Spin Trapping Method Research Articles

Study of the radical chemistry promoted by tributylborane

The structures of radicals generated in the oxidation process of trialkylborane were detected based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC/Q-ToF MS) combined with the spin trapping method. Structural identification of the spin adducts produced by 5,5-dimethyl-1-pyrroline-1-oxide with radicals could be carried out unambiguously by combining the data obtained by UPLC/Q-ToF MS analyses. Then the oxidation mechanism was described. To specify the inter-relationships between the oxidation process of trialkylborane and the concomitant radical chemistry, four kinds of alkanes providing different H-abstraction reactivity to alkoxy radical were chosen as radical capturers. The final oxidation products of trialkylborane were characterized by GC–MS and 11B-NMR. The results indicted that the radical content was not only affected by the oxidation degree of trialkylborane, but also done by the activity of alkane. Especially the hydrogen atom abstraction by n-butoxy radical played an important role in the oxidation process of tributylborane, which would promote the oxidizability of tributylborane and deepen the oxidation degree of tributylborane.

Comparative study on antioxidative and macrophage-stimulating activities of polyguluronic acid (PG) and polymannuronic acid (PM) prepared from alginate

The antioxidant and macrophage-stimulating activities of polyguluronic acid (PG) and polymannuronic acid (PM) prepared from alginate were examined. A chemiluminescence (CL) method using a luminol analog, L-012, showed that both PM and PG scavenge superoxide produced by hypoxanthine–xanthine oxidase system in a concentration-dependent manner. At 100μg/ml, PG showed slightly stronger superoxide scavenging activity than PM. In an electron spin resonance (ESR)-spin trapping method in which the Fenton reaction was used as hydroxyl radical generation system, we found that both PM and PG showed potent hydroxyl radical scavenging activity to a similar extent. Because PM and PG showed no chelating activity on Fe2+, it was confirmed that PM and PG can directly scavenge hydroxyl radical. No significant scavenging activity of PM and PG toward hydrogen peroxide was observed. Interestingly, the macrophage-stimulation activity of PG as measured by nitric oxide (NO)-production from mouse macrophage cell line RAW264.7 cells was evidently stronger than that of PM. Our results suggest that RAW264.7 cells might be able to distinguish the conformational differences between PM and PG, and respond differently to them, whereas the effects of such structural differences between PM and PG on the radical scavenging activities may not be so significant.

Multiple free-radical scavenging capacity in serum

We have developed a method to determine serum scavenging-capacity profile against multiple free radical species, namely hydroxyl radical, superoxide radical, alkoxyl radical, alkylperoxyl radical, alkyl radical, and singlet oxygen. This method was applied to a cohort of chronic kidney disease patients. Each free radical species was produced with a common experimental procedure; i.e., uv/visible-light photolysis of free-radical precursor/sensitizer. The decrease in free-radical concentration by the presence of serum was quantified with electron spin resonance spin trapping method, from which the scavenging capacity was calculated. There was a significant capacity change in the disease group (n = 45) as compared with the healthy control group (n = 30). The percent values of disease’s scavenging capacity with respect to control group indicated statistically significant differences in all free-radical species except alkylperoxyl radical, i.e., hydroxyl radical, 73 ± 12% (p = 0.001); superoxide radical, 158 ± 50% (p = 0.001); alkoxyl radical, 121 ± 30% (p = 0.005); alkylperoxyl radical, 123 ± 32% (p>0.1); alkyl radical, 26 ± 14% (p = 0.001); and singlet oxygen, 57 ± 18% (p = 0.001). The scavenging capacity profile was illustrated using a radar chart, clearly demonstrating the characteristic change in the disease group. Although the cause of the scavenging capacity change by the disease state is not completely understood, the profile of multiple radical scavenging capacities may become a useful diagnostic tool.

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

Abstract 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.

