The ESR study on the protective effect of grape seed extract on rat heart mitochondria from the injury of lipid peroxidation

The effect of grape seed extract (GSE) on the lipid peroxidation of rat heart mitochondria was studied using the spin trapping agent α-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) with electron spin resonance (ESR) spectrometer. Two fatty acid spin labels, 5-doxyl-stearic acid (5-DOXYL) and 16-doxyl-stearic acid (16-DOXYL) were used to study the effect of GSE on mitochondrial membrane fluidity. The results showed that the GSE could scavenge the lipid free radicals generated from the mitochondria of rat heart, and protect the mitochondrial membranes damaged by lipid peroxidation. Both the scavenging effect and the protective effect are better in water phase than in lipid phase. And the data indicate that perhaps triners and other polymers have better scavenging effect on lipid free radicals than monomers.     

Superoxide dismutase versus ferricytochrome C: determining rate constants for the spin trapping of superoxide by cyclic nitrones

Given that spin trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for spin trapping O(2)(*-) compared to the well-studied spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for spin trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known spin traps were also undertaken.     

Direct ESR detection and spin trapping of radicals generated by reaction of oxygen radicals with sulfonated poly(ether ether ketone) (SPEEK) membranes

The stability of membranes under the strong oxidizing conditions in fuel cells is one of the major challenges in the development of fuel cells based on proton exchange membranes (PEMs). This study is centered on the determination of the susceptibility to degradation of SPEEK membranes exposed to OH radicals, using both direct ESR and spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). In order to achieve a complete picture on SPEEK degradation, two types of experiments were performed: 1. UV irradiation at 77 K of SPEEK membranes swollen by aqueous solutions of H₂O₂; 2. UV irradiation of SPEEK membranes swollen by aqueous solutions of H₂O₂ in the presence of DMPO as a spin trap. UV irradiation without oxygen of SPEEK at 77 K in acid or basic form in the presence of H₂O₂/H₂O produced phenoxyl radicals as the predominant radicals detected by direct ESR or spin trapping methods. At pH 4, the oxygen radicals produced phenyl radicals as the predominant species detected by spin trapping methods. The hydroperoxyl radical, as DMPO/OOH adduct, was detected only when the DMPO/OH adduct was absent. The appearance of phenyl and phenoxyl radicals provides the evidence that OH radicals react with the aromatic ring of SPEEK or leading to the scission of its ether bridge.     

Detection of reactive free radicals derived from nucleosides by liquid chromatography coupled to tandem mass spectrometry of DMPO spin trapping adducts

In this study, reactive free radicals derived from several nucleosides were spin trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and then detected by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This method provides a specific detection of spin trapping adducts derived from nucleosides with a very high sensitivity: quantities as low as 0.5 picomoles of spin trapping adducts corresponding to concentrations of 2.5 10(-8) mol . L(-1) were detected. Different spin trapping adducts were characterized by HPLC/ESI-MS/MS in three well-known systems producing free radicals photochemically: the photolysis of 5-halo-2'-deoxyuridines, the photolysis of 5-thiophenylmethyl-2'-deoxyuridine and the photolysis of thymidine with menadione bisulfite as a photosensitizer. A new radical photoreactivity of uridine derivatives was also detected by this method both at the nucleoside and at the RNA level, showing that the method is also relevant for studying spin trapping adducts derived from DNA and RNA strands.     

Hydrogen atom abstraction from polypropylene and polystyrene by t-butoxy radical site of radical attack studied by spin trapping

In order to elucidate the site of radical attack on polypropylene and polystyrene, the abstractions of hydrogen atoms by t-butoxy radicals and phenyl radicals have been studied by using a spin trapping technique. The t-butoxy radical abstracted tertiary hydrogen atoms selectively from polypropylene, polystyrene and model compounds. On the other hand, the tertiary hydrogens in polypropylene and its model compounds were less reactive towards the phenyl radical than the secondary hydrogens within the same molecule and the secondary hydrogens in polystyrene were abstracted predominantly by the phenyl radical. The conformational effects on the reactivities of various hydrogens in polypropylene and model compounds were found to be similar.     

