Characteristics of molecular hydrogen and CH* radicals in a methane plasma in a magnetically enhanced capacitive RF discharge

The parameters of a methane-containing plasma in an asymmetric RF capacitive discharge in an external magnetic field were studied using optical emission spectroscopy. The power deposited in the discharge was 90 W and the gas pressure and magnetic field were varied in the ranges 1–5 Pa and 50–200 G, respectively. The vibrational and rotational temperatures of hydrogen molecules and CH* radicals were measured as functions of the magnetic field and methane pressure. The ratio between the densities of atomic and molecular hydrogen was estimated. The processes responsible for the excitation of molecular hydrogen and CH* radicals in a methane-containing plasma in an RF capacitive discharge are analyzed.     

Remote delivery of hydroxyl radicals via secondary chemistry of a nonthermal plasma effluent

Electron paramagnetic resonance spectroscopy is used to observe hydroxyl radicals produced by an atmospheric pressure nonthermal plasma device at distances greater than 1 m from the discharge. The plasma device is an indirect treatment setup with closed loop airflow and hydrogen peroxide additives that is effective in deactivating bacteria on time scales of seconds. The generation of the detected hydroxyl radicals is shown to occur in secondary chemical processes near the point of delivery of the plasma treated air stream. The production of hydroxyl radicals is correlated with humidity of the air stream and ability to lyse bacterial membranes. The overall mechanisms of bacteria inactivation are found to be a combinatorial effect of effluent species. The results indicate the feasibility of selective plasma induced free radical delivery for biomedical applications even in the case of short‐lived species like the hydroxyl radical. Biotechnol. Bioeng. 2013; 110: 1936–1944. © 2013 Wiley Periodicals, Inc.     

Free radical activity of natural and heat treated amphibole asbestos

The amphibole minerals amosite and crocidolite were subjected to calcination and to hydrothermal treatment in order to study the effect of these heat treatments on the ability of the minerals to trigger formation of free radicals, which is known to be a main factor causing asbestosis and other asbestos-induced diseases. Free radical activity of the natural and heat treated minerals was studied by using supercoiled DNA (pUC18 plasmid) as a target molecule, and also by means of EPR spectroscopy. It was shown that after calcination of the natural minerals at 1073 K their free radical activity was strongly decreased These results, which may have relevant consequences for asbestos technology, were correlated with concomitant alteration of the structure and surface chemistry of the minerals during calcination.     

In vivo spin trapping of free radicals generated in brain, spleen, and liver during gamma radiation of mice

Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur.     

Direct amination of carboxylic acids with ammonia by electric discharge against aqueous solution

Glow discharge was applied to the aqueous solution containing ammonia and aliphatic carboxylic acids. Several amino acids were synthesized by the direct amination reaction. Simultaneously stepwise oxidation reactions of the substrates and the products were also took place. The time courses of the amination reactions and also the oxidation reactions were studied. These results indicate that the amination and oxidation reactions induced by the electric discharge are initiated by hydroxyl radicals which are produced by the electric discharge through the decomposition of water molecules.     

Application of EPR Spectroscopy to Examination of the Effect of Sterilization Process on Free Radicals in Different Herbs

Free radicals in the original and sterilized caraway, curry, curcuma and cardamom were studied. An X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy was the experimental technique. Effect of microwave power in the range of 2.2–70 mW on amplitudes, linewidths, and lineshape parameters of the EPR spectra was tested. Free radicals concentrations in the non- and sterilized herb samples were compared. The aim of this work was to determine properties and concentration of free radicals in steam sterilized caraway, curry, curcuma and cardamom. It was pointed out that free radicals (~10¹⁸?spin/g) exist in both the original and sterilized herbs. Complex free radical system with oxygen and carbon paramagnetic centers characterizes the examined herbs. Homogeneously dipolar broadened EPR spectra were measured for all the tested herbs. Slow spin-lattice relaxation processes exist in the examined samples. Practical usefulness of EPR method in food technology was discussed.     

