The Experts below are selected from a list of 999 Experts worldwide ranked by ideXlab platform
Joseph S. Beckman - One of the best experts on this subject based on the ideXlab platform.
-
nitric oxide and peroxynitrite in health and disease
Physiological Reviews, 2007Co-Authors: Pál Pacher, Joseph S. Beckman, Lucas LiaudetAbstract:The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.
-
peroxynitrite induced cytotoxicity in pc12 cells evidence for an apoptotic mechanism differentially modulated by neurotrophic factors
Journal of Neurochemistry, 2002Co-Authors: Alvaro G Estevez, Rafael Radi, Luis Barbeito, Jordan T Shin, John A Thompson, Joseph S. BeckmanAbstract:: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 µM) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.
-
peroxynitrite mediated oxidation of dihydrorhodamine 123
Free Radical Biology and Medicine, 1994Co-Authors: Neil W Kooy, James A Royall, Harry Ischiropoulos, Joseph S. BeckmanAbstract:Nitric oxide reacts with superoxide to form peroxynitrite, which may be an important mediator of free radical-induced cellular injury. Oxidation of dihydrorhodamine to fluorescent rhodamine is a marker of cellular oxidant production. We investigated the mechanisms of peroxynitrite-mediated formation of rhodamine from dihydrorhodamine. Peroxynitrite at low levels (0–1000 nM) induced a linear, concentration-dependent, oxidation of dihydrorhodamine. Hydroxyl radical scavengers mannitol and dimethylsulfoxide had minimal effect (< 10%) on rhodamine production. Peroxynitrite-mediated formation of rhodamine was not dependent on metal ion catalyzed reactions because studies were performed in metal ion-free buffer and rhodamine formation was not enhanced in the presence of Fe3+ ethylenediaminetetraacetic acid (EDTA). Thus, rhodamine formation appears to be mediated directly by peroxynitrite. Superoxide dismutase slightly enhanced rhodamine production. L-cysteine was an efficient inhibitor (KI ∼ 25 μM) of dihydrorhodamine oxidation through competetive oxidation of free sulfhydryls. Urate was also an efficient inhibitor (KI ∼ 2.5 μM), possibly by reduction of an intermediate dihydrorhodamine radical and recycling of dihydrorhodamine. Under anaerobic conditions, nitric oxide did not oxidize dihydrorhodamine and inhibited spontaneous oxidation of dihydrorhodamine. In the presence of oxygen, nitric oxide induces a relatively slow oxidation of dihydrorhodamine due to the formation of nitrogen dioxide. We conclude that dihydrorhodamine is a sensitive and efficient trap for peroxynitrite and may serve as a probe for peroxynitrite production.
-
apparent hydroxyl radical production by peroxynitrite implications for endothelial injury from nitric oxide and superoxide
Proceedings of the National Academy of Sciences of the United States of America, 1990Co-Authors: Joseph S. Beckman, T W Beckman, Jiandong Chen, P A Marshall, Bruce A FreemanAbstract:Abstract Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.
Mark J S Miller - One of the best experts on this subject based on the ideXlab platform.