An application of spin trapping to radical polymerizations

Radical polymerizations and copolymerizations were carried out in the presence of phenyl tert-butyl nitrone(PBN) and tert-nitrosobutane (t-BuNO), and the structure of the spin adduct formed was investigated by ESR spectroscopy. PBN adducts gave the same ESR pattern, and the variation of the splitting constant was not large enough to warrant its use for the structure assignment. Therefore, the subsequent trapping experiments were performed with t-BuNO. The polymerization mixture of representative monomers showed esr patterns that are indicative of the propagating radical being trapped. These trapped radicals were not necessarily very stable and, in most cases, disappeared after long reaction periods. In the case of α-methyl substituted monomers, additional nine-line spectra were observed which were attributed to trapping of the radical species formed by hydrogen abstraction from the α-methyl group. The tert-butyl radical which was formed by decomposition of t-BuNo was probably responsible for the hydrogen abstraction. In the case of styrene, methyl acrylate, and methyl methacrylate, characteristic ESR patterns of the propagating radicals were observed with polymers which were prepared in the presence of t-BuNO and purified by reprecipitation. Simultaneous trapping of different propagating radicals was attempted in several copolymerization systems. However, this was generally unsuccessful, because of the large difference in reactivities of the propagating radical with t-BuNo.     

Electron spin resonance spin trapping of thiyl,radicals from the decomposition of thionitrites

Alkyl thiyl radicals produced by the homolytic decomposition of thionitrites are detected by ESR spin trapping using 5,5-dimethyl-1-Δ-pyrroline-N-oxide (DMPO).     

Spin trapping studies of poly(methyl methacrylate) degradation in solution

Free radicals produced either by γ or ultrasonic irradiation of poly(methyl methacrylate) (PMMA) in benzene solution were stabilized by spin trapping; they were identified by analysis of ESR spectra of the trapped radicals (the spin adducts). The radical species identified after γ-irradiation were methyl, ester (COOCH₃), a pair of the chain scission radicals, ～CH₂C(CH₃)(COOCH₃) and CH₂C(CH₃)(COOCH₃)～, and phenyl radical originating from the solvent. The chain scission radicals were also detected by spin trapping after ultrasonic irradiation of the benzene solution. Taking account of the difference in the trapping rate for two spin trapping agents, 2,4,6-tri-t-butylnitrosobenzene (BNB) and penta-methyl-nitrosobenzene (PMNB) the radical species trapped by PMNB are assumed to be precursors of those trapped by BNB. Based on the radical species found by the spin trapping method, plausible degradation processes for PMMA in benzene solution are proposed.     

pi-topology and spin alignment utilizing the excited molecular field: observation of the excited high-spin quartet (S = 3/2) and quintet (S = 2) states on purely organic pi-conjugated spin systems.

As a model system for the photoinduced/photoswitched spin alignment in a purely organic pi-conjugated spin system, 9-[4-(4,4,5,5-tetramethyl-1-yloxyimidazolin-2-yl)phenyl]anthracene (1a), 9-[3-(4,4,5,5-tetramethyl-1-yloxyimidazolin-2-yl)phenyl]anthracene (1b), 9,10-bis[4-(4,4,5,5-tetramethyl-1-yloxyimidazolin-2-yl)phenyl]anthracene (2a), and 9,10-bis[3-(4,4,5,5-tetramethyl-1-yloxyimidazolin-2-yl)phenyl]anthracene (2b) were designed and synthesized. In these spin systems, 9-phenylanthracene and 9,10-diphenylanthracene were chosen as photo spin couplers and iminonitroxide was chosen as a dangling stable radical. Time-resolved electron spin resonance (TRESR) spectra of the first excited states with resolved fine-structure splittings were observed for 1a and 2a in an EPA or a 2-MTHF rigid glass matrix. Using the spectral simulation based on the eigenfield method, the observed TRESR spectra for 1a and 2a were unambiguously assigned as an excited quartet (S = 3/2) spin state (Q) and an excited quintet (S = 2) spin state (Qu), respectively. The g value and fine-structure splitting for the quartet state of 1a were determined to be g(Q) = 2.0043, D(Q) = 0.0235 cm(-1), and E(Q) = 0.0 cm(-1). The relative populations (polarization) of each M(S)() sublevel in Q were determined to be P(+1/2') = P(-1/2') = 0.5 and P(+3/2') = P(-3/2') = 0.0 with an increasing order of energy in zero magnetic field. The spin Hamiltonian parameters for Qu are g = 2.0043, D = 0.0130 cm(-1), and E = 0.0 cm(-1), and the relative populations in Qu were determined to be P(0') = 0.30, P(-1') = P(+1') = 0.35 and P(-2') = P(+2') = 0.0. These are the first observations of a photoexcited quartet and a quintet high-spin state in pi-conjugated triplet-radical pair systems. In contrast high-spin excited states were not observed for 1b and 2b, the pi-topological isomers of 1a and 2a, showing the role of pi-topology in the spin alignment of the excited states. Since a weak antiferromagnetic exchange interaction was observed in the ground state of 2a, the clear detection of the excited quintet high-spin state shows that the effective exchange coupling between the two dangling radicals through the diphenylanthracene spin coupler has been changed from antiferromagnetic to ferromagnetic upon photoexcitation. Thus, a photoinduced spin alignment utilizing the excited triplet molecular field was realized for the first time in the purely organic pi-conjugated spin system. Furthermore, the mechanism for the generation of dynamic electron spin polarization was investigated for the observed quartet and quintet states, and a plausible mechanism of the enhanced selective intersystem crossing was proposed. Ab initio molecular orbital calculations based on density functional theory were carried out to determine the electronic structures of the excited high-spin states and to understand the mechanism of the spin alignment utilizing the excited molecular field. The role of the spin delocalization and the spin polarization mechanisms were revealed on the photoexcited state.     

