Effect of hydrogen peroxide in redox status estimation using nitroxyl spin probe

A procedure for estimating in vivo redox status using EPR and a hydrogen peroxide (H₂O₂)-dependent spin probe method is described. The mechanism of decreasing spin clearance in the selenium-deficient (SeD) rat is discussed. The in vivo decay constant of the nitroxyl spin probe in the liver region of SeD rats appeared to be slightly lower that of the selenium-adequate control (SeC) group, and was significantly smaller than that of normal rats. Bile H₂O₂ levels in normal rats were significantly lower than those in SeD rats. The in vivo decay constant of the spin probe in SeD rats depended on the bile H₂O₂ level. Furthermore, H₂O₂ was detected in the bile in all SeD rats, whereas bile H₂O₂ could be detected in only half of the normal rats. It was found that the in vivo decay constant of the spin probe in normal rats also depended on whether bile H₂O₂ was detected or not. In vivo decay constants were smaller in rats subjected to the surgical operation than in the nonoperated groups. The EPR signal of the nitroxyl radical in the liver homogenate was increased by addition of H₂O₂, which was administered 30 min before the rat was killed. It appears that H₂O₂ can oxidize the hydroxylamine formed following reduction of the spin probe in the liver.     

Solvent Effects in the Spin Trapping Of Lipid Oxyl Radicals

Studies documenting spin trapping of lipid radicals in defined model systems have shown some surprising solvent effects with the spin trap DMPO. In aqueous reactions comparing the reduction of H₂O₂ and methyl linoleate hydroperoxide (MLOOH) by Fe^z+, hydroxyl (HO·) and lipid alkoxyl (LO·) radicals produce identical four-line spectra with line intensities 1:2:2:1. Both types of radicals react with commonly-used HO· scavengers, e.g. with ethanol to produce ·C(CH₃)HOH and with dirnethylsulfoxide (DMSO)togive ·CH₃. However, DMSO radicals (either ·CH₃or ·OOCH₃) react further with lipids, and when radicals are trapped in these MLOOH systems, multiple adducts are evident. When acetonitrile is added to the aqueous reaction systems in increasing concentrations, ·CH₂CN radicals resulting from HO· attack on acetonitrile are evident, even with trace quantities of that solvent. In contrast, little, if any, reaction of LO· with acetonitrile occurs, even in 100% acetonitrile. A single four-line signal persists in the lipid systems as long as any water is present, although the relative intensity of the two center lines decreases as solvent-induced changes gradually dissociate the nitrogen and β-hydrogen splitting constants. Extraction of the aqueous-phase adducts into ethyl acetate shows clearly that the identical four-line spectra in the H₂0₂ and MLOOH systems arise from different radical species in this study, but the lack of stability of the adducts to phase transfer may limit the use of this technique for routine adduct identification in more complex systems. These results indicate that the four-line 1:2:2:1. a_N = a_H = 14.9G spectrum from DMPO cannot automatically be assigned to the HO· adduct in reaction systems where lipid is present, even when the expected spin adducts from ethanol or DMSO appear confirmatory for HO-. Conclusive distinction between HO· and LO· ultimately will require use of ¹³C-labelled DMPO or HPLC-MS separation and specific identification of adducts when DMPO is used as the spin trap.     

Iron Release from Metmyoglobin, Methaemoglobin and Cytochrome C by A System Generating Hydrogen Peroxide

The reaction of H₂O₂ with resting metmyoglobin (MetMb), methaemoglobin (MetHb) and cytochrome-c (Cyt-c) was studied in the Soret and visible regions. The differences between the original and the final peak heights of the native haemproteins at 408 nm was found to be directly proportional to the loss of iron from the molecule. The release of iron from haemproteins was studied in a system generating H₂O₂ continuously at a low rate by an enzymic system, or by addition of large amounts of H₂O₂. Cytochrome-c, methaemoglobin and metmyoglobin during interaction with H₂O₂ at a concentration of 200 μM release 40%, 20% and 3%, respectively, of molecular iron after l0min. The inhibition of haem degradation and iron release by enzymatically-generated H₂O₂ was determined using several hydroxyl radical scavengers, reducing agents and antioxienzymes, such as superoxide dismutase, catalase and caeruloplasmin.     