-
Antioxidant activity of the cruciferous vegetable Maca (Lepidium meyenii)
Food Chemistry, 2002Co-Authors: Manuel Sandoval, Nataly N. Okuhama, Fausto M. Angeles, Vanessa V. Melchor, Luis A. Condezo, Mark J S MillerAbstract:Maca (Lepidium meyenii) is a plant from the Andes of Peru. Maca is used as a food for its nutritional value and ethnomedicinal properties linked to fertility and vitality. The purpose of this study was to evaluate the antioxidant activity of Maca. For all experiments an aqueous extract of Maca was used. The antioxidant activity of Maca was assessed by the inhibition of peroxynitrite, 1,1-diphenyl-2-picrylhydrazyl (DPPH), peroxyls and deoxyribose degradation. The cytoprotection capacity of Maca was assessed using macrophages (RAW 264.7) treated with peroxynitrite or hydrogen peroxide (H2O2). Catechins were quantified by reversedphase HPLC. Addition of Maca extract (0.3–1 mg/ml) to peroxynitrite (300 mM) decreased peroxynitrite concentration by 15 and 41%, respectively (P
-
antioxidant activity of the cruciferous vegetable maca lepidium meyenii
Food Chemistry, 2002Co-Authors: Manuel Sandoval, Nataly N. Okuhama, Fausto M. Angeles, Vanessa V. Melchor, Luis A. Condezo, Mark J S MillerAbstract:Maca (Lepidium meyenii) is a plant from the Andes of Peru. Maca is used as a food for its nutritional value and ethnomedicinal properties linked to fertility and vitality. The purpose of this study was to evaluate the antioxidant activity of Maca. For all experiments an aqueous extract of Maca was used. The antioxidant activity of Maca was assessed by the inhibition of peroxynitrite, 1,1-diphenyl-2-picrylhydrazyl (DPPH), peroxyls and deoxyribose degradation. The cytoprotection capacity of Maca was assessed using macrophages (RAW 264.7) treated with peroxynitrite or hydrogen peroxide (H2O2). Catechins were quantified by reversedphase HPLC. Addition of Maca extract (0.3–1 mg/ml) to peroxynitrite (300 mM) decreased peroxynitrite concentration by 15 and 41%, respectively (P <0.01). The IC50 for scavenging DPPH and peroxyls was 0.61 and 0.43 mg/ml, respectively. Deoxyribose protection by Maca (1–3 mg/ml) against hydroxyl radicals was in the order of 57 and 74%. Maca (1 mg/ml) protected RAW 264.7 cells against peroxynitrite-induced apoptosis (P< 0.01), and increased ATP production in cells treated with H2O2 (1 mM). The concentration of catechins in Maca was lower than in green tea (2.5 mg/g vs 145 mg/g). Collectively, our results indicate that Maca has the capacity to scavenge free radicals and protect cells against oxidative stress. # 2002 Elsevier Science Ltd. All rights reserved.
Rafael Radi - One of the best experts on this subject based on the ideXlab platform.
-
Peroxynitrite decay in the presence of hydrogen peroxide, mannitol and ethanol: a reappraisal.
Free Radical Research, 2009Co-Authors: Beatriz Alvarez, Rafael RadiAbstract:We have reported previously that the apparent rate of peroxynitrite (ONOO-) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331–1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing Peroxynitrate (O2NOO-) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458–3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695–10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxid...
-
Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals.
Free Radical Biology and Medicine, 2004Co-Authors: Silvina Bartesaghi, Lisa K Folkes, Peter Wardman, Madia Trujillo, Ana Denicola, Rafael RadiAbstract:The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3−) and nitrogen dioxide (NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3− and NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 × 109 and 7.6 × 106 M−1 s−1, respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 × 106 M−1 s−1. However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3− and NO2 radicals.
-
peroxynitrite induced cytotoxicity in pc12 cells evidence for an apoptotic mechanism differentially modulated by neurotrophic factors
Journal of Neurochemistry, 2002Co-Authors: Alvaro G Estevez, Rafael Radi, Luis Barbeito, Jordan T Shin, John A Thompson, Joseph S. BeckmanAbstract:: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 mM) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC50 = 850 µM) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.