Effect of the phosphoryl substituent in the linear nitrone on the spin trapping of superoxide radical and the stability of the superoxide adduct: combined experimental and theoretical studies

A new phosphorylated linear nitrone N-(4-hydroxybenzyliene)-1-diethoxyphosphoryl-1-methylethylamine N-oxide (4-HOPPN) was synthesized, and its X-ray structure was determined. The spin trapping ability of various kinds of free radicals by 4-HOPPN was evaluated. Kinetic study of decay of the superoxide spin adduct (4-HOPPN-OOH) shows the half-life time of 8.8 min. On the basis of the X-ray structural coordinates, theoretical analyses using density functional theory (DFT) calculations at the B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level were performed on spin-trapping reactions of superoxide radical with 4-HOPPN and PBN and three possible decay routes for their corresponding superoxide adducts. The comparative calculations on the spin-trapping reactions with superoxide radical predicted that both spin traps share an identical reaction type and have comparable potency when spin trapping superoxide radical. Analysis of the optimized geometries of 4-HOPPN-OOH and PBN-OOH reveals that an introduction of the phosphoryl group can efficiently stabilize the spin adduct through the intramolecular H-bonds, the intramolecular nonbonding attractive interactions, as well as the bulky steric protection. Examination of the decomposition thermodynamics of 4-HOPPN-OOH and PBN-OOH further supports the stabilizing role of the phosphoryl group to a linear phosphorylated spin adduct.     

High-performance liquid chromatographic determination of linoleic acid peroxide-derived radicals using electrochemical detection

High-performance liquid chromatography-electrochemical detection (HPLC-ED) was applied to detect 13-hydroperoxide octadecadienoic acid (13-HPODE)-derived radicals such as the pentyl radical and octanoic acid radical. The 13-HPODE-derived radicals were successfully detected using HPLC-ED by the combined use of the spin-trapping technique with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN). The 4-POBN-pentyl radical adduct was detected at the retention time of 18.2 +/- 0.3 min on the elution profile of HPLC-ED with an ODS column (15 cm x 4.6 mm I.D.) using a flow-rate of 1.0 ml/min with 50 mM ammonium acetate in 29% (v/v) aqueous acetonitrile. The 4-POBN-octanoic acid radical adduct was also detected at the retention time of 13.7 +/- 0.7 min using a flow-rate of 1.0 ml/min with 50 mM ammonium acetate in 14% (v/v) aqueous acetonitrile. The concentrations of the 4-POBN radical adducts were determined using HPLC-ED without an internal standard. HPLC-ED is 100 times as sensitive as HPLC-electron spin resonance (ESR) under the ESR and ED conditions employed here. Even 1.8 pmol of the 4-POBN-pentyl (or octanoic acid) radical adduct was detectable using     

Studies on toxicological mechanisms of gas-phase cigarette smoke and protection effects of GTP

Gas-phase cigarette smoke (GPCS) was able to induce lipid peroxidation in lecithin liposomes, rat liver microsomes, and rat lung cells (RLC), and change the membrane fluidity of RLCs. Lipid free radicals were trapped in a GPCS-treated microsomal suspension by using 4-POBN as the spin trap. In addition, it was found that GPCS-peroxidized liposomes in appropriate degree of lipid peroxidation had the ability to increase the generation of superoxide anions in rat peritoneal neutrophils (RPN). Effects of green tea polyphenols (GTP) on the GPCS-induced damages were investigated The results showed that GTP was capable of inhibiting the GPCS-induced damages.     

