Diethoxyphosphoryl ) -5-methyl-1-pyrroline-N-oxide Research Articles

Reactive oxygen species formation at Pt nanoparticles revisited by electron paramagnetic resonance and electrochemical analysis

A combined electrochemical and electron paramagnetic resonance (EPR) procedure for the study of oxygen reduction reaction (ORR) intermediates generated at Pt nanoparticles (NPs) is validated in this work. Using spin-trap EPR complemented by electrochemical analysis, we show that we can detect and identify the free radicals that are produced during the ORR through trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) which are otherwise difficult to detect. Experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as spin trap show no evidence of DEPMPO-OOH and indicate that only OH radicals are trapped during the ORR. The results of this study serve as a functional proof-of-concept for further research on the identification of radical ORR intermediates in solution. We propose that our procedure can be used for a more rigorous quantification of free radicals involved in other electrochemical reactions.

Cell‐based studies of the first‐in‐class half‐sandwich Ir(III) complex containing histone deacetylase inhibitor 4‐phenylbutyrate

We report on a cytotoxic half‐sandwich iridium(III) complex [Ir(η5‐Cpph)(phen)(PB)]PF6 (1‐PB), containing a monodentate coordinated O‐donor 4‐phenylbutyrato ligand (PB) belonging to the family of histone deacetylase inhibitors (HDACi); HCpph = (2,3,4,5‐tetramethylcyclopenta‐2,4‐dien‐1‐yl)benzene, phen = 1,​10‐phenanthroline. The solution behaviour studies indicated that complex 1‐PB partially hydrolysed in the mixture of methanol and water (1:4, v/v), resulting in the release of the PB ligand. The extent of the PB ligand release increased in the presence of 2 molar equiv. of the reduced glutathione (GSH). Complex 1‐PB exhibited comparable in vitro cytotoxicity against the cisplatin‐sensitive (IC50 = 15.8 μM) and ‐resistant (IC50 = 13.0 μM) variants of the A2780 human ovarian carcinoma cells, while its potency against the MRC‐5 human normal fibroblast cells was markedly lower (IC50 = 124.1 μM). The cytotoxicity studies revealed an ability of complex 1‐PB to overcome the acquired resistance against cisplatin, with the resistance factor (RF = 0.8) being markedly lower than for complex 1‐Cl (RF = 1.8) and cisplatin (RF = 2.9). The A2780 cell‐based flow cytometry experiments showed different cell cycle modification induced by complex 1‐PB and cisplatin, induction of production of reactive oxygen species, and higher mitochondria membrane potential depleted cell populations after the treatment by complex 1‐PB as compared with cisplatin. In the cell‐free assay, complex 1‐PB inhibited the HDAC activity to ca 66% as compared to ca 74% valid for NaPB. The [Ir(η5‐Cpph)(phen)(H2O)]2+ species (1‐OH2), representing the hydrolysis product of both complexes 1‐PB and 1‐Cl, induced hydroxyl radical from the hydrogen peroxide, as proved by the EPR spin trapping studies with the 5‐(diethoxyphosphoryl)‐5‐methyl‐1‐pyrroline‐N‐oxide (DEPMPO) spin trap.

Radical Chemistry and Cytotoxicity of Bioreductive 3-Substituted Quinoxaline Di-N-Oxides.

