Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.     

Pathways of dopamine oxidation mediated by nitric oxide

This study was aimed at establishing the interaction between dopamine and nitric oxide and elucidating the mechanistic aspects inherent in this interaction. At high (*) NO concentrations (microM range), dopamine underwent nitrosation with subsequent nitration. Nitrosation is proposed to occur via a nucleophilic attack to N(2)O(3) by dopamine. At low (*) NO concentrations (microM range), dopaminochrome was formed. EPR spin stabilization studies showed the occurrence of two o-semiquinone intermediates during dopaminochrome formation. Heats of formation obtained by AM1 semiempirical calculations supported the formation of the two o-semiquinone species. Hydroxyl radicals were detected by spin trapping EPR, and experiments performed with superoxide dismutase and catalase suggested that peroxynitrite was the source of HO(*). A mechanism is presented that considers the several factors influencing these reactions.     

Nadph-Cytochrome-P450 Reductase Promotes Hydroxyl Radical Production by the Iron Complex of ADR-925, the Hydrolysis Product of ICRF-187 (Dexrazoxane)

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectropho-tometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.     

Antioxidant and prooxidant action of nitric oxide donors and metabolites

It has been shown that various nitric oxide donors and metabolites have similar effects on lipid peroxidation in rat myocardium homogenate. The formation of malondialdehyde, a secondary product of lipid peroxidation, was inhibited in a dose-dependent manner by PAPA/NONO (a synthetic nitric oxide donor), S-nitrosoglutathione, nitrite, and nitroxyl anion. The inhibition of lipid peroxidation was provided most efficiently by the administration of dinitrosyl-iron complexes with dextran and PAPA/NONO. S-nitrosoglutathione also inhibited the destruction of coenzymes Q9 and Q10 during free radical oxidation of myocardium homogenate. Low-molecular-weight dinitrosyl iron complexes with cysteine also promoted lipid peroxidation, which is probably due to iron release during the destruction dinitrosyl iron complexes. It is likely that the antioxidant action of nitric oxide derivatives is related to the reduction of ferry forms of hemoproteins and interaction of nitric oxide with lipid radicals.     

Cautionary note for DMPO spin trapping in the presence of iron ion

2-Hydroxy-5,5-dimethyl-1-pyrrolidinyloxy (DMPO-OH), which is known to be produced by spin trapping of hydroxyl radicals (.OH) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and has been a good monitor for detecting .OH in biological systems, has been examined by EPR for its production scheme in the presence of iron ion. In an aqueous DMPO solution containing ferric ion (Fe3+), DMPO-OH was produced and addition of methanol, a good scavenger for .OH, to this solution led to an aminoxyl radical, DMPO-OCH3, instead of DMPO-CH2OH which is produced by DMPO spin trapping of .CH2OH arising from H-abstraction by .OH. Also EPR measurements at 77K indicated the formation of a chelate between DMPO and Fe3+. Based on these, it has been elucidated that DMPO-OH as well as DMPO-OCH3 is formed by the nucleophilic attack of water and methanol to the chelating DMPO, respectively.     

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.     

Radical-Induced Damage to Bovine Serum Albumin: Role of the Cysteine Residue

The reactions of cerium(IV) and the hydroxyl radical [generated from iron(ii)/H₂O₂] with bovine serum albumin (BSA) have been investigated by EPR spin trapping. With the former reagent a protein-derived thiyl radical is selectively generated; this has been characterized via the anisotropic EPR spectra observed on reaction of this radical with the spin trap DMPO. Blocking of the thiol group results in the loss of this species and the detection of a peroxyl radical, believed to be formed by reaction of oxygen with initially-generated, but undetected, carbon-centred radicals from aromatic amino acids. Experiments with a second spin trap (DBNBS) confirm the formation of these carbon-centred species and suggest that damage can be transferred from the thiol group to carbon sites in the protein. A similar transfer pathway can be observed when hydroxyl radicals react with BSA.

Further experiments demonstrate that the reverse process can also occur: when hydroxyl radicals react with BSA, the thiol group appears to act as a radical sink and protects the protein from denaturation and fragmentation through the transfer of damage from a carbon site to the thiol group. Thiol-blocked BSA is shown to be more susceptible to damage than the native protein in both direct EPR experiments and enzyme digestion studies. Oxygen has a similar effect, with more rapid fragmentation detected in its presence than its absence.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.

In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.     

Iron and free radical oxidations in cell membranes.

