Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

Superoxide anion and hydroxyl radical scavenging activities of vegetable extracts measured using electron spin resonance.

Radical scavenging by reconstituted lyophilized powders of water extracts from 16 common vegetables was measured using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals, (.OH) or superoxide anion radicals (O2.-), as DMPO-OH or DMPO-OOH spin adducts. On a dry weight basis, eggplant, and red, yellow and green bell pepper extracts showed potent superoxide anion radical scavenging activities (SOD-like activities). Ascorbate oxidase- or heat-treatments, decreased SOD-like activities in bell pepper extracts suggesting that ascorbate accounts for much of their free radical scavenging activity. Eggplant epidermis extract exhibited the most potent hydroxyl radical scavenging and SOD-like activities. Eggplant SOD-like activity did not decrease after ascorbate oxidase treatment, but decreased following ultrafiltration demonstrating that SOD-like activity is partially due to high molecular weight substances. Nasunin, an anthocyanin in eggplant epidermis, showed markedly potent superoxide anion radical scavenging activity, while it inhibited hydroxyl radical generation probably by chelating ferrous ion.     

Is oxidative stress primarily involved in reperfusion injury of the ischemic heart?

Reperfusion injury of ischemic organs is suggested to result from metabolic derangements initiating an imbalanced formation of free oxygen radicals. Most investigators in this field have used the spin-trap 5,5'-dimethyl-N-pyrroline-N-oxide (DMPO) to stabilize these short-lived radicals and make them visible by means of the electron spin resonance (ESR) technique. ESR signals obtained from intravascular DMPO were reported to indicate the formation of free OH. radicals and, in some cases, also carbon-centered radicals. We were unable to confirm these findings. Carbon-centered radicals were not obtained irrespectively of conditions studied, while oxygen-centered DMPO-adducts could only be detected in minor amounts. Instead, we observed an ascorbyl-related ESR signal. The addition of ethylenediaminetetraacetic acid (EDTA), which was used by many investigators in this field, was found to greatly influence ESR-spectra of the reperfusion fluid. The ascorbyl radical concentration was clearly reduced and the DMPO-OH. adduct became more prominent. The addition of iron further stimulated this change eliciting a Fenton-type reaction responsible for DMPO-OH.-related ESR spectra in the perfusate after ischemia. Accordingly, we observed the release of iron and ascorbic acid into the perfusate as a consequence of ischemia. We could demonstrate that iron in the presence of ascorbate and EDTA causes both types of radicals detected in the perfusate. DMPO-OH. generation in the presence of EDTA was found to result from free OH. radicals that were not generated in the absence of EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydroxyl radical generation by red tide algae.

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-t-butyl-alpha-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O2-) (DMPO-OOH) and that of hydroxyl radical (.OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.     

Conversion of Nitroxide Radicals by Phenolic and Thiol Antioxidants

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyI-piperi-dine-N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.     

Ozone exposure generates free radicals in the blood samples in vitro. Detection by the ESR spin-trapping technique

Generation of free radicals in the reaction of ozone with blood samples and related salt solutions was investigated in vitro by using ESR spin-trapping technique with DMPO. In the reactions of low levels of ozone, a carbon-centered radical was spin-trapped with DMPO, giving rise to the 6-line ESR signal in both whole blood and blood plasma. In the blood plasma, DMPO-spin adduct of hydroxyl radical (DMPO-OH) was detected together with the spin adduct of carbon-centered radical. The present spin-trapping study demonstrates that, when exposed to ozone, 0.9% NaCl solution in the presence of DMPO gives rise to the formation of DMPO-OH. The addition effects of ethanol, which is a ^·OH scavenger, into the NaCl solution reveal that DMPO-OH is produced by the reaction of DMPO with both ^·OH and unidentified oxidants originated from the reaction of Cl^- and ozone. Based on these observations, we consider that ^·OH is generated similarly in the blood plasma exposed to ozone. The ESR study of DMPO-spin adducts in the ozone-exposed aqueous solution of NaOCl indicates that Cl^- reacts with ozone to give ClO^-. Presumably, further oxidation of ClO^- by ozone leads to the formation of ^·OH and the unidentified oxidants.     

Electron spin resonance studies of the reaction of hypochlorite with 5,5-dimethyl-1-pyrroline-N-oxide

The reaction of hypochlorous acid with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was found to yield 5,5-dimethyl-2-pyrrolidone-N-oxyl (DMPOX). In addition to DMPOX, 5,5-dimethyl-2-hydroxypyrrolidine-N-oxyl (DMPO-OH) and an unidentified chlorine-containing radical species were also observed under neutral and near-neutral conditions. Through the use of [17O]HOCl and the hydroxyl radical scavengers ethanol and formate, it was established that DMPO-OH was derived from hydration of DMPO rather than the spin-trapping of hydroxyl radical. Furthermore, kinetic studies and the incorporation of 17O showed that DMPO-OH was readily oxidized to DMPOX and that this reaction was acid and base catalyzed. Under strongly alkaline conditions, DMPOX reversibly formed another species, presumably the enolate, that had a four-line ESR signal identical to that of DMPO-OH. Eventually, carbon-centered adducts appeared whose ESR signals were consistent with the formation of DMPO condensation products.     

