Evaluation of superoxide anion radicals generated from an aqueous extract of particulate phase cigarette smoke by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide

The reactive oxygen species generated by an aqueous extract of the particulate phase of cigarette smoke were evaluated by an electron spin resonance (ESR) analysis, using spin-trapping agents, and by comparing with model reaction systems. The ESR signals of DMPO-OH were detected from the extract by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). These signals were eliminated by adding superoxide dismutase, but hardly by catalase. These responses of the ESR signals to the scavengers were similar to those of a hypoxanthine-xanthine oxidase system. The results indicate that the signals of DMPO-OH from the extract were derived from a reaction product of DMPO with superoxide anion radicals and clarify the mechanism by which the extract generated superoxide anion radicals.     

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells

Objective and Methods Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO^· production from endothelium has been extensively characterized, little is known about endothelium-derived O^-·₂. In the present study, we determined the O^-·₂ production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy.

Results An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O^-·₂, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O^-·₂ production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O^-·₂ production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO^· did not alter O^-·₂ production. Finally, the amount of O^-·₂ generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.     

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.     

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.     

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.     

The Effects of Myoglobin and Apomyoglobin on the Formation and Stability of the Hydroxyl Radical Adduct of 5,5'-Dimethyl-1-Pyrroline-N-Oxide

When aqueous solutions of the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) are treated with hydrogen peroxide in the presence of either Fe or light, the hydroxyl radical adduct DMPO-OH is formed, with a characteristic 4 line ESR spectrum. When oxy- or metmyoglobin is added to such a system the initial yield and the halife of DMPO-OH are reduced, and at high myoglobin concentrations (about 0.1 mmol dm ^-l3) DMPO-OH becomes undetectable. Using the stable nitroxide 2,2,6,6-tetramethyl-1-piperidinyloxy-N-oxyl (TMPO) for comparison it was found that neither hydrogen peroxide nor myoglobin alone caused a loss of signal, but together a marked loss of signal was induced. From the evidence of these and other experiments it was concluded that the DMPO-OH adduct reacts with hydrogen peroxide and myoglobin to give non-paramagnetic products, and hence that the use of the DMPO spin trap to detect hydroxyl or other active radicals in systems containing physiological concentrations of myoglobin may give misleading results.     

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)     

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).     

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.     

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.     

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.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes

The passive permeation rates of DMPO and DEPMPO spin traps and their hydroxyl radical adducts through liposomal membranes were measured using ESR spectroscopy. For the spin traps, we measured the time-dependent change in the signal intensity of the OH-adduct, which is formed by a reaction between the penetrated spin trap and hydroxyl radicals produced by the UV-radiolysis of H(2)O(2) inside the liposomes. The hydroxyl radicals produced outside the liposomes were quenched with polyethylene glycol. For the OH-adduct, pre-formed adduct was mixed with liposomes and the time-dependent change of the ESR signal was measured in the presence of a line-broadening reagent outside the liposomes to make the signal outside the liposomes invisible. Both the spin traps and their OH-adducts diffused across the lipid membranes rapidly and reached equilibrium within tens of seconds. These findings suggest that if used for the detection of free radicals inside cells, these spin traps should be well distributed in cells and even in organelles.     

Detection of free radicals during the cellular metabolism of adriamycin

Experiments were conducted to determine which free radicals are generated during the metabolism of adriamycin (ADM) by canine tracheal epithelial (CTE) cells, guinea pig enterocytes, and rat hepatocytes. The technique employed in this study was spin trapping; the spin trap utilized was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed during the metabolism of ADM by CTE cells. However, the addition of dimethyl sulfoxide to the in vitro system suggested that superoxide is initially spin trapped by the nitrone, and that the adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) is rapidly bioreduced to afford DMPO-OH. The addition of superoxide dismutase to the system indicated that superoxide generation was primarily intracellular. The adriamycin semiquinone free radical (ADM-SQ) was produced during the metabolism by enterocytes and hepatocytes. The rate of the production of ADM-SQ was enhanced under anaerobic conditions, suggesting that molecular oxygen was responsible for the degradation of this carbon-centered free radical. However, spin trapping of oxygen radicals was not observed; this observation suggests that these reactive intermediates are not produced at concentrations sufficient for detection by spin-trapping experiments.     

Detection of phagocyte-derived free radicals with spin trapping techniques: effect of temperature and cellular metabolism

Human neutrophils activated with either particulate or soluble stimuli generate oxygen-centered free radicals which are detected by spin trapping in conjunction with electron spin resonance (ESR) spectroscopy. We investigated the effect of temperature on ESR spectra resulting from stimulation of human neutrophils with phorbol myristate acetate (PMA) or opsonized zymosan in the presence of the spin trap, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO). At 20 degrees C with either stimuli, neutrophil superoxide production was manifested predominantly as the superoxide spin-trapped adduct, 5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy (DMPO-OOH). In contrast, at 37 degrees C, the hydroxyl spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH) was dominant. No evidence of hydroxyl radical (defined as the methyl spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy, DMPO-CH3) was observed, suggesting that elevated temperatures increased the rate of DMPO-OOH conversion to DMPO-OH. In addition, the elevated temperature activated a neutrophil reductase which accelerated the rate of DMPO-OH reduction to its corresponding hydroxylamine, 2,2-dimethyl-5-hydroxy-1-hydroxypyrrolidine. This bioreduction was dependent upon the presence of both superoxide and a phagocyte-derived factor (possibly a thiol) released into the surrounding media.     

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.     

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells

Objective and Methods Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO^· production from endothelium has been extensively characterized, little is known about endothelium-derived O^-·₂. In the present study, we determined the O^-·₂ production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy.

Results An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O^-·₂, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O^-·₂ production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O^-·₂ production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO^· did not alter O^-·₂ production. Finally, the amount of O^-·₂ generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

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.