Brain tissue being rich in polyunsaturated fatty acids, is very susceptible to lipid peroxidation. Iron is well known to be an important initiator of free radical oxidations. We propose that the principal route to iron-mediated lipid peroxidations is via iron-oxygen complexes rather than the reaction of iron with hydrogen peroxide, the Fenton reaction. To test this hypothesis, we enriched leukemia cells (K-562 and L1210 cells) with docosahexaenoic acid (DHA) as a model for brain tissue, increasing the amount of DHA from approximately 3 mole % to 32 mole %. These cells were then subjected to ferrous iron and dioxygen to initiate lipid peroxidation in the presence or absence of hydrogen peroxide. Lipid-derived radicals were detected using EPR spin trapping with alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). As expected, lipid-derived radical formation increases with increasing cellular lipid unsaturation. Experiments with desferal demonstrate that iron is required for the formation of lipid radicals from these cells. Addition of iron to DHA-enriched L1210 cells resulted in significant amounts of radical formation; radical formation increased with increasing amount of iron. However, the exposure of cells to hydrogen peroxide before the addition of ferrous iron did not increase cellular radical formation, but actually decreased spin adduct formation. These data suggest that iron-oxygen complexes are the primary route to the initiation of biological free radical oxidations. This model proposes a mechanism to explain how catalytic iron in brain tissue can be so destructive.     

Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy

The formation of hydroxyl radicals in beta-glucan solutions treated with ascorbic acid and iron(II) was demonstrated by ESR spin trapping based methods. Two different spin traps were tested, namely DMPO which is commonly used to detect hydroxyl radicals, and POBN often used to detect carbon centered radicals. The experiments performed showed that the presence of iron(II) with DMPO led to low DMPO-OH adduct stability and further to DMPO dimerization. The level of hydroxyl radicals formed during the beta-glucan radical mediated degradation was evaluated using two ESR spin trapping methods based on the use POBN together with either 2% (v/v) EtOH or DMSO. The addition of ascorbic acid together with iron(II) in beta-glucan solution led to an immediate maximal production of hydroxyl radicals while the presence of ascorbic acid alone led to a progressive production of radical. Further hydroxyl radicals were found to be formed when iron(II) was added alone in beta-glucan solutions. The viscosity loss observed in the three last mentioned beta-glucan solutions were found to relate with the formation of hydroxyl radicals. These data confirm the involvement of hydroxyl radical in the beta-glucan degradation.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

Evidence against transition metal-independent hydroxyl radical generation by xanthine oxidase

It is shown by the use of EPR spectroscopy that formation of the hydroxyl radical adduct with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the xanthine-xanthine oxidase system is hydrogen peroxide-independent. Production of the DMPO-hydroxyl radical adduct is inhibited by superoxide dismutase but is unaffected by purified grades of catalase. Hydroxyl radicals are a secondary product of the decomposition of the DMPO-superoxide radical adduct and are also formed as a result of trace metals such as iron present in the buffer. These results are in contrast with a recent report (Kuppusamy, P., and Zweier, J. W. (1989) J. Biol. Chem. 264, 9880-9884) in which the assertion is made that the hydroxyl radical adduct arises from the trapping of hydroxyl radicals generated via the direct reduction of hydrogen peroxide by xanthine oxidase. It is demonstrated here that treatment of phosphate buffer with the chelator deferoxamine mesylate is not in itself sufficient to suppress the effect of contaminating adventitious metal ions in xanthine-xanthine oxidase incubations.     

EPR detection of glutathiyl and hemoglobin-cysteinyl radicals during the interaction of peroxynitrite with human erythrocytes

Peroxynitrite, which is formed by the fast reaction between nitric oxide and superoxide anion, has been receiving increasing attention as a mediator of human diseases. An initial controversy about the possibility of free radical production from peroxynitrite in test tubes has been resolved, and presently it is important to establish whether peroxynitrite produces radicals in cells. Here we employed the EPR spin trapping methodology with 5,5-dimethylpyrroline N-oxide (DMPO) to study the interaction of peroxynitrite with human erythrocytes. The results confirmed previous findings in demonstrating that oxyhemoglobin is the main target of peroxynitrite in erythrocytes. As we first show here, the produced ferryl-hemoglobin oxidizes its own amino acids and, most probably, amino acids from other hemoglobin monomers to produce hemoglobin-tyrosyl and hemoglobin-cysteinyl radicals. In parallel, ferryl-hemoglobin also oxidizes intracellular glutathione to produce the glutathiyl radical. The EPR spectrum of both DMPO/(*)cysteinyl-hemoglobin (a(beta)(H) = 15.4 G) and DMPO/(*)tyrosyl-hemoglobin (a(beta)(H) = 8.8 G) radical adducts was characterized. It is proposed that erythrocytes can be efficient peroxynitrite scavengers in vivo through the coupled action of oxyhemoglobin and glutathione. Overall, the results indicate that, through the intermediacy of carbon dioxide and/or hemoproteins, oxidation of glutathione to the glutathiyl radical is likely to be an important consequence of peroxynitrite production in vivo.     