Epr Spectra of Dmpo Spin Adducts of Superoxide and hydroxyl Radicals in Pyridine

Electron spin resonance spectroscopy and the spin trapping technique were used to study the formation of the superoxide radical in pyridine. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was employed as a trapping agent. Superoxide radical was generated using chemical (potassium superoxide) and photochemical methods with anthralin, benzanthrone, rose bengal, 1,8-dihydroxyanthraquinone and zinc tetraphenylporphyrine as photoactive pigments. Hyperfine coupling (hf) constants for DMPO/O₂- were determined to be a_N = 12.36 G, a^β_H= 9.85G, a^γ_H = 1.34 G. The a_N and a^β_H constants are in good agreement with values calculated from a previously determined relationship between hf constants and solvent acceptor number (Reszka et al., (1992) Free Radical Res. Commun., in press). When concentrated hydrogen peroxide was added to DMPO in pyridine a similar EPR spectrum was observed. It is suggested that in this case the DMPO/'O₂H adduct is formed by nucleophilic addition of H₂O₂ to DMPO to give a hydroxylamine, followed by oxidation to the respective nitroxide. The EPR spectrum observed when tetrapropylammonium hydroxide and H₂O₂ were added to DMPO in pyridine had hf couplings a_N = 13.53 G, a^β_H = 11.38 G, a^γ_H = 0.79 G and it was assigned to a DMPO/'OH adduct. This assignment was based on similarity of this spectrum to the one produced by UV photolysis of hydrogen peroxide and DMPO in aqueous solution and subsequent transfer to pyridine.     

Dmpo Spin Trapping in the Presence of Fe Ion

Aminoxyl radical formation from DMPO in the presence of Fe ion was studied to clarify the ambiguous ESR signals obtained by spin trapping with DMPO. It was found that when DMPO was used in a Fenton system, a Fe-DMPO complex was formed immediately. This complex was subsequently attacked by oxidative species originating from H₂O₂ and thus oxidative degradation of DMPO was induced in the Fenton system. On the other hand, in the case of M, PO, the degradation was found to be very slow, indicating that the 3 position of DMPO was favorably attacked by the oxidative species. Some of the degradation products are probably aminoxyl radicals. This series of the degradation products are probably aminoxyl radicals. This series of reactions may compete with spin trapping and make it difficult to analyze ESR spectra obtained in the presence of Fe ion.     

The haemolytic reactions of 1-acetyl-2-phenylhydrazine and hydrazine: A spin trapping study

Free radical production from the reaction of hydrazine and 1-acetyl-2-phenylhydrazine (AcPhHZ) with oxyhaemoglobin and with human red blood cells, has been observed by the electron spin resonance technique of spin trapping. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the free radical intermediates detected depended on the hydrazine derivative, oxyhaemoglobin and the oxyhaem/hydrazine derivative concentration ratio.

The reaction of hydrazine with oxyhaemoglobin in the presence of DMPO gave a nitroxide which was identified as a reduced dimer of DMPO. Whereas hydrazine-treated red blood cells, in the presence of DMPO, gave a nitroxide spin adduct which was identified as the hydroxyl radical spin adduct of DMPO, 5,5-dimethyl-1-pyrrolidino-1-oxyl (DMPO-OH).

The reaction of AcPhHZ with oxyhaemoglobin, in the presence of DMPO, gave DMPO-OH, the phenyl radical spin adduct of DMPO, 5,5-dimethyl-2-phenylpyrrolidino-1-oxyl (DMPO-Ph) and an oxidised derivative of DMPO, 5,5-dimethyl-2-pyrrolidone-1-oxyl (DMPOX). The amounts of DMPO-Ph, DMPO-OH and DMPOX observed depended on the 1-acetyl-2-phenyl-hydrazine/oxyhaemoglobin concentration ratio; DMPOX replaced DMPO-OH as the concentration of AcPhHZ was decreased. DMPOX production has been previously associated with the production of highly oxidised haem iron-oxygen intermediates. AcPhHZ treated red blood cells gave DMPO-Ph and DMPO-OH spin adducts in the presence of DMPO.