Spin-trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production

The spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used to investigate oxy-radical production in post-ischemic rat hearts previously exposed to 20, 30, or 40 minutes of global ischemia. A hydroxyl spin adduct (DMPO-OH) was identified in coronary effluent during the initial seconds of reperfusion by Electron Spin Resonance (ESR) Spectroscopy. The intensity of the ESR signal in post-ischemic effluent increased as ischemic duration was prolonged; however, regardless of the duration of ischemia, maximal spin adduct detection occurred 3 minutes after initiation of reperfusion. Superoxide dismutase inhibited the formation of DMPO-OH, suggesting that superoxide anion was initially generated and is the principle source for the production on the hydroxyl adduct. Our investigations indicate that superoxide anion is produced during the early moments of reperfusion and that its production in the post-ischemic heart is related to the severity of ischemia.     

Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.     

Use of a Low-Frequency Esr Spectrometer: Implications for Spin-Trapping Free Radicals, In Situ

We have adapted the low-frequency ESR spectrometer, designed and built by H.J. Halpern, to the physiologic needs of organ preparations operating at 250 MHz. Initial studies have allowed us to detect nitroxides in an isolated perfused heart. These in siru measurements were made with nitroxides specifically designed to mimic the lipophilic nature of 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) and 2.2-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH). These spin labels provided information about the influence of dynamic factors of the heart, such as flow rate, different cell populations and unequal distribution between compartments on our ability to conduct and interpret spin trapping experiments. They also clarified the sacrifice in sensitivity involved in operating at the lower frequencies. To deal with this later problem. we have increased the sensitivity of the spin trapping method by synthesizing a family of ¹⁵N-and deuterium-containing DMPO analogs and by determining their ability to spin trap free radicals generated by the model superoxide system of xanthinelxanthinc oxidase. Finally, since activated neutrophils are one of the few cells known to generate free radicals as part of their physiologic function, we used these phagocytic cells, as a source of superoxide.     

Toxic Effects and Detection of Oxygen Free Radicals on Cultured Articular Chondrocytes Generated by Menadione

The aim of this work was to study the proliferation pathological perturbations of cultured chondrocytes in response to menadione, an oxygen free radicals producing drug. Rabbit articular chondrocytes in monolayer culture were treated with 10^-5, 1.5.M^-5 and 2.10^-5M of menadione during three days. A dose dependent decrease of the proliferative capacity was observed. Flow cytometry analysis revealed a perturbation of the cell cycle progression consisting in an accumulation of cells in the S and G2 + M phases. This growth perturbation was due to oxygen radicals production since a treatment with catalase suppressed these toxic effects. Furthermore, to identify oxygen derived radicals in the cellular suspension of cultures treated with menadione, we used a technique of spin-trapping coupled with electron spin resonance (ESR). The ESR signal corresponding to the DMPO hydroxyl radical adduct (DMPO-OH) has been detected. The spectra observation indicated the actual production of hydroxyl radical. However, superoxide anions have not been identified; this fact can be explained by the low reactivity of these anions with DMPO and by the decomposition of signal DMPO-OOH to DMPO-OH.     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5.5-dimethyl-I-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However. in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-0; which rapidly decomposes forming DMPO-OH.     

Reaction of Vanadyl with Hydrogen Peroxide. An ESR and Spin Trapping Study

Vanadyl reacts with hydrogen peroxide forming hydroxyl radicals in a Fenton-like reaction. The hydroxyl radicals were spin trapped and identified using 5.5-dimethyl-I-pyrroline-N-oxide (DMPO). The quantity of hydroxyl radicals spin trapped during the reaction between vanadyl and hydrogen peroxide are equal to half of the hydroxyl radicals spin trapped during the reaction between ferrous ions and hydrogen peroxide. Experiments in the presence of formate show that this hydroxyl radical scavenger effectively competes with DMPO preventing the formation of the DMPO-OH adduct. However. in experiments using ethanol as the hydroxyl radical scavenger it was not possible to completely prevent the formation of DMPO-OH. The formation of this additional DMPO-OH in the presence of ethanol does not depend on the concentration of dissolved oxygen, but does depend on the concentration of hydrogen peroxide added to the vanadyl solution. The results suggest that the additional DMPO-OH formed in the presence of ethanol originates from a vanadium (V) intermediate. This intermediate may oxidize DMPO leading to the formation of DMPO-0; which rapidly decomposes forming DMPO-OH.     