-
SLOWING OF PEROXYNITRITE DECOMPOSITION IN THE PRESENCE OF MANNITOL AND ETHANOL
Free Radical Biology and Medicine, 1998Co-Authors: Beatriz Alvarez, Gerardo Ferrer-sueta, Rafael RadiAbstract:Abstract The kinetics of peroxynitrite decomposition in the presence of the hydroxyl radical scavengers mannitol and ethanol were studied by stopped-flow spectrophotometry. Mannitol and ethanol decreased the rate of peroxynitrite decomposition in a concentration-dependent manner, following a hyperbolic function. The decreases in peroxynitrite decay rates were observed all throughout the pH range 5.8 to 8.0. In the presence of 100 mM mannitol or ethanol, the first-order rate constant for peroxynitrite decomposition changed from 1.25 ± 0.01 s −1 at 25°C, to values of 0.83 ± 0.01 s −1 and 0.95 ± 0.01 s −1 , respectively. One explanation for this decrease in the rate of peroxynitrite decay with mannitol and ethanol could be a stabilizing effect of the substrate by hydrogen bonding with peroxynitrite, analogous to what has been recently proposed for hydrogen peroxide (Alvarez, B., Denicola, A. and Radi, R. Chem. Res. Toxicol. 8: 859–869; 1995). In this sense, kinetic data fitted a mechanism implying fast equilibria between peroxynitrite anion and peroxynitrous acid with the substrates to form the corresponding complexes. The equilibrium constants of complex dissociation were estimated to be (6.7 ± 0.9) × 10 −3 M and (9.6 ± 1.5) × 10 −3 M for mannitol and ethanol, respectively. When bonded to mannitol or ethanol, peroxynitrous acid could ionize, too, or decompose at a slower rate than in the absence of substrate, in part to a reactive intermediate which performs oxidations. While mannitol and ethanol inhibit oxidation and nitration processes that occur through the reaction of secondary reactive intermediates of peroxynitrite with target molecules, up to 0.5 M mannitol or ethanol failed to inhibit cysteine oxidation by peroxynitrite at pH 7.4 and 25°C. Thus, the formation of stabilizing complexes would not divert the reaction yield of direct, second order reactions such as thiol oxidation, but highlights the importance of hydrogen bonding and solvent effects on peroxynitrite stability.
-
desferrioxamine inhibition of the hydroxyl radical like reactivity of peroxynitrite role of the hydroxamic groups
Free Radical Biology and Medicine, 1995Co-Authors: Ana Denicola, Josem Souza, Reynaldo M Gatti, Ohara Augusto, Rafael RadiAbstract:Abstract Nitric oxide reacts with superoxide to form peroxynitrite, a strong oxidizing species. Peroxynitrite can either directly oxidize molecules such as thiols or protonate to peroxynitrous acid, which can yield an oxidant with a reactivity similar to that of hydroxyl radical in a transition metal-independent mechanism. This oxidative chemistry of peroxynitrite, however, is inhibited by the metal chelator desferrioxamine. Indeed, desferrioxamine, was a potent inhibitor of dimethylsulfoxide, hydrogen peroxide, 5,5-dimethyl-l-pyrroline-N-oxide, and luminol oxidation, whereas the metal chelator diethylenetriaminepentaacetic acid, and ferrioxamine, the iron complex of desferrioxamine, were not. Two other hydroxamates, acetohydroxamate and salicylhydroxamate, were also effective inhibitors. Stopped-flow experiments showed that there is no direct reaction between peroxynitrite anion or cis-peroxynitrous acid with desferrrioxamine. Electron paramagnetic resonance (EPR) studies showed the formation of the desferrioxamine nitroxide radical in incubations containing desferrioxamine, but not ferrioxamine, indicating that the hydroxamic group acts as a one-electron donor to peroxynitrite-derived oxidants. Taken together, our results led us to propose that desferrioxamine can inhibit the oxidative chemistry of peroxynitrite by reaction of the hydroxamic acid moieties with trans-peroxynitrous acid.
Herbert De Groot - One of the best experts on this subject based on the ideXlab platform.
-
The pathobiochemistry of nitrogen dioxide.
Biological Chemistry, 2002Co-Authors: Michael Kirsch, Hansgert Korth, Reiner Sustmann, Herbert De GrootAbstract:Nitrogen dioxide (NO 2 ) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed NO 2 . Peroxynitrite (ONOO-/ONOOH) is believed to be an important endogenous source of NO 2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of NO 2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and β-carotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O 2 _ - )-dependent formation of Peroxynitrate (O 2 NOO - ) and the central role of vitamin C are of special importance.