The radical chemistry and cytotoxicity of a series of quinoxaline di-N-oxide (QDO) compounds has been investigated to explore the mechanism of action of this class of bioreductive drugs. A series of water-soluble 3-trifluoromethyl (4-10), 3-phenyl (11-19), and 3-methyl (20-21) substituted QDO compounds were designed to span a range of electron affinities consistent with bioreduction. The stoichiometry of loss of QDOs by steady-state radiolysis of anaerobic aqueous formate buffer indicated that one-electron reduction of QDOs generates radicals able to initiate chain reactions by oxidation of formate. The 3-trifluoromethyl analogues exhibited long chain reactions consistent with the release of the HO(•), as identified in EPR spin trapping experiments. Several carbon-centered radical intermediates, produced by anaerobic incubation of the QDO compounds with N-terminal truncated cytochrome P450 reductase (POR), were characterized using N-tert-butyl-α-phenylnitrone (PBN) and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) spin traps and were observed by EPR. Experimental data were well simulated for the production of strongly oxidizing radicals, capable of H atom abstraction from methyl groups. The kinetics of formation and decay of the radicals produced following one-electron reduction of the parent compounds, both in oxic and anoxic solutions, were determined using pulse radiolysis. Back oxidation of the initially formed radical anions by molecular oxygen did not compete effectively with the breakdown of the radical anions to form oxidizing radicals. The QDO compounds displayed low hypoxic selectivity when tested against oxic and hypoxic cancer cell lines in vitro. The results from this study form a kinetic description and explanation of the low hypoxia-selective cytotoxicity of QDOs against cancer cells compared to the related benzotriazine 1,4-dioxide (BTO) class of compounds.

Assays for Detecting Biological Superoxide

Superoxide is produced from various endogenous and exogenous sources and has been shown to play an important role in diverse physiological and pathophysiological processes. Thus, detection and measurement of this ubiquitous reactive oxygen species (ROS) are instrumental in understanding its role in biology and medicine. This ROS protocol article describes two well-established assays for detecting superoxide, namely, superoxide dismutase-inhibitable ferricytochrome c reduction and electron paramagnetic resonance (EPR) spectrometry in conjunction with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline- N -oxide (DEPMPO)-spin trapping. The article provides a step-by-step, recipe-type protocol for each of the two assays and discusses their advantages and limitations.

Ultraviolet-B radiation applied to detached peach fruit: A study of free radical generation by EPR spin trapping

In peaches, phenolic compounds are the major sources of antioxidants, and cyanidin-3-O-glucoside is the main anthocyanin present, above all in the skin. Anthocyanin content has been shown to increase after UV-B irradiation, which may be very harmful for all biological organisms due to the induction of the generation of reactive oxygen species (ROS). Peach fruits (cv. ‘Suncrest’) were exposed during post-harvest to supplemental ultraviolet-B radiation. A spin-trapping technique was used to monitor the generation of free radicals under UV-B, and 5-(diethoxy-phosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was used as the spin trap. The flesh of peaches was essentially unaffected by the treatment, whereas the skin was responsive at the end of the treatment, accumulating ascorbate, flavonoids, cyanidin-3-O-glucoside, and showing a higher antioxidant activity. The levels of stable free radicals were also lower at the end of treatment. Carbon-centred radicals contributed the most to the total amounts of free radicals, whereas hydroxyl radicals and oxygen-centred free radicals contributed minimally. The carbon-centred free radical identified was the same as the one obtained after irradiation of authentic cyanidin-3-O-glucoside. During UV-B treatment cyanidin-3-O-glucoside increased and was capable of radicalization protecting the other organic molecules of the cell from oxidation. ROS, among which hydroxyl radicals, were thus maintained to minimal levels. This ability of cyanidin-3-O-glucoside displayed the mechanism underlined the tolerance to UV-B irradiation indicating that shelf life can be prolonged by the presence of anthocyanins. Thus, UV-B technique results a good approach to induce antioxidant production in peach fruits increasing their nutraceutical properties.