Free radicals induced by adriamycin-sensitive and adriamycin-resistant cells: a spin-trapping study

The radicals generated by adriamycin-sensitive (CHO-AB) and adriamycin-resistant (CHO-C5) Chinese hamster ovary cells as well as by adriamycin-sensitive and -resistant human breast cancer cells (MCF7-WT and MCF7-ADR) have been studied with spin-trapping and ESR spectroscopy. During anoxic exposure to adriamycin (ADR) both pairs of cell lines produced the broad ESR singlet characteristic of ADR semiquinone (AQ.). By use of tris(oxalato)chromate (CrOx) as an extracellular line-broadening agent, the distribution of AQ. between the intra- and extracellular compartments was studied. For cell densities of (1-3) X 10(7) cells/mL, CrOx eliminated most, though not all, of the ESR signal, indicating that the AQ. radicals freely diffuse and partition between the intra- and extracellular compartments proportionally to their respective volumes. Similar behavior was exhibited by all four cell lines studied. Upon introduction of oxygen to anoxic cells in the presence of the spin trap 5,5-dimethylpyrroline N-oxide (DMPO), the AQ. signal was replaced by that of the DMPO-OH spin adduct. Metal chelators such as desferrioxamine had no effect on DMPO-OH or AQ. formation. Superoxide dismutase, not catalase, totally eliminated the ESR signal, indicating that DMPO-OH produced by ADR-treated cells originates from superoxide rather than from .OH produced from H2O2. In the presence of CrOx, the DMPO-OH signal was not distinguishable from the background noise, thus excluding any contribution to the signal by intracellular spin adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

A novel method of measuring hydroxyl radical-scavenging activity of antioxidants using γ-irradiation

A new method using ESR spin trapping was proposed for measuring the scavenging activity of antioxidants for the hydroxyl (OH) radical. (-)-Epigallocatechin gallate (EGCg) and 5,5-dimethyl-1-pyrrolline N-oxide (DMPO) were used as the antioxidant and spin trapping agent, respectively. The conventional method using a Fenton reaction had problems associated with the estimation of activity, because the antioxidant disturbs the system for generating OH radical by coordinating on Fe²⁺ and by consuming H₂O₂, besides scavenging the spin adduct (DMPO-OH). Intense γ-irradiation was therefore used to generate OH radicals, and the intensity decrease in DMPO-OH after irradiation was followed to obtain the rate constant for the scavenging of DMPO-OH by EGCg. The intensities were extrapolated to zero time to estimate the quantity of DMPO-OH formed during γ-irradiation. By using these values, the reaction rate constant between OH radical and EGCg was calculated as a ratio to that of DMPO. It was shown that this method is useful for comparing the OH radical-scavenging activity of various antioxidants.     

Irradiation of titanium dioxide generates both singlet oxygen and superoxide anion.

Although photoexcited TiO2 has been known to initiate various chemical reactions, such as the generation of reactive oxygen species, precise mechanism and chemical nature of the generated species remain to be elucidated. The present work demonstrates the generation of singlet oxygen by irradiated TiO2 in ethanol as measured by ESR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) as a 1O2-sensitive trapping agent. Under identical conditions, the superoxide ion was also detected by spin trapping agent 5,5-dimethyl-pyrroline-N-oxide (DMPO). Kinetic analysis in the presence of both 4-oxo-TMP and DMPO revealed that singlet oxygen is produced directly at the irradiated TiO2 surface but not by a successive reaction involving superoxide anion. The basis for this view is the fact that DMPO added in the mixture increased the signals responsible for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), a reaction product of 4-oxo-TMP and 1O2. The detailed mechanism for the generation of 1O2 and superoxide ion by irradiated TiO2 and reactions between these species and DMPO are discussed.     

Hydroxyl radical production by stimulated neutrophils reappraised

Release of active oxygen species during the human neutrophil respiratory burst is thought to be mandatory for effective defense against bacterial infections and may play an important role in damage to host tissues. Part of the critical bacterial and host tissue damage has been attributed to hydroxyl radicals produced from superoxide and hydrogen peroxide. Because of the short life time of the very reactive hydroxyl radical, direct study of hydroxyl radical production is not possible; therefore, indirect detection methods such as electron spin resonance (ESR) coupled with appropriate spin-trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been used. Superoxide production during the oxidative burst has been unambiguously demonstrated. Recent reports claim that hydroxyl radicals are not made during neutrophil stimulation and offer as an explanation the presence of granular components that interfere with hydroxyl radical production. When using the spin-trap agent DMPO, absence of the relatively long-lived adducts DMPO-OH and DMPO-CH3 has been assumed to be prima facie evidence for lack of hydroxyl radical participation. We show that high superoxide flux produced during stimulation of human neutrophils rapidly destroys both DMPO-OH and DMPO-CH3. In accord with previous implications, our results provide an alternative explanation for the absence of .OH adduct in spin-trapping studies and corroborate results obtained using other methods that implicate hydroxyl radical production during neutrophil stimulation.