Spin Trapping Studies of Amine-Stabilized Trialkylborane Free Radical Initiator Systems: Kinetics and Mechanism

The free radical initiation chemistry of highly reactive trialkylboranes (R3B, R = n-butyl), stabilized by complexation to amines (A–R3B), has been investigated using spin trapping methods and electron paramagnetic resonance spectroscopy. Protonation of A–R3B leads to release of R3B and its subsequent autooxidation in the presence of molecular oxygen. This reaction provides highly reactive alkyl and alkoxy radicals for initiation of α–olefin polymerization. Kinetic traces of the R3B autooxidation reaction were obtained by monitoring the relative concentrations of trapped radical species as a function of time for several different initial conditions (acid concentrations). Analysis of a representative kinetic run is discussed in terms of several proposed mechanisms from the literature.

Characterization of inclusion complex of vitamin E compound with 2,6-di-O-methylated β-cyclodextrin as the solubility enhancer and its kinetic determination for radical scavenging ability

The structure of the inclusion complex of α-tocopherol (vitamin E compound) with 2,6-di-O-methylated β-cyclodextrin (DM-β-CD) was characterized by 2D ROESY NMR measurements, suggesting that DM-β-CD includes the side-chain moiety of α-tocopherol. The inclusion complexation of DM-β-CD showed the usefulness of water solubilizer for the radical scavenging assay of vitamin E compounds in aqueous solution. Using the electron paramagnetic resonance (EPR) competitive spin trapping method, we determined the oxygen radical (RO∙) scavenging abilities of seven vitamin E compounds (tocopherols and tocotrienols), which were solubilized by DM-β-CD in water. The order of the RO∙ radical scavenging abilities for vitamin E compounds solubilized by DM-β-CD are α- > β- ≈ γ- > δ-, which is in agreement with the oxidation potential values of antioxidants. It is noted that the RO∙ radical scavenging abilities of tocotrienols are comparable to those of tocopherols. Based on the results, the mechanism of the antioxidant reaction of vitamin E compounds with the RO∙ radical is discussed.

Detection and Imaging of Superoxide in Roots by an Electron Spin Resonance Spin-Probe Method

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.

A comparative study of antioxidative activities of cell-wall polysaccharides

Oxidative burst in plants is elicited by biotic and abiotic stressors. Analogously to some monosaccharides which act as intracellular antioxidants, cell-wall polysaccharides may be in charge of buffering free-radical production in the extracellular compartment under pronounced prooxidative settings. Although a wide range of plant polysaccharides have been examined for their antioxidative properties, this usually has not been done in a coherent and comparative manner and against biologically relevant reactive species. Here we show that different cell-wall polysaccharides, cellulose, pectin, d-galacto- d-mannan, arabinogalactan, and xylan, exhibit distinctive antioxidative activities against the hydroxyl radical ( OH)-generating Fenton reaction and superoxide. We found, using an EPR spin-trapping method, that the main carriers of ‘anti-Fenton’ activity in the plant cell wall are pectin and xylan. They most likely act by binding metal ions in such a manner to allow the Fenton reaction, after which they scavenge OH. Such a mode of action is preferred by cells resulting in a safe degradation of H 2O 2. On the other hand, the polysaccharides examined showed similar superoxide scavenging capacities. We propose that plants may employ different antioxidative characteristics of polysaccharides to regulate their redox status by modifying the composition of the cell wall.

Effect of magnetic field on the zero valent iron induced oxidation reaction

The magnetic field (MF) effect on the zero valent iron (ZVI) induced oxidative reaction was investigated for the first time. The degradation of 4-chlorophenol (4-CP) in the ZVI system was employed as the test oxidative reaction. MF markedly enhanced the degradation of 4-CP with the concurrent production of chlorides. The consumption of dissolved O 2 by ZVI reaction was also enhanced in the presence of MF whereas the competing reaction of H 2 production from proton reduction was retarded. Since the ZVI-induced oxidation is mainly driven by the in situ generated hydroxyl radicals, the production of OH radicals was monitored by the spin trap method using electron spin resonance (ESR) spectroscopy. It was confirmed that the concentration of trapped OH radicals was enhanced in the presence of MF. Since both O 2 and Fe 0 are paramagnetic, the diffusion of O 2 onto the iron surface might be accelerated under MF. The magnetized iron can attract oxygen on itself, which makes the mass transfer process faster. As a result, the surface electrochemical reaction between Fe 0 and O 2 can be accelerated with the enhanced production of OH radicals. MF might retard the recombination of OH radicals as well.