DMPO had little to no effect on the rate of oxygen consumption by oxyhaemoglobin with hydrazine and AcPhHZ. Moreover, the rate of oxyhaemoglobin oxidation induced by hydrazine, was not decreased by DMPO whereas the rate of oxyhaemoglobin oxidation induced by AcPhHZ was decreased approx. 40% by DMPO. DMPO (10 mM) gave a small decrease in haemolysis and lipid peroxidation induced by 1 mM hydrazine and AcPhHZ in a 1% suspension of red blood cells.     

Synthesis and Evaluation of DMPO-Type Spin Traps

The spin traps substituted with some groups at the 4-position of dimethyl-1-pyrroline N-oxide(DMPO) were compared with DMPO itself regarding their abilities as spin traps and their physical properties. 4,5,5-Trimethyl-l-pyroHine N-oxide (4MDMPO) and 5,5-dimethyl- 4-phenyl-l-pyrolline N-oxide (4PDMPO) were synthesized by the Bonnett method, and 5,5-dimethyl-4-hydroxymethyl-l-pyrolline N-oxide (4HMDMPO) was made by a unique method from 2(5H)-furanone. The melting points of 4MDMPO, 4PDMPO and 4HMDMPO were higher than that of DMPO. The magnitude of hydrophilicity was in the order of 4HMDMPO, DMPO, 4MDMPO, and 4PDMPO based on the partition coefficient experiments in a 1-octanol-water system. Several radicals, O₂, HO-, -CH₃, -CH₂OH, -CH(CH₃)OH, (CH₃)₃ CO and H radicals, were trapped with these DMPO derivatives for comparison with the trapping by DMPO itself. Spin adducts of O J with the three DMPO derivatives showed ESR spectra similar to that of DMPO. In spite of the formation of diastereomers arising from spin trapping, the line-width enlargement was very small. The intensities and the decay rates of the spectra of 4MDMPO-O₂^-, 4PDMPO-O₂^- 4HMDMPO-O₂^- and DMPO-O₂^- were almost equal. In the trapping of the OH radical by 4MDMPO, 4PDMPO and 4HMDMPO, the eight-line ESR spectra observed were different from the well-known four-line spectrum of DMPO-OH.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Oxygen Radical Formation in Well-Washed Rat Liver Microsomes: Spin Trapping Studies

The generation of hydroxyl radicals by rat liver microsomes was monitored by spin trapping with 5, 5-dimethylpyrroline N-oxide (DMPO). The results confirm and extend previous data which demonstrated that hydroxyl radicals are produced by microsomes in the presence of NADPH and O₂, and without the exogenous addition of iron. No EPR signals could be detected unless catalase activity which was associated with the microsomes could be substantially diminished. Addition of azide was the most effective means of eliminating catalase activity, but azide also reacted rapidly with hydroxyl radicals, forming azidyl radicals which were in turn trapped by DMPO. Extensive washing and preincubation of microsomes with 3-amino-1, 2,4-triazole in the presence of H₂O₂ were evaluated as alternative methods of decreasing the catalase contamination of microsomes. Although neither method completely eliminated microsomal catalase activity, addition of azide was no longer necessary for hydroxyl radical detection with DMPO. When highly washed microsomal preparations were tested, weak signals of the superoxide radical adduct of DMPO could also be detected. These data indicate that the sensitivity of spin trapping in microsomal systems can be improved substantially when care is taken to eliminate cytosolic contaminants such as catalase.     