低温氧等离子体杀灭铜绿假单胞菌的效果及机理

【目的】本文采用低温氧等离子体,在自行设计的远程等离子体反应装置中,对位于不同放电区域（放电区,余辉区,远程区）的模拟染菌载体聚对苯二甲酸乙二醇酯（PET）表面的铜绿假单胞菌(Pseudomonas aeruginosa)的杀灭效果和机理进行了研究。【方法】采用扫描电子显微镜观察了等离子体处理前后细菌细胞的形貌变化,采用考马斯亮蓝法测定了等离子体处理后细菌蛋白质泄漏量,采用双悬浮探针对氧等子体的电子温度和离子浓度以及电子自旋共振波谱对自由基浓度进行了测定。【结果】强放电区、余辉区和远程区处理30s后的灭菌效果分别为4.2 ,3.8和2.6;扫描电镜观察结果和蛋白泄漏量测定结果证明细菌细胞被损毁,在强放电区是电子、离子、自由基和紫外光子的协同作用,而在余辉区和远程区的灭杀作用主要因自由基所为。【结论】证明该反应装置可有效实现活性粒子的分离,在远程等离子体场中揭示了等离子体灭菌的规律和机理。     

An e.s.r. and spin-trapping study of the reactions of the SO4- radical with protein and nucleic acid constituents.

Reactions of the SO4- radical, generated by U.V. photolysis of Na2S2O8, were studied in aqueous solutions of amino acids, dipeptides, nucleic acid bases, nucleosides and nucleotides. The transient free radicals so formed were spin-trapped by t-nitrosobutane and identified by e.s.r. spectroscopy. The amino acids primarily undergo oxidative decarboxylation. The pKs of the ammonium groups of the spin-trapped decarboxylated radicals of glycine and alanine in D2O were determined to be 8.3 +/- 0.2. An oxidation product, which is the precursor of the decarboxylated radical, is tentatively identified for alanine, valine and isoleucine. Radicals formed by hydrogen abstraction by SO-4 are identified for leucine, serine, phenylalanine and 4-hydroxyproline. In dipeptides, SO-4 produces decarboxylation of the amino acid located at the carboxylate terminal residue. For gly-ala and ala-ala, radicals generated by hydrogen abstraction from the carboxylate terminal residue alanine were also characterized. Radicals centered on the C(5) carbon were observed for uracil, cytosine and thymine. For nucleosides and nucleotides, radicals situated on the base and/or the sugar moiety were assigned.     

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.     

Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor

Ascorbate is readily oxidized in aqueous solution by ascorbate oxidase. Ascorbate radicals are formed, which disproportionate to ascorbate and dehydroascorbic acid. Addition of erythrocytes with increasing intracellular ascorbate concentrations decreased the oxidation of ascorbate in a concentration-dependent manner. Concurrently, it was found, utilizing electron spin resonance spectroscopy, that extracellular ascorbate radical levels were decreased. Control experiments showed that these results could not be explained by leakage of ascorbate from the cells, inactivation of ascorbate oxidase, or oxygen depletion. Thus, this means that intracellular ascorbate is directly responsible for the decreased oxidation of extracellular ascorbate. Exposure of ascorbate-loaded erythrocytes to higher levels of extracellular ascorbate radicals resulted in the detection of intracellular ascorbate radicals. Moreover, efflux of dehydroascorbic acid was observed under these conditions. These data confirm the view that intracellular ascorbate donates electrons to extracellular ascorbate free radical via a plasma membrane redox system. Such a redox system enables the cells to effectively counteract oxidative processes and thereby prevent depletion of extracellular ascorbate.     

Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes

Exposure of red blood cells to oxygen radicals can induce hemoglobin damage and stimulate protein degradation, lipid peroxidation, and hemolysis. To determine if these events are linked, rabbit erythrocytes were incubated at 37 degrees C with various oxygen radical-generating systems and antioxidants. Protein degradation, measured by the production of free alanine, increased more than 11-fold in response to xanthine (X) + xanthine oxidase (XO). A similar increase in proteolysis occurred when the cells were incubated with acetaldehyde plus XO, with ascorbic acid plus iron (Asc + Fe), or with hydrogen peroxide (H2O2) alone. Upon addition of XO, increased proteolysis was evident within 5 min and was linear for up to 5 h. In contrast, lipid peroxidation, as shown by the production of malonyldialdehyde, conjugated dienes, or lipid hydroperoxides was observed only after 2 h of incubation with X + XO, acetaldehyde + XO, or H2O2. Ascorbate plus Fe2+ induced both protein degradation and lipid peroxidation; however, the addition of various antioxidants (urate, xanthine, glucose, or butylated hydroxytoluene) decreased lipid peroxidation without affecting proteolysis. Thus, these processes seem to occur by distinct mechanisms. Furthermore, at low concentrations of XO, protein degradation was clearly increased in the absence of detectable lipid peroxidation products. Hemolysis occurred only in a small number of cells (9%) and followed the appearance of lipid peroxidation products. Thus, an important response of red cells to oxygen radicals is rapid degradation of damaged cell proteins. Increased proteolysis seems to occur independently of membrane damage and to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation.     

Reduction of Antitumour Mitosenes in Non-Aqueous and Aqueous Environment. An Electron Spin Resonance and Cyclic Voltammetry Study

Chemical reduction of mitosenes under aerobic conditions in DMSO showed characteristic ESR signals of the mitosene derived semiquinone free radicals. However, these signals diminished strongly upon addition of water to the reaction mixture, indicating a short lifetime of the mitosene semiquinone free radicals under aqueous conditions. In addition, enzymatic one-electron reduction of these mitosenes with either xanthine oxidase or purified NADPH cytochrome P450 reductase under anaerobic conditions showed no signals of the mitosene semiquinone free radicals. Subsequent cyclic voltammetry measurements demonstrated facilitation of the further one-electron reduction of the mitosene semiquinone free radicals in the presence of water in comparison with non-aqueous conditions. The present results strongly suggest that in the presence of water relatively stable hydroquinones are formed upon reduction of mitosenes. Consequently, the steady state concentrations of mitosene semiquinone free radicals will be lowered substantially in aqueous environment. Thus under physiological conditions, two-electron reduction and formation of the mitosene hydroquinone might be important in processes leading to DNA alkylation by these mitosenes.     

Free radicals and neuronal cell death

Production of oxygen free radicals is a natural consequence of aerobic metabolism and they are constantly generated in vivo by chemical reactions and metabolic processes. Antioxidant defence systems scavenge and minimise the formation of oxygen-radical-derived biochemical products, however, these defences are not completely effective even under normal physiological conditions. In pathologic situations, oxygen free radicals can be generated in excess of a cell's antioxidant capacity resulting in severe damage to cellular constituents including proteins, DNA and lipids. The inherent biochemical and physiological charateristics of the brain, including high lipid concentrations and energy requirements, make it particularly susceptible to free radical mediated insult. Increasing evidence indicates that many neurological disorders may have components of free radical and oxidative stress induce injury.     

Application of electron spin resonance for evaluation of the level of free radicals in the myometrium in full-term pregnancy with normal labour and uterine inertia

In order to identify and quantify free radicals in the tissues of patients with normal physiological and pathological states of births, we developed a method to evaluate the amount of free radicals in myometrium of subplacental area and from body of uterus, using electron spin resonance spectroscopy. Analysis of the concentration of free radicals in the myometrium in full-term pregnancy with normal labour and during uterine inertia was studied. The activities of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase in samples of these tissues were tested too. Low free radical concentrations in these tissues were associated with disturbances in contractile activity of myometrium along with reduction of Ca2+-ATPase, cytochrome c oxidase and succinate dehydrogenase activity. There proved to be an association between the level of free radicals in the tissues and alteration in the physiological processes.     

