧自由基可能是黄芪抗衰老的主要机理之一,TFA和TSA是黄芪抗氧化作用的主要药理活性成分．