Kinetic analysis of reactive oxygen species generated by the in vitro reconstituted NADPH oxidase and xanthine oxidase systems

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the xanthine oxidase (XOD) systems generate reactive oxygen species (ROS). In the present study, to characterize the difference between the two systems, the kinetics of ROS generated by both the NADH oxidase and XOD systems were analysed by an electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide (DEPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). As a result, two major differences in ROS kinetics were found between the two systems: (i) the kinetics of (•)OH and (ii) the kinetics of hydrogen peroxide. In the NADH oxidase system, the interaction of hydrogen peroxide with each component of the enzyme system (NADPH, NADH oxidase and FAD) was found to generate (•)OH. In contrast, (•)OH generation was found to be independent of hydrogen peroxide in the XOD system. In addition, the hydrogen peroxide level in the NADPH-NADH oxidase system was much lower than measured in the XOD system. This lower level of free hydrogen peroxide is most likely due to the interaction between hydrogen peroxide and NADPH, because the hydrogen peroxide level was reduced by ~90% in the presence of NADPH.

α-Tocopherol impact on oxy-radical induced free radical decomposition of DMSO: Spin trapping EPR and theoretical studies

EPR spin trapping and theoretical methods such as density functional theory (DFT) as well as combined DFT and quadratic configuration interaction approach (DFT/QCISD) were used to identify the radicals produced in the reaction of oxy-radicals and dimethyl sulfoxide (DMSO) in the presence and absence of α-tocopherol. Additionally, the mixtures of α-tocopherol with linolenic acid and glyceryl trilinoleate as well as bioglycerols (glycerol fractions from biodiesel production) were tested. α-Tocopherol inhibited oxidation of the main decomposition product of DMSO, •CH 3 to •OCH 3 but did not prevent the transformation process of N-t-butyl- α-phenylnitrone (PBN) into 2-methyl-2-nitrosopropane (MNP). Theoretical investigations confirmed the structures of proposed spin adducts and allowed to correlate the EPR parameters observed in the experiment with the spin adducts electronic structure.

Development of a method based on ESR spectroscopy for the identification of irradiated beef, pork, and chicken meats

The electron spin resonance (ESR) spintrapping method for the detection of irradiated beef, pork, and chicken was studied using a α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) spin trapper in the dose of 0.5–7 kGy. Irradiation caused a significant increase in the ESR signal intensity of samples with hyperfine coupling constants of aN=1.57 mT and aH=0.25 mT, which correspond to lipid-derived radicals. In contrast, un-irradiated samples exhibited a weak ESR signal with no hyperfine coupling constants. The irradiation-induced lipid radical stability vs. temperature was also studied at room temperature, −4 and −18°C using 3 kGy irradiated beef. Temperature did not affect ESR signal intensity or the hyperfine coupling constants. To investigate the applicability of the proposed procedure for pork and chicken, a comparison of the spectra at the hyperfine coupling constants confirmed the presence of lipid-derived radicals in the samples.

Understanding the radical mechanism of lipoxygenases using 31P NMR spin trapping

In this paper, we use our quantitative 31P NMR spin trapping methods, already developed for simple oxygen- and carbon-centered radicals, to understand the radical intermediates generated by enzymatic systems and more specifically lipoxygenases. Our methodology rests on the fact that free radicals react with the nitroxide phosphorus compound, 5-diisopropoxy-phosphoryl-5-methyl-1-pyrroline- N-oxide (DIPPMPO), to form stable radical adducts, which are suitably detected and accurately quantified using 31P NMR in the presence of a phosphorus containing internal standard. This system was thus applied to better understand the mechanism of enzymatic oxidation of linoleic acid by soybean lipoxygenases-1 (LOX). The total amount of radicals trapped by DIPPMPO was detected by 31P NMR at different experimental conditions. In particular the effect of dioxygen concentration on the amount of radicals being trapped was studied. At low dioxygen concentration, a huge increase of radicals trapped was observed with respect to the amount of radicals being trapped at normal dioxygen concentrations.

