Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms.

Nitroxide stable radicals generally serve for probing molecular motion in membranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging. Recently nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which are depleted while attenuating oxidative damage, nitroxides can be recycled. In many cases the antioxidative activity of nitroxides is associated with switching between their oxidized and reduced forms. In the present work, superoxide radicals were generated either radiolytically or enzymatically using hypoxanthine/xanthine oxidase. Electron paramagnetic resonance (EPR) spectrometry was used to follow the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxidation. The results indicate that: a) The rate constant of superoxide reaction with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of superoxide reaction with nitroxides; b) The oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superoxide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simulation of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxides in vitro can be distinguished from that of common LMWA due to hydroxylamine oxidation by superoxide, which allows nitroxide recycling and enables its catalytic activity.     

Potential of albumin labeled with nitroxides as a contrast agent for magnetic resonance imaging and spectroscopy

The biological and physical properties of albumin and nitroxides make them attractive candidates as special purpose MRI contrast agents which could be used to study the intravascular compartment or specific targets in tissues. In this study, albumin-nitroxide complexes were prepared by reduction and alkylation of the disulfide bonds of the protein and characterized by electron spin resonance and ultraviolet absorption spectroscopy. An average of six nitroxides were bound covalently to each molecule of human serum albumin. The water proton relaxivity of the protein-bound nitroxide (at 20 MHz and 37 degrees C) was 4-fold greater than that of the free nitroxide. The digestion of the nitroxide-albumin complexes by cells or by trypsin decreased the relaxivity of the nitroxide-protein complex. The rate of reduction of albumin-bound nitroxide by cells was much slower than that of the free nitroxide but still was oxygen-sensitive (2-3-fold increase in the rate of reduction in the absence of oxygen).     

Optimization of labile esters for esterase-assisted accumulation of nitroxides into cells: a model for in vivo EPR imaging

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.     

Cellular metabolism of water-soluble nitroxides: Effect on rate of reduction of cell/nitroxide ratio,oxygen concentrations and permeability of nitroxides

In order to interpret more accurately studies that have used nitroxides and to improve the efficacy of the use of nitroxides in both basic studies of cells and as contrast agents for in vivo NMR, we have initiated a systematic study of the distribution and metabolism of nitroxides in biological systems. Overall, the results provide a reasonably coherent picture of some aspects of the interactions between nitroxides and cells. Reduction of the nitroxides appears to be an intracellular process, so that one of the principal variables that affects the rate of reduction is the ability of a nitroxide to enter cells. The entrance of nitroxides into cells shows considerable variability and ranges from essentially no penetration (e.g., 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylamine) through rates that are comparable to rates of reduction (e.g., 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-carboxylic acid), to rates that are so fast that there is complete equilibrium between intracellular and extracellular compartments (e.g., Tempone). The presence of a charged group on the nitroxide appears to be the important variable that affects their ability to enter cells. Once a nitroxides enters the cell, the structure of the nitroxide, e.g., piperidine vs. pyrrolidine ring, is major factor that affects the rate of reduction. The rates of reduction increase with increasing concentrations of nitroxides. This indicates that the principal mechanism(s) of reduction do not saturate in the concentration range we studied. We observed no abrupt changes in the rates of reduction over the entire concentration range of cells and nitroxides that we studied, which suggests that the mechanism(s) of nitroxide reduction did not change. The presence of oxygen decreased the observed rate of reduction of many of the nitroxides and this effect was independent of the concentration of nitroxide.     

Nitroxide-doped liposomes containing entrapped oxidant: an approach to the "reduction problem" of nitroxides as MRI contrast agents

The purpose of this investigation is to test the feasibility of a nitroxide regeneration system involving liposomes as an approach toward solving the "reduction problem" when nitroxides are used as contrast enhancing agents in MRI applications. It is shown that the inclusion of an entrapped oxidant (K3Fe(CN)6) in the aqueous compartment of nitroxide-doped liposomes causes a 4-5-fold increase in the duration of the nitroxide ESR signal in the presence of the external reductant sodium ascorbate. Confirmation was obtained by monitoring the concentration of the internalized Fe(CN)6(3-) ion versus time by visible spectroscopy at 410 nm. Trans bilayer (flip-flop) motion of the long chain nitroxide ester is the likely mechanism of this nitroxide regeneration system.     

Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Exchange and shuttling of electrons by nitroxide spin labels

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.     

Superoxide Reaction with Nitroxides

Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoprmetric (Cyt C) and chemilu-minescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemi-luminescence clearly demonstrated that the six-membered piperidine-I-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 × 10⁴ to 1.2 × 10^-5M^-1s^-l. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/ xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides. they may ultimately be more useful as superoxide oxide dismutase mimetic agents.     

Reduction and destruction rates of nitroxide spin probes

A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemical, and two biological systems by ESR assays. In all cases, piperidine and hydropyridine nitroxides were reduced consistently more rapidly than pyrroline and pyrrolidine nitroxides. Substituents on the nitroxides also affected reduction rates, although not as greatly as ring structure. One of the reduction systems, consisting of the photosensitizer FMN and the photoreductant EDTA, was used to study both anaerobic reduction and O2-dependent reoxidation of some of the nitroxides. Reduced piperidine and hydropyridine nitroxides were also oxidized more rapidly than the reduced pyrroline and pyrrolidine nitroxides. Reoxidation subsequent to reduction was partially inhibited by superoxide dismutase, indicating that superoxide radicals are involved in the process. Even after prolonged reoxidation, not all of the probe molecules were returned to their oxidized form, implying an irreversible "destruction" of the spin probe concomitant with its chemical reduction. Probe destruction was studied more specifically with a photochemical system for generating methyl radicals, which showed that these carbon-centered radicals destroyed different nitroxides at rates which were much less influenced by the nitroxide structures than one-electron reduction was.     

The chemistry and biology of nitroxide compounds

Cyclic nitroxides are a diverse group range of stable free radicals that have unique antioxidant properties. Because of their ability to interact with free radicals, they have been used for many years as biophysical tools. During the past 15-20 years, however, many interesting biochemical interactions have been discovered and harnessed for therapeutic applications. Biologically relevant effects of nitroxides have been described, including their ability to degrade superoxide and peroxide, inhibit Fenton reactions, and undergo radical-radical recombination. Cellular studies defined the activity of nitroxides in vitro. By modifying oxidative stress and altering the redox status of tissues, nitroxides have been found to interact with and alter many metabolic processes. These interactions can be exploited for therapeutic and research use, including protection against ionizing radiation, as probes in functional magnetic resonance imaging, cancer prevention and treatment, control of hypertension and weight, and protection from damage resulting from ischemia/reperfusion injury. Although much remains to be done, many applications have been well studied and some are currently being tested in clinical trials. The therapeutic and research uses of nitroxide compounds are reviewed here with a focus on the progress from initial development to modern trials.     

Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte

Nitroxides were used as models of persistent free radicals to study the antioxidant function of ascorbic acid in the human erythrocyte. It was concluded that: 1) ascorbate and other reductant(s) derived from dehydroascorbic acid (DHA) in the presence of thiols are the only significant reducing agents for nitroxides, 2) glutathione and DHA reduce nitroxides by a process that cannot be inhibited by ascorbic acid oxidase, 3) erythrocytes can be depleted of ascorbic acid by exhaustive washing in the presence of membrane-permeable cationic nitroxides such as N,N-dimethylamino-Tempo, 4) ascorbate-depleted cells do not reduce nitroxides; however, nitroxide reduction is restored when the cells are incubated with DHA, 5) reduction of nitroxides in ascorbate-depleted, DHA-treated cells is significantly faster than in buffered solutions of DHA and glutathione, 6) several equivalents of nitroxide are reduced relative to the intracellular ascorbate pool, 7) sustained nitroxide reduction is observed even when most of the intracellular ascorbate is oxidized, 8) spin trapping of oxyradicals in tert-butyl hydroperoxide-treated cells is accelerated with ascorbate depletion and inhibited with ascorbate loading, 9) ascorbate can be quantified within intact cells by analyzing the initial reduction rates of membrane-permeable cationic nitroxides, and 10) DHA-stimulated reduction of cationic nitroxides is slower and less extensive in erythrocytes deficient in glucose-6-phosphate dehydrogenase than in normal erythrocytes.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