Preparation and use as spin trapping agents of new ester-nitrones

The synthesis of two new nitrones, N-benzylidene-1,1-bis(ethoxycarbonyl)ethylamine N-oxide (DEEPN) and N-[(1-oxidopyridin-1-ium-4-yl)methylidene]-1-ethoxycarbonyl-1- methylethylamine N-oxide (EPPyON), is described. Measurement of their n-octanol-phosphate buffer partition coefficient permitted evaluation of their lipophilicity. Their capacity to act as spin trapping agents was investigated in aqueous media. Although these nitrones were unsuitable for detecting hydroxyl radical, they efficiently trapped various carbon- and oxygen-centred radicals, including superoxide, in aqueous media. The half-lives of their superoxide adducts were determined at pH 5.8 and 7.2. At neutral pH, the superoxide spin adduct of DEEPN was found to be as persistent as that of 5-diethoxyphosphoryl-5-methyl-3,4-dihydropyrrole N-oxide (DEPMPO). Consequently, DEEPN was believed to be an efficient trap for superoxide detection in aqueous media.     

Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate

Spin trapping using a nitrone and electron paramagnetic resonance (EPR) spectroscopy is commonly employed in the identification of transient radicals in chemical and biological systems. There has also been a growing interest in the pharmacological activity of nitrones, and there is, therefore, a pressing need to develop nitrones with improved spin trapping properties and controlled delivery in cellular systems. The beta-cyclodextrin (beta-CD)-cyclic nitrone conjugate, 5-N-beta-cyclodextrin-carboxamide-5-methyl-1-pyrroline N-oxide (CDNMPO) was synthesized and characterized. 1-D and 2-D NMR show two stereoisomeric forms (i.e., 5S- and 5R-) for CDNMPO. Spin trapping using CDNMPO shows distinctive EPR spectra for superoxide radical anion (O2(*-)) compared to other biologically relevant free radicals. Kinetic analysis of O2(*-) adduct formation and decay using singular value decomposition and pseudoinverse deconvolution methods gave an average bimolecular rate constant of k = 58 +/- 1 M(-1) s(-1) and a maximum half-life of t(1/2) = 27.5 min at pH 7.0. Molecular modeling was used to rationalize the long-range coupling between the nitrone and the beta-CD, as well as the stability of the O2(*-) adducts. This study demonstrates how a computational approach can aid in the design of spin traps with a relatively high rate of reactivity to O2(*-), and how beta-CD can improve adduct stability via intramolecular interaction with the O2(*-) adduct.     

Addition of radicals to quinones—II. ESR spectra of radicals derived from fluoranil

In systems consisting of thermally decomposing benzoyl peroxide in alkylbenzenes, fluoranil behaves as a selective spin trap. As a result of charge transfer (CT) complex formation between alkylbenzenes and fluoranil, the latter traps secondary radicals formed via H-atom abstraction by phenyl radicals from the alkyl group of the aromatic solvents. Weaker CT interaction leads to the trapping of phenyl radicals. The kinetic isotope effect in H-atom abstraction leads to the same result.     

芳基重氮盐及其冠醚络合物光解活泼自由基的ESR研究

本文用自由基捕捉剂2,3,5,6-四甲基亚硝基苯(ND)及苯亚甲基叔丁基氮氧化物(PBN)与ESR相结合的方法研究了芳基重氮盐RC_6H_4N~+_2BF~-_4(R=F, Cl, Br, I, NO_2, COOH,OCH_3及H)及其与18-冠-6、二苯并-24-冠-8的络合物在苯中的光解过程。结果表明它们光解产生了活泼自由基RC_6H_4并可被ND及PBN捕捉....     