Degradation of pentachlorophenol by a novel peroxidase-catalyzed process in the presence of reduced nicotinamide adenine dinucleotide

A novel horseradish peroxidase (HRP)-catalyzed H 2O 2 process in the presence of reduced nicotinamide adenine dinucleotide (NADH) was applied to remove aqueous pentachlorophenol (PCP). Parameters (pH, H 2O 2 concentration, HRP activity and NADH dosage) on PCP removal were investigated. It was found that initial 0.05 mM PCP was removed by 98% in HRP–NADH–H 2O 2 system at pH 5.0 and 30 °C for 1 h. Addition of O 2 in HRP–NADH–H 2O 2 system enhanced the removal rate of PCP due to promoting hydroxyl radicals ( OH) and superoxide anion radical ( O 2 - ) generation, which were confirmed by electron paramagnetic resonance (EPR)-spin trapping method. PCP removal efficiency decreased when O 2 - and H 2O 2 were scavenged by superoxide dismutase and catalase in HRP–NADH–O 2 system, indicating that OH/ O 2 - played a great role in the degradation of PCP. Gas chromatography–mass spectrometry (GC–MS) analysis revealed that octachlorinated dibenzodioxin (OCDD) in residual solution was reduced after treated by the HRP–NADH–O 2 process, resulting in lower toxicity of treated solution than conventional enzymatic process. Two enzymatic-catalysis pathways were proposed for PCP removal in HRP–NADH–H 2O 2/O 2 system: (i) OH/ O 2 - free radical oxidation (ii) conventional phenoxy polymerization.

Antioxidant effects of antioxidant biofactor on reactive oxygen species in human gingival fibroblasts

The purpose of this study was to investigate the effects of antioxidant biofactor (AOB) on reactive oxygen species (ROS). Generation of superoxide radical (O2•−) and hydroxyl radical (•OH) was determined using an electron spin resonance (ESR) spin-trapping method. AOB was added at different concentrations to these free radical generating systems. The generation of both O2•− and •OH was scavenged by the addition of AOB in a dose-dependent manner. These results indicate that AOB has strong antioxidant properties against these radicals. We further investigated the anti-oxidative effect of AOB on human gingival fibroblasts (HGFs). HGFs were treated for 3 h with α-MEM containing a combination of AOB and H2O2 (AOB + H2O2 group), containing H2O2 (H2O2 group), or containing AOB alone (AOB group). Non-stimulated HGFs were used as a control group. The number of surviving cells was in the order of the AOB group > control group > AOB + H2O2 group > H2O2 group. The level of expression of type I collagen mRNA and production of collagen were also in the order of the AOB group > control group > AOB + H2O2 group > H2O2 group. In conclusion, our results suggest that AOB may protect HGFs against oxidative stress by reducing stress-induced ROS.

Free radical scavenging abilities of flavonyl‐thiazolidine‐2,4‐dione compounds

Free radical scavenging activity of flavonyl-thiazolidine-2,4-dione compounds has been evaluated using chemiluminescence, electron spin resonance spectroscopy with 5,5-dimethyl-1-pyrroline-1-oxide as spin trap and DPPH (2,2'-diphenyl-1-picrylhydrazyl) method. The examined compounds exhibited 28-50% scavenging superoxide anion radical O₂⁻ⁱ16.7-76.7% hydroxyl radical (HO•) and 9-40% DPPH radical. Compounds containing carbonyl group in their structure can be considered as antioxidants with high relevance and great biological importance.

Estimation of the postmortem duration of mouse tissue by electron spin resonance spectroscopy.

Electron spin resonance (ESR) method is a simple method for detecting various free radicals simultaneously and directly. However, ESR spin trap method is unsuited to analyze weak ESR signals in organs because of water-induced dielectric loss (WIDL). To minimize WIDL occurring in biotissues and to improve detection sensitivity to free radicals in tissues, ESR cuvette was modified and used with 5,5-dimethtyl-1-pyrroline N-oxide (DMPO). The tissue samples were mouse brain, hart, lung, liver, kidney, pancreas, muscle, skin, and whole blood, where various ESR spin adduct signals including DMPO-ascorbyl radical (AsA∗), DMPO-superoxide anion radical (OOH), and DMPO-hydrogen radical (H) signal were detected. Postmortem changes in DMPO-AsA∗ and DMPO-OOH were observed in various tissues of mouse. The signal peak of spin adduct was monitored until the 205th day postmortem. DMPO-AsA∗ in liver (y = 113.8–40.7 log (day), R1 = −0.779, R2 = 0.6, P < .001) was found to linearly decrease with the logarithm of postmortem duration days. Therefore, DMPO-AsA∗ signal may be suitable for detecting an oxidation stress tracer from tissue in comparison with other spin adduct signal on ESR spin trap method.