Cellular uptake of electron paramagnetic resonance imaging probes through endocytosis of liposomes

Electron paramagnetic resonance imaging (EPRI) allows detection and localization of paramagnetic spin probes in vivo and in real time. We have shown that nitroxide spin probes entrapped in the intracellular milieu can be imaged by EPRI. Therefore, with the development of a tumor-targetable vehicle that can efficiently deliver nitroxides into cells, it should be possible to use nitroxide spin probes to label and image cells in a tumor. In this study, we assess the potential of liposomes as a delivery vehicle for imaging probes. We demonstrate that liposomes can stably encapsulate nitroxides at very high concentrations (> 100 mM), at which nitroxides exhibit concentration-dependent quenching of their EPR signal—a process analogous to the quenching of fluorescent molecules. The encapsulating liposomes thus appear spectroscopically “dark”. When the liposomes are endocytosed and degraded by cells, the encapsulated nitroxides are liberated and diluted into the much larger intracellular volume. The consequent relief of quenching generates a robust intracellular nitroxide signal that can be imaged. We show that through endocytosis of nitroxide-loaded liposomes, CV1 cells can achieve intracellular nitroxide concentrations of ∼ 1 mM. By using tissue phantom models, we verify that this concentration is more than sufficient for in vivo EPR imaging.     

Application of Spin Traps to Biological Systems

Since 1971. when nitroxides were first reported to be bioreduced, several cellular enzymes, in addition to ascorbic acid. have been found to catalyze the reduction of nitroxides to their corresponding hydroxylami-nes. Numerous studies have demonstrated that cellular bioreduction of nitroxides are both dependent upon the structure of the nitroxide and cell type. For example, pyrrolidinyloxyls are considerably more resistant to bioreduction than their corresponding piperidinyloxyls. In addition, cellular levels of reductases present in freshly isolated rat hepatocytes are considerably greater than concentrations found in freshly isolated rat enterocytes. Thus, through the proper selection of a cell type and an appropriate nitroxide. one can study cellular-mediated free radical processes.

With the discovery that α-hydrogen-containing nitroxides, including 2, Z-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH) decompose rapidly in the presence of superoxide and thiols, the ability to determine if hydroxyl radical is generated during stimulation of human neutrophils, is in doubt. To explore the limits of spin trapping in this context. we have studied the effect of varying the rates of superoxide production. in the presence and absence of thiols, on the decomposition of DMPO-OH. In parallel studies, we have found that t-butyl α-methyl-4-pyridinyl-N-oxide nitroxide (4-POBN-CH₃) will not degrade in the presence of superoxide and a thiol. From these studies. we have determined that if hydroxyl radicals were generated as an isolated event in the presence of a continual flow of superoxide. spin trapping might not be able to detect its formation. Otherwise. spin trapping should be able to measure hydroxyl radicals. if continually generated, during activation of human neutrophils.     

Cellular sites of H2O2-induced damage and their protection by nitroxides

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

Abstract

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Abstract

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50?nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4?mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.     