Chemical composition and biological activity of Gaultheria procumbens L. essential oil

The aim of this study was to examine chemical composition and biological activity of Gaultheria procumbens L. essential oil (EO) against food spoilage and oral microorganisms. The components of EO were identified by GC–MS. Antimicrobial activity was determined against food spoilage (five bacteria and six fungal species) and oral microorganisms (eight bacteria and thirty two fungal species) by microdilution and microplate biofilm assay, antioxidant activity was tested using the persistent free radical 2,2-diphenyl-1-picryl hydrazyl (DPPH), while antiradical activity was examined by fluorescence spectroscopy and electron paramagnetic resonance spectroscopy (EPR). GC–MS.analysis showed that methyl salicylate (96.90%) was the main component of the oil. Essential oil inhibited the growth of all microorganisms tested, i.e. food and oral bacteria and fungi, respectively (MIC 0.18–3.00mg/ml and MBC 1.25–4.00mg/ml; MIC 0.73–5.00mg/ml and MFC 2.92–26.67mg/ml); The oil effectively inhibited the biofilm formation of oral Streptococcus mutans and Candida albicans as well (MIC 25.00 MBC 50.00mg/ml; MIC 12.50, MFC 50.00mg/ml). In addition, oil exhibited a dose-dependent DPPH-radical-scavenging activity with IC50 value of 30.61mg/ml. The specific fluorescence probe 2-[6-(4′-amino)phenoxy-3H-xanten-3-on-9-yl] benzoic acid (APF) and the the spin trap 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), capable for simultaneous detection of different free radical specie were used in antiradical activity of the oil measurements. Oil showed a moderate antiradical activity, reducing quantity of produced hydroxyl radicals to about 20% of initial value. This study succeeds in creating directly comparable and quantitative data for the oil unsufficiently examined so far.

Detailed analysis of reactive oxygen species induced by visible light in various cell types

Light in the visible and near infrared region stimulates various cellular processes, and thus has been used for therapeutic purposes. One of the proposed mechanisms is based on cellular production of reactive oxygen species (ROS) in response to illumination. In the present study, we followed visible light (VL)-induced hydroxyl radicals in various cell types and cellular sites using the electron paramagnetic resonance (EPR) spin-trapping technique. Fibroblasts, sperm cells, cardiomyocytes, and skeletal muscle cells were irradiated with broadband (400-800 nm) VL. To detect ROS, the EPR spin-trapping technique coupled with the spin-traps 5,5-dimethyl pyrroline-N-oxide (DMPO) or 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) were used. To investigate the cellular sites of ROS formation, the cell-permeable molecule, isopropanol, or the nonpermeable proteins, bovine serum albumin (BSA) and superoxide dismutase (SOD), were introduced to the cells before irradiation. ROS production in mitochondria was measured using the fluorescent probe, MitoTracker Red (MTR). The concentration of .OH increased both with illumination time and with cell concentration, and decreased when N(2) was bubbled into the cell culture, suggesting that VL initiates a photochemical reaction via endogenous photosensitizers. VL was found to stimulate ROS generation both in membrane and cytoplasm. In addition, fluorescent measurments confirmed the mitochondria to be target for light-cell interaction. The findings support the hypothesis that ROS are generated in various cellular sites following light illumination.

Comparison of superoxide detection abilities of newly developed spin traps in the living cells

This study compared the superoxide detection abilities of four spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(diphenylphosphinoyl)-5-methyl-1pyrroline N-oxide (DPPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in living cells. Electron spin resonance (ESR) signals of the superoxide adducts were observed when spin traps were added to a suspension of human oral polymorphonuclear leukocytes (OPMNs) stimulated by phorbol 12-myristate 13-acetate. The ESR signal of the CYPMPO-superoxide adduct (CYPMPO-OOH) increased for 24 min after the initiation of the reaction, whereas the signals from DMPO-OOH and DPPMPO-OOH peaked at 6 and 10 min, respectively. The maximum concentrations of DMPO-OOH, DPPMPO-OOH and CYPMPO-OOH in OPMNs were 1.9, 6.0 and 10.7 µM, respectively. Furthermore, CYPMPO could more efficiently trap superoxide in blood PMNs compared with DEPMPO. From these results, it was concluded that CYPMPO performs better than DMPO, DPPMPO and DEPMPO for superoxide measurements in living cell systems because it has lower cytotoxicity and its superoxide adduct has a longer lifetime.

Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis

The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline- N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria–neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.

Early specific free radical-related cytotoxicity of gas phase cigarette smoke and its paradoxical temporary inhibition by tar

Electron paramagnetic resonance (EPR) spin trapping studies demonstrated aqueous tar particulate matter (TPM) and gas phase cigarette smoke (GPCS) to behave as different sources of free radicals in cigarette smoke (CS) but their cytotoxic implications have been only assessed in CS due to its relevance to the natural smoking process. Using a sensitive spin trapping detection with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), this study compared the respective roles of CS- and GPCS-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes yielding a wide range of TPM yields. In buffer bubbled with CS the DEPMPO/superoxide spin adduct was the major detected nitroxide. Use of appropriate control experiments with nitric oxide radical (NO*) or carbonyl sulfide, and a computer analysis of spin adduct diastereoisomery showed that the hydroxyl radical (HO*) adduct of DEPMPO seen in GPCS-bubbled was rather related to metal-catalyzed nucleophilic synthesis than to direct HO* trapping. Unexpectedly a protective effect of TPM on murine 3T3 fibroblasts was observed in early (<3h) free radical-, GPCS-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with GPCS. These results highlight an acute, free radical-dependent, harmful mechanism specific to the GPCS phase, possibly involving NO* chemistry, whose physical or chemical control may be of great interest with the aim of reducing the toxicity of smoke.

Inefficient spin trapping of superoxide in the presence of nitric-oxide: Implications for studies on nitric-oxide synthase uncoupling

Uncoupling of nitric-oxide synthase (NOS) by deficiency of the substrate l-arginine or the cofactor (6 R)-5,6,7,8-tetrahydrobiopterin (BH4) is known to generate the reactive oxygen species H 2O 2 and superoxide. Discrimination between these two compounds is usually achieved by spin trapping of superoxide. We measured superoxide formation by uncoupled rat neuronal NOS, which contained one equivalent of tightly bound BH4 per dimer, using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline- N-oxide (DEPMPO) as a spin trap. As expected, the Ca 2+-stimulated enzyme exhibited reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity that was accompanied by generation of superoxide and H 2O 2 in the absence of added l-arginine and BH4. Addition of BH4 (10 μM) did not significantly affect the rate of H 2O 2 formation but almost completely inhibited the apparent formation of superoxide, suggesting direct formation of H 2O 2. Although l-arginine (0.1 mM) increased the rate of NADPH oxidation about two-fold, the substrate largely attenuated apparent formation of both superoxide and H 2O 2, indicating that the spin trap did not efficiently outcompete the reaction between NO and superoxide. The efficiency of DEPMPO to scavenge superoxide in the presence of NO was studied by measuring free NO with a Clark-type electrode under conditions of NO/superoxide cogeneration. Neuronal NOS half-saturated with BH4 and the donor compound 3-morpholinosydnonimine (SIN-1) were used as enzymatic and nonenzymatic sources of NO/superoxide, respectively. Neither of the two systems gave rise to considerable NO signals in the presence of 50–100 mM DEPMPO, and even at 400 mM the spin trap uncovered less than 50% of the NO release that was detectable in the presence of 5000 U/ml superoxide dismutase. These results indicate that DEPMPO and all other currently available superoxide spin traps do not efficiently outcompete the reaction with NO. In addition, the similar behavior of nNOS and SIN-1 provides further evidence for NO as initial product of the NOS reaction.