Radical-Induced Damage to Proteins: E.S.R. Spin-Trapping Studies

The reactions of hydroxyl radicals generated from Fe¹¹/H₂O₂ and Cu¹¹/H₂O₂ redox couples with a variety of proteins (BSA, histones, cytochrome c, lysozyme and protamine) have been investigated by e.s.r. spin trapping. The signals obtained, which are generally anisotropic in nature, characterize the formation of partially-immobilized spin-adducts resulting from attack of the HO- radicals on the protein and subsequent reaction of the protein-derived radicals with the spin trap. Similar spin adducts are observed on incubation of two haem-proteins (haemoglobin and myoglobin) with H₂O₂ in the absence of added metal ions implying a reaction at the haem centre followed by internal electron transfer reactions.

Two strategies have been employed to obtain information about the site(s) of radical damage in these proteins. The first involves the use of a variety of spin traps and in particular DMPO: with this particular trap the broad spectra from largely immobilized radicals show characteristic a(β-H) values which enable carbon-, oxygen- and sulphur-centred radicals to be distinguished. The second involves the use of enzymatic cleavage of first-formed adducts to release smaller nitroxides, with isotropic spectra, which allow the recognition of β-proton splittings and hence information about the sites of radical damage to be obtained. These results, which allows backbone and side-chain attack to be distinguished, are in agreement with random attack of the HO^. radical on the protein and are in accord with studies carried out on model peptides. In contrast the use of less reactive attacking radicals [N₃·, ·CH(CH₃)OH] and oxidising agents (Ce⁴⁺) provides evidence for selective attack on these proteins at particular residues.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Reactions of captopril and epicaptopril with transition metal ions and hydroxyl radicals: An EPR spectroscopy study

In the present study, using the technique of EPR spin trapping with DMPO a spin trap, we demonstrated formation of thiyl radicals from thiol-containing angiotensin converting enzyme (ACE) inhibitor captopril (CAP) and from its stereoisomer epicaptopril (EPICAP), a non-ACE inhibitor, in the process of ^.OH radical scavenging. Splitting constants of DMPO/thiyl radical adducts were identical for both thiols and were a_N = 15.3 G, and a_H = 16.2 G. Bimolecular rate constants for the reaction of CAP and EPICAP with ^.OH radicals were close to a diffusion-controlled rate (≈ 2 × 10¹⁰ M⁻¹s⁻¹). Our data also show that both CAP and EPICAP reduce Fe(III) ions and that their respective thiyl radicals are formed in this reaction. In the presence of Fe(III), H₂O₂, and CAP, or EPICAP, ^.OH radicals were produced by a thiol-driven Fenton mechanism. Copper(II) ions were also reduced by these thiols, but no thiyl radicals could be detected in these reactions, and no ^.OH or other Fenton oxidants were observed in the presence of H₂O₂. Our data show direct evidence that thiol groups of CAP and EPICAP are involved in scavenging of ^.OH radicals. The direct ^.OH radical scavenging, together with the reductive “repair” of other sites of ^.OH radical attack, may contribute to the known protective effect of CAP against ischemia/reperfusion-induced arrhythmias. The formation of reactive thiyl radicals in the reactions of the studied compounds with ^.OH radicals and with Fe(III) ions may play a role in some of the known adverse effects of CAP.     

Reaction of human myoglobin and H2O2. Involvement of a thiyl radical produced at cysteine 110

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H(2)O(2) (protein/H(2)O(2) = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO( small middle dot)) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g approximately 2. 036) was detected after decay of Trp-OO( small middle dot) that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios /=1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H(2)O(2) demonstrated the formation of a homodimer (mass of 34,107 +/- 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H(2)O(2) differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.     

Esr Spin Trapping Studies Into of Nature of the Oxidizing Species Formed in the Fenton Reaction: Pitfalls Associated With of Use Of 5,5-Dimethyl-1-Pyrroline-n-Oxide in the Detection of the Hydroxyl Radical

Several investigators have challenged the widely held view that the hydroxyl radical is the primary oxidant formed in the reaction between the ferrous ion and hydrogen peroxide. In recent studies, using the ESR spin trapping technique, Yamazaki and Piette found that the stoichiometry of oxidant formation in the reaction between Fe²⁺ and H₂O₂ often shows a marked deviation from the expected value of 1:1 (I. Yamazaki and L. H. Piette (1990) J. Am. Chem. Soc. 113, 7588-7593). In order to account for these observations, it was suggested that additional oxidizing species are formed, such as the ferryl ion (FeO²⁺), particularly when iron is present at high concentration and chelated to EDTA.