Synthesis and spin-trapping behavior of 5-ChEPMPO, a cholesteryl ester analogue of the spin trap DEPMPO

[structure: see text] 5-(Cholesteryloxyethoxyphosphoryl)-5-methylpyrroline N-oxide (5-ChEPMPO), a DEPMPO analogue bearing a cholesterol group on the phosphorus atom, has been prepared and used to trap peroxyl-, alkoxyl-, thiyl-, and carbon-centered radicals in organic solvent. The important steric hindrance in 5-ChEPMPO does not affect the properties of 5-ChEPMPO in comparison to DEPMPO for the spin trapping of an enantiopure linoleic acid hydroperoxide. The 5-ChEPMPO-OOL spin adduct was observed by ESR and confirmed by ESI-MS/MS experiments. The relaxation terms of the 5-ChEPMPO-lipid peroxyl spin adduct were compared with those of other peroxyl spin adducts, and it was shown that the cholesteryl group has only a weak influence on the exchange rate between adduct conformers.     

利用ESR技术研究α-蒎烯,β-蒎烯光敏氧化反应机理

本文主要利用电子顺磁共振(ESR)自旋捕获技术研究9,10 二氰基蒽(DCA)敏化α-蒎烯(αP),β-蒎烯(βP)光氧化反应.提供了在乙腈中α-蒎烯和β-蒎烯的光氧化反应过程中存在超氧负离子基(O₂^-)和单重态氧(¹O₂)的直接证据;在四氯化碳溶剂中只捕获到¹O₂;在正己烷中没有捕获到O₂^-或¹O₂.ESR实验结果进一步证明在乙腈中光敏氧化反应的¹O₂可能来自O₂^-和反应底物α、β-蒎烯正离子自由基之间的电荷复合(CR).     

NO spin trapping and EPR studies on the photochemistry of aliphatic aldehydes

Radicals, such as acyl, hydrated acyl, alkyl and ketyl radicals, from aliphatic aldehyde photochemistry were detected by NO spin trapping and EPR techniques. Deuterium effects on EPR spectra and the generation of radicals by 2-amido-2-propyl radical attack on substrate molecules in aqueous solution via hydrogen-atom abstraction were applied to identify radicals produced photochemically from aldehydes. Aliphatic aldehydes used in the present investigation were formaldehyde, acetaldehyde, acetaldehyde-d4, propionaldehyde, isobutyraldehyde, isopentanal and tert-pentanal. Possible reaction mechanisms are suggested.     

ESR investigation of the oxidative damage in lungs caused by asbestos and air pollution particles

Exposure to asbestos and air pollution particles can be associated with increased human morbidity and mortality. However, the molecular mechanism of lung injuries remains unknown. It has been postulated that the in vivo toxicity results from the catalysis of free radical generation. Using electron spin resonance (ESR) in conjunction with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) we previously investigated in vivo free radical production by rats treated with intratracheal instillation of asbestos (crocidolite fibers) and an emission source air pollution particle (oil fly ash). In this report we compare the effect of two different exposures on the type of free radicals they induce in in vivo animal model. Twenty-four hours after the exposure, ESR spectroscopy of the chloroform extract from lungs of animals exposed to either asbestos or oil fly ash gave a spectrum consistent with a carbon-centered radical adduct (aN = 15.01 G and aH = 2.46 G). To test whether free radical formation occurred in vivo and not in vitro, a number of control experiments were performed. Combinations (both individually and together) of asbestos or oil fly ash and 4-POBN were added to lung homogenate of unexposed rats prior to chloroform extraction. No detectable ESR signal resulted. To exclude the possibility of ex vivo free radical generation, asbestos or oil fly ash was added to lung homogenate of an animal treated with 4-POBN. Also, 4-POBN was added to lung homogenate from rats instilled with asbestos or oil fly ash. Neither system produced radical adducts, indicating that the ESR signal detected in the lung extracts of the treated animals must be produced in vivo and not ex vivo or in vitro. In conclusion, ESR analysis of lung tissue demonstrated that both exposures produce lipid-derived radical metabolites despite their different composition and structure. Analogously, both exposures provide evidence of in vivo enhanced lipid peroxidation. Furthermore, it is concluded that without the presence of a spin-trapping agent, no free radical metabolites could be detected directly by ESR in either exposure.