Characterization of the high-resolution ESR spectra of superoxide radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Analysis of conformational exchange

It has been previously reported that the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) can form stable radical adducts with superoxide radical. However, the presence of diastereomers of DEPMPO radical adducts and the appearance of superhyperfine structure complicates the interpretation of the ESR spectra. It has been suggested that the superhyperfine structure in the ESR spectrum of DEPMPO/⋅OOH is a result of conformational exchange between conformers. The analysis of the temperature dependence of the ESR spectrum of DEPMPO/⋅OOH and of its structural analog DMPO/⋅OOH have demonstrated that both ESR spectra contain exchange effects resulting from conversion between two conformers. Computer simulation calculates a conformer lifetime on the order of 0.1 μs for DMPO/⋅OOH at room temperature. However, temperature dependence of the ESR spectrum of DEPMPO/⋅OOH suggests that superhyperfine structure does not depend on the conformational exchange. We have now found that the six-line ESR spectrum with superhyperfine structure should be assigned to a DEPMPO-superoxide-derived decomposition product. Therefore, ESR spectra previously assigned to DEPMPO/⋅OOH contain not only the two diastereomers of DEPMPO/⋅OOH but also the decomposition product, and these spectra should be simulated as a combination of four species: two conformers of the first diastereomer, one conformer of the second diastereomer and the superoxide-derived decomposition product. The presence of four species has been supported by the temperature dependence of the ESR spectra, nucleophilic synthesis of radical adducts, and isotopic substitution experiments. It is clear that to correctly interpret DEPMPO spin trapping of superoxide radicals, one must carefully consider formation of secondary radical adducts.

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.

Formation of Superoxide Anion during Ferrous Ion-Induced Decomposition of Linoleic Acid Hydroperoxide under Aerobic Conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.

Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis.

The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.

Characterization of the High Resolution ESR Spectra of the Methoxyl Radical Adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)

Spin-trapping investigators are largely limited by the instability of the radical adducts. Spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) forms very stable alkoxyl radical adducts. However, the presence of two chiral centers in the DEPMPO alkoxyl radical adduct results in two diastereomers with distinctive ESR spectra, which complicates the interpretation of the ESR spectra. We have analyzed the high resolution ESR spectra of the DEPMPO/•OCH3 radical adduct. DEPMPO/•OCH3 has been synthesized by the nucleophilic addition of alcohols to DEPMPO. The electron spin resonance (ESR) spectrum of DEPMPO/•OCH3 in oxygen-free methanol solution reveals superhyperfine structure with hyperfine coupling constants as small as 0.3 G. In order to simplify the analysis of the electron spin resonance (ESR) spectrum, we synthesized the DEPMPO/•OCD3 radical adduct. Computer simulation of the DEPMPO/•OCD3 ESR spectrum revealed two diastereomers. Hyperfine coupling constants of γ-protons and 17O from the –OCH3 group were also determined. ESR spectra of DEPMPO/•OCH3 in phosphate buffer have also been characterized. The presence of specific hyperfine couplings from the –OCH3 group can be used for the unambiguous identification of the DEPMPO/•OCH3 radical adducts. We suggest that the analysis of high resolution ESR spectra can be used for the unambiguous characterization of DEPMPO radical adducts.

Skin protection against ultraviolet induced free radicals with ascorbyl palmitate in microemulsions

UV irradiation induces free radical formation in the skin. UV filters and antioxidants can be used for protection. In the present work, the amphiphilic antioxidant ascorbyl palmitate has been investigated and its effectiveness against free radical formation in porcine skin determined with electron paramagnetic resonance (EPR) spectroscopy with a spin trapping technique. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline- N-oxide (DEPMPO) was used as spin trap. In this study, three different radicals were identified in UV irradiated porcine ear skin: two originated from sulphur centred radicals (SO 3 ), while the third was the carbon-centred acyl (CO ) radical. Ascorbyl palmitate applied on the skin decreased the level of formation of free radicals. Its effectiveness depended significantly on the carrier system – the type of microemulsion and its concentration, while the time of application had no influence on its effectiveness. Oil in water microemulsions delivered ascorbyl palmitate to the skin significantly better than water in oil microemulsions. In both types of microemulsions, the effectiveness increases at higher concentrations of ascorbyl palmitate.

Singlet oxygen-mediated hydroxyl radical production in the presence of phenols: whether DMPO-*OH formation really indicates production of *OH?

