Trapping of Short-Lived Free Radicals as Nitroxide Radicals Detectable by ESR Spectroscopy. The Radicals Formed in the Reaction between OH-Radicals and Some Sulphoxides and Sulphones.

Lagercrantz, Carl; Forshult, Stig; Lemmich, John; Lindblad, Carl-Gunnar; Lindberg, Alf A.; Jansen, Gert; Lamm, Bo; Samuelsson, Benny

doi:10.3891/acta.chem.scand.23-0811

Cited by 71 publications

(20 citation statements)

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“…The rate constant for reaction of -OH with DMSO (5.8 x 109 M-1 s-1) indicates rapid reaction of the two compounds (16,45). -CH3 + RH -* CH4 + R This reaction is consistent with the observation that -OH attacks dialkyl sulfoxides at sulfur rather than by hydrogen abstraction (46). Furthermore, our mass spectral studies of the CH4 formed by stimulated phagocytes (and by Fe++/EDTA/H202, data not presented) showed that d3-CH4 was produced from d6-DMSO.…”

Section: Discussionsupporting

confidence: 71%

Generation of Hydroxyl Radical by Enzymes, Chemicals, and Human Phagocytes In Vitro

Repine¹,

Eaton²,

Anders³

et al. 1979

J. Clin. Invest.

265

101

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A B S T R A C T Methane (CH4) production from the anti-inflammatory agent, dimethyl sulfoxide (DMSO), was used to measure *OH from chemical reactions or human phagocytes. Reactions producing * OH (xanthine/xanthine oxidase or Fe++/EDTA/H202) generated CH4 from DMSO, whereas reactions yielding primarily 0r or H202 failed to produce CH4. Neutrophils (PMN), monocytes, and alveolar macrophages also produced CH4 from DMSO. Mass spectroscopy using cJ-DMSO showed formnation of d3-CH, indicating that CH4 was derived from DMSO. Methane generation by normal but not chronic granulomatous disease or heat-killed phagocytes increased after stimulation with opsonized zymosan particles or the chemical, phorbol myristate acetate. Methane production from DMSO increased as the number of stimulated PMN was increased and the kinetics of CH4 production approximated other metabolic activities of stimulated PMN. Methane production from stimulated phagocytes and DMSO was markedly decreased by purportedly potent -OH scavengers (thiourea or tryptophane) and diminished to lesser degrees by weaker -OH scavengers (mannitol, ethanol, or Indeed, stimulated neutrophils (PMN), monocytes (MN) or alveolar marcophages (AM) do produce C2H4 from methional or KMB (6-10). However, the strict dependence of C2H4 production upon -OH is in doubt (8,(10)(11)(12)(13)(14). Methional spontaneously forms C2H4, especially during prolonged incubations with tissues (8), and can also react with H202 to form C2H4 (10). The specificity of KMB as a detector of -OH is also in question because C2H4 production from KMB may reflect, at least in part, reactions with 02 or hypochlorous acid (10).In Ca++ and Mg++ (26). Contaminating erythrocytes were lysed by adding 6 ml of ice-cold sterile distilled water and mixing gently for 35 s. Tonicity was rapidly restored with 2 ml of hypertonic (4x) Ca++ and Mg++ free HBSS. The mixture was then centrifuged at 170 g for 10 min. Cells in the pellet were then washed once more, resuspended in HBSS with Ca++ and Mg++, counted, and used immediately. PMN preparations contained >98% PMN with only a few lymphocytes and rare erythrocytes, platelets or MN. Suspensions of MN were similarly prepared. The percentages of MN were determined by differential counting of 600 cells on Wright's stained smears and cytochemically confirmed by esterase stain. MN preparations contained -30% MN, <1% PMN, and the remainder lymphocytes. AM were obtained by bronchoscopic sterile saline lavage of the unaffected subsegments of the lungs of patients undergoing evaluations for localized pulmonary disease or from healthy volunteers (9). AM were recovered from lavage fluids, washed once, and counted. The final preparations contained >90% AM and <2% PMN. More than 85% of the AM excluded trypan blue. Concentrations of lymphocytes or platelets that were comparable to those remaining in phagocyte preparations did not produce significant amounts of CH4 from DMSO.Preparation of pooled human serum, opsonized zymosan, or PMA. Pooled human serum was prepared from clotted ...

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Section: Discussionsupporting

confidence: 71%

Generation of Hydroxyl Radical by Enzymes, Chemicals, and Human Phagocytes In Vitro

Repine¹,

Eaton²,

Anders³

et al. 1979

J. Clin. Invest.

265

101

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“…First, it is known that quinones can be biologically reduced to semiquinones, which then autoxidize yielding superoxide (15). Second, previous studies document the reaction of hydroxyl radical with Me2SO, giving a methyl radical (16)(17)(18). Finally, the EPR scans shown in Fig.…”

Section: Resultsmentioning

confidence: 70%

Detection of superoxide generated by endothelial cells.

Rosen¹,

Freeman²

1984

Proc. Natl. Acad. Sci. U.S.A.