Cell-penetrating nitroxides as molecular sensors for imaging of cancer in vivo, based on tissue redox activity

The present study shows that hydrophobic and cell-penetrating piperidine-type nitroxide radicals SLENU and TEMPOL, but not hydrophilic and partially penetrating or non-penetrating pyrrolidine-type nitroxides carbamoyl-PROXYL and carboxy-PROXYL, are appropriate contrast agents for magnetic resonance imaging (MRI) of cancer, based on its functionality - tissue redox activity. The experiments were conducted on anesthetized mice: healthy and neuroblastoma-bearing in a moderate stage of cancer development. The method is based on the nitroxide redox cycle, coupled with appearance or disappearance of the MRI signal. The half-life (τ(1/2)) of a nitroxide-enhanced MRI signal in the respective tissue was used as a marker to assess tissue redox activity to the nitroxide radical. In the case of SLENU and TEMPOL, there were large differences in the histograms between control and cancer-bearing mice. All tissues (cancer and non-cancer) of cancer-bearing organisms were characterized by a long-lived MRI signal (τ(1/2) > 14 min), indicating a high oxidative activity. The tissues of healthy organisms were characterized by a short-lived MRI signal (τ(1/2) = 1-3 min), indicating a high reducing activity. In the case of carbamoyl-PROXYL and carboxy-PROXYL, there was no difference in the histograms between control and cancer-bearing mice. The data show that the penetration of nitroxide in cells and tissues is obligatory for imaging of cancer, based on its redox activity. The principle of the method is applicable also to biopsy specimens, using MRI or EPR spectroscopy. We provide direct evidence that the nitroxide redox cycle could be used as a sensing platform for functional imaging of different pathologies, based on changes in cellular and tissue redox activity, as in the case of cancer.     

Nitroxide-Stimulated H2O2 Decomposition by Peroxidases and Pseudoperoxidases

Nitroxide free radicals interact with Hb/metHb, Mb/metMb and with peroxidases/phenols to induce a catalase-like conversion of H₂O₂ to O₂ (catalatic activity), without being substantially consumed in the process. The mechanism of this reaction is postulated to involve a one-electron oxidation of the nitroxide to the immonium oxene, which then reacts further to release oxygen and the nitroxide. An involvement of the immonium oxene in the reaction mechanism is consistent with ferryl heme reduction by nitroxides and a detection of the reduced nitroxide when the reaction mixture is supplemented with the two-electron reductant sodium borohydride. The nitroxide-induced catalatic activity is completely inhibited when the reaction mixture is supplemented with glutathione. Nitroxides suppress free radical formation by hydroperoxide-activated heme proteins, as inferred from their inhibition of the spin-trapping of glutathionyl radicals. H₂O₂ decomposition and a suppression of reactive free radical formation by heme proteins appears to be an antioxidant activity of nitroxides, which is distinct from their previously reported superoxide dismutating activity and which may be a factor in their protective action in models of cardiac reperfusion injury.     

Modulation of oxidative damage by nitroxide free radicals

Piperidine nitroxides like 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) are persistent free radicals in non-acidic aqueous solutions and organic solvents that may have value as therapeutic agents in medicine. In biological environments, they undergo mostly reduction to stable hydroxylamines but can also undergo oxidation to reactive oxoammonium compounds. Reactions of the oxoammonium derivatives could have adverse consequences including chemical modification of vital macromolecules and deleterious effects on cell signaling. An examination of their reactivity in aqueous solution has shown that oxoammonium compounds can oxidize almost any organic as well as many inorganic molecules found in biological systems. Many of these reactions appear to be one-electron transfers that reduce the oxoammonium to the corresponding nitroxide species, in contrast to a prevalence of two-electron reductions of oxoammonium in organic solvents. Amino acids, alcohols, aldehydes, phospholipids, hydrogen peroxide, other nitroxides, hydroxylamines, phenols and certain transition metal ions and their complexes are among reductants of oxoammonium, causing conversion of this species to the paramagnetic nitroxide. On the other hand, thiols and oxoammonium yield products that cannot be detected by ESR even under conditions that would oxidize hydroxylamines to nitroxides. These products may include hindered secondary amines, sulfoxamides and sulfonamides. Thiol oxidation products other than disulfides cannot be restored to thiols by common enzymatic reduction pathways. Such products may also play a role in cell signaling events related to oxidative stress. Adverse consequences of the reactions of oxoammonium compounds may partially offset the putative beneficial effects of nitroxides in some therapeutic settings.