The reaction of singlet oxygen (1O2) generated by ultraviolet-A (UVA)-visible light (lambda > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-*OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO-*OH decreased in the presence of either a *OH scavenger or a quencher of *O2 and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both 1O2- and DMPO-mediated formation of free *OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that 1O2, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO-*OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of *OH. These observations indicate that 1O2 reacts first with DMPO, and the resulting DMPO-1O2 intermediate is immediately decomposed/reduced to give *OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of 1O2 to *OH. Furthermore, DEPMPO did not cause the spin-trapping agent-mediated generation of *OH like DMPO did.

Inhibition of superoxide generation from fMLP-stimulated leukocytes by high concentrations of nitric oxide or peroxynitrite: characterization by electron spin resonance spectroscopy

A Corrigendum has been published for this article in Redox Report, Volume 17, Number 1, January 2012, page 47: http://www.ingentaconnect.com/content/maney/rer/2012/00000017/00000001/art00006 (please copy and paste this URL into your web browser in order to link to it)This present study examined the effects of high concentrations of nitric oxide (NO•) and peroxynitrite (ONOO-) on superoxide (O2•-) production from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leukocytes (PMNs) by using electron spin resonance (ESR) and spin trapping with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). We demonstrated that ONOO- (100 μM) decreased the ESR signal of DEPMPO-OOH from fMLP-activated PMNs, indicating the inhibition of O2•- generation, while it enhanced the signal of DEPMPO-OH. Inhibition of the respiratory burst was also observed when PMNs were pre-exposed to high concentrations of NO• (100 μM), generated by the NO• donor NOR-1, 30 min prior to stimulation with fMLP. NOR-1 inhibited O2•- generation more effectively under conditions in which ONOO- was formed concurrently. The ability of high concentrations of either ONOO- or NO• to inhibit O2•- generation from fMLP-stimulated PMNs is relevant to pathophysiological conditions, such as severe inflammation, in which NO• or ONOO- production can be significantly elevated.

An EPR study of free radical generation during maceration of uncooked vegetables

AbstractElectron paramagnetic resonance (EPR) spectroscopy has been used to study free radicals formed in a range of plant tissues as a result of physical damage, with the objective of gaining some insight into the free radical chemistry that is initiated when uncooked vegetable products (eg salads) are eaten. Chemical spin traps were used to aid the detection of unstable free radicals; more stable radicals were detected directly. Commonly observed ‘stable’ species were the monodehydroascorbate radical, which has a characteristic doublet spectrum, and a single‐peak resonance, which is presumed to come from free radical centres stabilised in macromolecules. In mushrooms (Agaricus spp), spin‐trapping experiments using either α‐(4‐pyridyl‐1‐oxide)‐N‐t‐butyl‐nitrone (4‐POBN) or phenyl‐t‐butyl‐nitrone (PBN) showed the formation of large quantities of adducts of the radical from 4‐hydroxymethylbenzene diazonium salts. Pleurotus species, in contrast, gave signals consistent with the formation of unidentified C‐centred radicals. With other foodstuffs, reaction with 4‐POBN was complex and signals from 4 − POBN· and ${\bf CO_{2}^{\cdot -}}$ adducts were observed along with the t‐butylhydronitroxide radical (an adduct breakdown product). Investigation of carrot hypocotyl rootstock in the presence of 5‐(diethoxyphosphoryl),5‐methyl‐1‐pyrroline‐N‐oxide (DEPMPO) revealed adducts of ·OH and unidentified C‐centred radicals. Free radical interactions between food components were demonstrated by the suppression of the signal from the 4‐POBN adducts of lettuce by onion, garlic, satsuma or vinaigrette, but not by olive oil. In addition, an appreciable decrease in spectral intensity of the 4‐POBN adduct from lettuce was observed in the presence of saliva, which suggests that saliva contains free radical scavengers which are able to compete successfully with the spin trap.© 2002 Society of Chemical Industry