Platinized WO3 as an Environmental Photocatalyst that Generates OH Radicals under Visible Light

This study aims to understand the visible light photocatalytic activities of platinized WO3 (Pt/WO3) on the degradation of aquatic pollutants and the role of main photooxidants. The presence of Pt on WO3 is known to facilitate the multielectron reduction of O2, which enables O2 to serve as an electron acceptor despite the insufficient reduction potential of the conduction band electrons (in WO3) for the one-electron reduction of O2. The concurrent oxidative reactions occurring on WO3 were markedly enhanced in the presence of Pt and accompanied the production of OH radicals under visible light, which was confirmed by both a fluorescence method (using a chemical trap) and a spin trap method. The generation of OH radicals mainly comes from the reductive decomposition of H2O2 that is produced in situ from the reduction of O2 on Pt/WO3. The rate of in situ production of H2O2 under visible light was significantly faster with Pt/WO3 than WO3. Six substrates that were tested for the visible light (lambda>420 nm) induced degradation on Pt/WO3 included dichloroacetate (DCA), 4-chlorophenol (4-CP), tetramethylammonium (TMA), arsenite (As(III)), methylene blue (MB), and acid orange 7 (AO7). The degradation (or conversion) of all six substrates was successfully achieved with Pt/WO3 and the role of OH radicals in Pt/WO3 photocatalysis seemed to be different depending on the kind of substrate. In the presence of tert-butyl alcohol (TBA: OH radical scavenger), the photocatalytic degradation was markedly reduced for 4-CP or completely inhibited for DCA and TMA whereas that of As(III), MB, and AO7 was little affected. Pt/WO3 photocatalyst that oxidizes various substrates under visible light with a sufficient photostability can be applied for solar water treatment.

ESR study of spin adducts of the direct electrocatalytic decomposition of light aliphatic alcohols in a polymer electrolyte fuel cell

Spin adducts of methanol and ethanol electrocatalytic oxidation were detected by the spin trap method using a tiny ?2/O2 fuel cell (FC) designed for ESR in situ with a Nafion/Pt membrane electrode assembly. Spin adducts of intermediates of the direct electrooxidation of ethanol, which have not been observed earlier, were obtained by the variation of oxidation conditions, in particular, the FC potential. The work of the FC was controlled by monitoring the diagnostic curves potential3-current density, power density3-current density, and efficiency—power density.

Structure Effect on Antioxidant Activity of Catecholamines toward Singlet Oxygen and Other Reactive Oxygen Species in vitro

The reactivity of catecholamine neurotransmitters and the related metabolites were precisely investigated toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and reactive oxygen species. Catecholamines reacted immediately with DPPH radicals, their reactivity being stronger than that of ascorbic acid as a reference. Superoxide scavenging activities of catecholamines determined by WST-1 and electron spin resonance (ESR) spin trapping methods were also high. Whereas tyrosine, the dopamine precursor showed no reactivity toward superoxide. The reactivity toward singlet oxygen was evaluated by observing specific photon emission from singlet oxygen. The results revealed that reactivity of catecholamines was markedly higher than that of sodium azide, and catechin as catechol reference. The reaction of catecholamines and singlet oxygen was further studied by ESR using 55-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping reagent and rose bengal as photosensitizer. DMPO-OH signal of epinephrine was significantly small compared to other catecholamines, catechin, and 4-methylcatechol as a reference compound and was as small as that of tyrosine. The signal formation was totally dependent on singlet oxygen, and the presence of catechol compounds. These results indicated that epinephrine is the most potent singlet oxygen quencher than other catecholamines, and the secondary amino group in its alkyl side chain could play a role in unique singlet oxygen quenching property of epinephrine.