Detection and characterisation of radicals in biological materials using EPR methodology

Background

Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, spectroscopy) is widely considered to be the “gold standard” for the detection and characterisation of radicals in biological systems.

Scope of review

The article reviews the major positive and negative aspects of EPR spectroscopy and discusses how this technique and associated methodologies can be used to maximise useful information, and minimise artefacts, when used in biological studies. Consideration is given to the direct detection of radicals (at both ambient and low temperature), the use of spin trapping and spin scavenging (e.g. reaction with hydroxylamines), the detection of nitric oxide and the detection and quantification of some transition metal ions (particularly iron and copper) and their environment.

Major conclusions

When used with care this technique can provide a wealth of valuable information on the presence of radicals and some transition metal ions in biological systems. It can provide definitive information on the identity of the species present and also information on their concentration, structure, mobility and interactions. It is however a technique that has major limitations and the user needs to understand the various pitfalls and shortcoming of the method to avoid making errors.

General significance

EPR remains the most definitive method of identifying radicals in complex systems and is also a valuable method of examining radical kinetics, concentrations and structure. This article is part of a Special Issue entitled Current methods to study reactive oxygen species — pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Electron Paramagnetic Resonance and Spin Trapping Study of Radicals Formed During Reaction of Aromatic Amines with isoAmyl Nitrite Under Aprotic Conditions

Diazotization of primary aromatic amines with isoamyl nitrite in benzene at room temperature was studied employing EPR and spin trapping techniques. Nitrosodurene (ND). 2-methyl-2-nitrosopropane (MNP). and 5,5-dimethyl-pyrroline N-oxide (DMPO) were used as spin trapping agents. Aryl radicals were detected employing ND and MNP. Using DMPO as a spin trap most of the amines produced EPR spectra ascribed to adducts with aniline-type radicals (N-centred radicals). The assignments were verified using ¹⁵JN-labeled anilines. Similar spectra of DMPO adducts were recorded from amines treated with benzoyl peroxide or benzophenone plus UV. Possible mechanisms of formation of these adducts (radical trapping versus nucleophilic addition to DMPO followed by oxidation) during treatment of the amines with isoamyl nitrite are discussed.     

Involvement of inducible nitric oxide synthase in hydroxyl radical-mediated lipid peroxidation in streptozotocin-induced diabetes

Free radical production is implicated in the pathogenesis of diabetes mellitus, where several pathways and different mechanisms were suggested in the pathophysiology of the complications. In this study, we used electron paramagnetic resonance (EPR) spectroscopy combined with in vivo spin-trapping techniques to investigate the sources and mechanisms of free radical formation in streptozotocin-induced diabetic rats. Free radical production was directly detected in the diabetic bile, which correlated with lipid peroxidation in the liver and kidney. EPR spectra showed the trapping of a lipid-derived radical. Such radicals were demonstrated to be induced by hydroxyl radical through isotope-labeling experiments. Multiple enzymes and metabolic pathways were examined as the potential source of the hydroxyl radicals using specific inhibitors. No xanthine oxidase, cytochrome P450s, the Fenton reaction, or macrophage activation were required for the production of radical adducts. Interestingly, inducible nitric oxide synthase (iNOS) (apparently uncoupled) was identified as the major source of radical generation. The specific iNOS inhibitor 1400W as well as L-arginine pretreatment reduced the EPR signals to baseline levels, implicating peroxynitrite as the source of hydroxyl radical production. Applying immunological techniques, we localized iNOS overexpression in the liver and kidney of diabetic animals, which was closely correlated with the lipid radical generation and 4-hydroxynonenal-adducted protein formation, indicating lipid peroxidation. In addition, protein tyrosine nitration occurred in the diabetic target organs. Taken together, our studies support inducible nitric oxide synthase as a significant source of EPR-detectable reactive intermediates, which leads to lipid peroxidation and may contribute to disease progression as well.