Natural-abundance 13C nuclear magnetic resonance studies of regulation and overproduction of L-lysine by Brevibacterium flavum

Natural-abundance 13C NMR spectroscopy has been used to study the metabolism of the L-lysine-producing bacterium, Brevibacterium flavum. Relationships of biomass formation, precursor uptake, and product excretion, as a function of culture medium, oxygen supply and specific cell membrane permeability, were rapidly determined using 67.89-MHz 13C NMR. The induction of lysine production throughout the growth cycle was studied. Intracellular and extracellular levels of free metabolites and unconsumed precursor were quantitatively measured as a function of growth culture conditions. Limited availability of oxygen resulted in accumulation and excretion of unfavorable products: lactate, succinate, alanine and valine. However, under optimal aeration conditions L-lysine was the sole metabolite detected extracellularly. Various important long-lived intermediates and storage compounds were detected in the intact cells (by NMR measurements). Carbon resonances of carbohydrates and amino acids were resolved and easily identified. Of particular interest are those of trehalose carbons, a storage carbohydrate. Natural-abundance 13C NMR spectroscopy seems most suitable for biotechnological processes where high concentrations of intermediates and end-products can be observed. We anticipate that this approach will be employed to screen overproducing bacterial strains.     

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism

The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.     

Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing and oxidising radicals

The human skin and eye melanin is commonly viewed as an efficient photoprotective agent. To elucidate the molecular mechanism of the melanin-dependent photoprotection, we studied the interaction of two synthetic melanins, dopa-melanin and cysteinyldopa-melanin, with a wide range of oxidising and reducing free radicals using the pulse radiolysis technique. We have found that although both types of free radicals could efficiently interact with the synthetic melanins, their radical scavenging properties depended, in a complex way, on the redox potential, the electric charge and the lifetime of the radicals. Repetitive pulsing experiments, in which the free radicals, probing the polymer redox sites, were generated from four different viologens, indicated that the eumelanin model had more reduced than oxidised groups accessible to reaction with the radicals. Although with many radicals studied, melanin interacted via simple one-electron transfer processes, the reaction of both melanins with the strongly oxidising peroxyl radical from carbon tetrachloride, involved radical addition. Our study suggests that the free radical scavenging properties of melanin may be important in the protection of melanotic cells against free radical damage, particularly if the reactive radicals are generated in close proximity to the pigment granules.     

Pulse radiolysis study of the interaction of retinoids with peroxyl radicals

Vitamin A (retinol) and its derivatives-retinal and retinoic acid-are known for their ability to inhibit lipid peroxidation. Antioxidant actions of retinoids have been attributed to chain-breaking by scavenging of peroxyl radicals. Based on chemical analysis of retinoic acid degradation products formed during microsomal lipid peroxidation, it was previously suggested that retinoids interact with peroxyl radicals forming free carbon-centered radical adducts. However, it can be argued that such a mode of antioxidant action of retinoids is not sufficient to fully explain their effectiveness at inhibiting lipid peroxidation, which in many systems is comparable to, or even exceeds, that of alpha-tocopherol. In order to elucidate the mechanism of interaction of retinoids with peroxyl radicals, (trichloromethyl)peroxyl radical was generated by pulse radiolysis, and its interactions with retinoids solubilized in Triton X-100 micelles were followed by kinetic absorption spectroscopy. All retinoids--retinol, retinal, and retinoic acid--interacted with the peroxyl radical, and at least two transient products were detected. One of these products, absorbing at 590 nm, was identified as retinoid cation radical. Therefore, we postulate that, apart from formation of radical adducts, retinoids may also scavenge peroxyl radicals by electron transfer.     

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

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

Lipid Free Radical Generation and Brain Cell Membrane Alteration Following Nitric Oxide Synthase Inhibition During Cerebral Hypoxia in the Newborn Piglet

Abstract: Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N ^ω-nitro- l -arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO₂ and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na⁺,K⁺-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.