336

138

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Superoxide and lipid free-radical generation in cultured endothelial cells treated with menadione or nitrazepam were measured using electron paramagnetic resonance spectroscopy. Superoxide was detected both intracellularly and extracellularly. Extracellular generation of superoxide and hydrogen peroxide was also measured, either by spectrophotometric measurement of succinoylated cytochrome c reduction or by polarography. Extracellular superoxide was generated due to reduced menadione diffusing across the plasma membrane and reacting with oxygen to generate superoxide in the medium. Increased intracellular oxygen tension favored intracellular oxidation of reduced menadione, thus decreasing diffusion of reduced menadione from the cells and, hence, decreasing extracellular superoxide production. The nitro anion free radical of reduced nitrazepam, which cannot cross the plasma membrane, did not generate detectable extracellular superoxide. Our results show that intracellular superoxide can be spin-trapped using 5,5-dimethyl-1-pyrroline-1-oxide and that secondary free-radical injury to membrane lipids, due to excess production of partially reduced species of oxygen by intact cells, can be detected by spin-trapping lipid free radicals with phenyl N-tert-butylnitrone.In 1968, McCord and Fridovich (1) showed that superoxide could be produced by the catalytic action of xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2). The superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) activity of a copper/zinc-containing protein, erythrocuprein, was subsequently described (2). The biological generation and reactions of superoxide are of considerable interest in studies of cellular metabolism and of the pathogenesis of diverse cytotoxic phenomena (3-6). The most difficult obstacle to the examination of biologically generated free radicals has been the identification and quantitation of these highly reactive species.One method that has been used successfully to identify biologically generated free radicals is spin trapping. This technique consists of using a nitrone or a nitroso compound to "trap" the initial unstable free radical as a "long-lived" nitroxide free radical that can be observed at room temperature using conventional EPR spectrometric techniques. The hyperfine splitting of the spin-trapped adduct provides information that can aid in identification of the original free radical. Because the stable free radical accumulates, spin trapping is an integrative method of measurement and is inherently more sensitive than procedures that measure instantaneous or steady-state levels of free radicals.We report that superoxide can be spin-trapped in suspensions of intact endothelial cells during the uncoupling of cellular reductases by menadione, a quinone capable of undergoing redox cycling, or by the aromatic nitro compound nitrazepam. Additionally, secondary free-radical injury to cell membranes was observed by spin-trapping lipid free radicals. Finally, no evidence was found for direct cel...

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“…43,44 Ethanol, for example, gives three intermediate radicals, *CH 2 -CH 2 -OH, CH 3 -CH*-OH, and CH 3 -CH 2 -O* upon reaction with HO*, which decay to form a multiplicity of products. 22 The unique chemical properties of dimethyl sulfoxide that give it special advantages as a molecular probe for HO* are complemented by its benign biological effects. The median lethal dose (LD50) of intravenous DMSO in animals ranges from about 4 to 10 g/kg body weight, depending on the species.…”

Section: Discussionmentioning

confidence: 99%

“…19 Widespread acceptance of pathogenic mechanisms invoking formation of oxygen radicals has been limited by their inability of investigators to convincingly detect and quantify such radicals in isolated tissues or in animal models. 3,20 This paper describes a new approach to the measurement of HO* radicals in biologic systems by trapping them with DMSO and applying a newly developed and simple colorimetric assay 21 to detect the methane sulfinic acid produced by the trapping reaction, described by Lagercrantz 22 and Cohen, 23 for which the rate constant is k = 7 × 10 9 1/(M-sec). 24 DMSO has the advantages of being a hydroxyl radical trap that distributes rapidly to all body fluid compartments 25 and that can be tolerated in high concentrations in vivo.…”

Section: Introductionmentioning

confidence: 99%

Scatchard analysis of methane sulfinic acid production from dimethyl sulfoxide: A method to quantify hydroxyl radical formation in physiologic systems

Babbs

Griffin

1989

Free Radical Biology and Medicine

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A major impediment to the confirmation of free radical mechanisms in pathogenesis is a lack of direct, chemical evidence that oxygen centered free radicals actually arise in living tissues in quantities sufficient to cause serious damage. This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO*) under physiologic conditions. Reaction of HO* with DMSO produces methane sulfinic acid (MSA) as a primary product, which can be detected by a simple colorimetric assay. To develop a method for estimating total HO* production, we studied two model systems: the superoxide driven Fenton reaction in vitro, using xanthine oxidase as the source of superoxide, and a computer model of Fenton chemistry. Measured MSA production both in vitro and in the computer model was a predictable function of the concentrations of DMSO and competing scavengers of HO*, according to the principle of competition kinetics. Both experimental results and model calculations showed that Scatchard analysis may be used to infer total HO* generation, despite the presence of scavengers other than DMSO, such as mannitol. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO* formation in biologic systems pretreated with DMSO, and Scatchard analysis of repeated experiments with varying DMSO concentrations can yield an estimate of total HO* generation.

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Trapping of Short-Lived Free Radicals as Nitroxide Radicals Detectable by ESR Spectroscopy. The Radicals Formed in the Reaction between OH-Radicals and Some Sulphoxides and Sulphones.

Cited by 71 publications

References 0 publications

Generation of Hydroxyl Radical by Enzymes, Chemicals, and Human Phagocytes In Vitro

Generation of Hydroxyl Radical by Enzymes, Chemicals, and Human Phagocytes In Vitro

Detection of superoxide generated by endothelial cells.

Scatchard analysis of methane sulfinic acid production from dimethyl sulfoxide: A method to quantify hydroxyl radical formation in physiologic systems

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