In this paper it is shown that secondary reactions, involving the redox cycling of iron and the oxidation of the hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by iron, operate under the reaction conditions employed by Yamazaki and Piette. Consequently, the stoichiometry of oxidant formation can be rationalized without the need to envisage the formation of oxidizing species other than the hydroxyl radical. It is also demonstrated that the iron(III) complex of DETAPAC can react directly with DMPO to form the DMPO hydroxyl radical adduct (DMPO/OH) in the absence of hydrogen peroxide. Therefore, to avoid the formation of (DMPO/OH) as an artefact, it is suggested that DETAPAC should not be used as a reagent to inactivate containating adventitious iron in experiments using DMPO.     

Does hydrogen peroxide exist “free” in biological systems?

Hydrogen peroxide (H₂O₂) can diffuse far from the site of production to intracellular locations where biological effects may be greater. The diffusion range is extended by H₂O₂ carriers formed spontaneously by hydrogen bonding with monomeric and polymeric compounds, including amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides. Hydrogen peroxide adducts (HPAs) are readily synthesized, e.g., crystalline histidine (His)-H₂O₂ adducts. An equilibrium exists between an adduct-forming compound and H₂O₂. The detection and relative stabilities of HPAs are measured by the degree of decomposition of H₂O₂ as influenced by test compounds in buffered solution competing with glucose or fructose for H₂O₂. The HPAs delay decomposition of H₂O₂ up to several hundredfold. The overall charge on an HPA, i.e., its ability to penetrate cell membranes, influences the cytotoxic and clastogenic effects of H₂O₂. Growth inhibition of Salmonella typhimurium LT₂ by H₂O₂ is enhanced by neutral HPAs but decreased by anionic HPAs. Addition of catalase 1, 10, or 30 min after inoculation of S. typhimurium LT₂ reduces or nearly eliminates partial growth inhibition by H₂O₂, but a neutral HPA, expecially his-H₂O₂, transported H₂O₂ into the cells within 1 min, and in about 10 min completely inhibited growth. The stability of HPAs decreases with increasing pH or increasing temperature, while added Fe(II) in the presence and absence of EDTA accelerates H₂O₂ and HPA decomposition. Calculations indicate H₂O₂ hydrogen bonds with nucleic acid-base pairs with no apparent bond strain and energy stabilization comparable to normal hydrogen bonding.     

Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (_•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against _•OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H₂O₂), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a _•OH-trapping agent after UVB exposure (305 nm) to H₂O₂ for 1 min in the presence of rebamipide. The signal intensity of _•OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 10₁₀, 8.16 × 10₉ and 1.65 × 10₁₀ M_-1 s_-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the _•OH generated by UVB/H₂O₂. Specific formation of this product explained the molecular characteristics of rebamipide as a potential _•OH scavenger.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

On the Reaction of Superoxide With DMPO/*OOH

A kinetic model has been used to estimate the rate constant for the reaction of superoxide (O₂/OOH) with the superoxide spin adduct of 5.5-dimethylpyrroline-N-oxide. DMPO/OOH. This rate constant is estimated to be 4.9 (± 2.2) × 10⁶ M^-1 s^-1, pH 7.4 and 25°C.     

In vitro nonenzymatic glycation enhances the role of myoglobin as a source of oxidative stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO₂ and GMbO₂, respectively. H₂O₂-induced iron release was significantly higher from GMbO₂ than that from MbO₂. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H₂O₂ was studied. GMbO₂ degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO₂. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H₂O₂ with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO₂. Compared to MbO₂, GMbO₂ was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO₂. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H₂O₂. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.