-
Inhibition of Peroxynitrite‐Induced Nitration of Tyrosine by Glutathione in the Presence of Carbon Dioxide through both Radical Repair and Peroxynitrate Formation
Chemistry: A European Journal, 2001Co-Authors: Michael Kirsch, Hansgert Korth, Reiner Sustmann, Manfred Lehnig, Herbert De GrootAbstract:: Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2*) and trioxocarbonate(1-) (CO3*-) radicals. We have studied the mechanism by which glutathione (GSH) inhibits the NO2*/CO3*--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent tyrosine consumption (IC50 = 660 microM) as well as 3-nitrotyrosine formation (IC50) = 265 microM). From the determination of the level of oxygen produced or consumed under various initial conditions, it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS*) through reduction of dioxygen to superoxide (O2*-) and reaction with NO2* to form Peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effective in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2* radicals is highly unlikely at physiological levels of GSH.
-
inhibition of peroxynitrite induced nitration of tyrosine by glutathione in the presence of carbon dioxide through both radical repair and Peroxynitrate formation
Chemistry: A European Journal, 2001Co-Authors: Michael Kirsch, Hansgert Korth, Reiner Sustmann, Manfred Lehnig, Herbert De GrootAbstract:: Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2*) and trioxocarbonate(1-) (CO3*-) radicals. We have studied the mechanism by which glutathione (GSH) inhibits the NO2*/CO3*--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent tyrosine consumption (IC50 = 660 microM) as well as 3-nitrotyrosine formation (IC50) = 265 microM). From the determination of the level of oxygen produced or consumed under various initial conditions, it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS*) through reduction of dioxygen to superoxide (O2*-) and reaction with NO2* to form Peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effective in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2* radicals is highly unlikely at physiological levels of GSH.
-
hydrogen peroxide formation by reaction of peroxynitrite with hepes and related tertiary amines implications for a general mechanism
Journal of Biological Chemistry, 1998Co-Authors: Michael Kirsch, Elena Lomonosova, Hansgert Korth, Reiner Sustmann, Herbert De GrootAbstract:Abstract Organic amine-based buffer compounds such as HEPES (Good’s buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimineN-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (⋅NO) and superoxide (O⨪2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mmSIN-1 was low (∼15 μm), incubation of SIN-1 with Good’s buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mm SIN-1 with 20 mm HEPES about 190 μm H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mm SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration ofp-hydroxyphenylacetic acid were strongly inhibited by 20 mm HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H2O2. Likewise, authentic peroxynitrite (1 mm) also induced the formation of both O⨪2 and H2O2 upon addition to HEPES (400 mm)-containing solutions in a pH (4.5–7.5)-dependent manner. In accordance with previous reports it was found that at pH ≥5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mm)-induced H2O2 formation (∼80 μm at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mm/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good’s buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate α-amino alkyl radicals with molecular oxygen leads to the formation of O⨪2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H2O2formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.
William A Pryor - One of the best experts on this subject based on the ideXlab platform.
-
BIPHASIC SYNTHESIS OF HIGH CONCENTRATIONS OF PEROXYNITRITE USING WATER-INSOLUBLE ALKYL NITRITE AND HYDROGEN PEROXIDE
Methods in Enzymology, 2004Co-Authors: Rao M. Uppu, William A PryorAbstract:Publisher Summary This chapter discusses the biphasic synthesis of high concentrations of peroxynitrite using water-insoluble alkyl nitrite and hydrogen peroxide. A method that allows the preparation of peroxynitrite solutions up to 1 M is reviewed. These solutions have hydrogen peroxide as an impurity; however this can be reduced to less than about 1 mM after passage through a manganese dioxide column. This synthesis is carried out in a two-phase system based on a displacement reaction by the hydroperoxide anion on isoamyl nitrite. When expressed as a fraction of the concentration of peroxynitrite present, these peroxynitrite solutions contain less nitrite than those prepared by the ozonation of azide, the reaction of nitrite with acidified H 2 O 2 , or the autooxidation of hydroxylamine. This method is a practical alternative to the azide–ozone method in situations in which high concentrations of peroxynitrite are needed and trace levels of azide. Isoamyl alcohol and the unreacted isoamyl nitrite form a separate organic phase, which can be easily removed from the aqueous phase containing peroxynitrite, using a separatory funnel after a brief centrifugation.
-
DIRECT AND SIMULTANEOUS ULTRAVIOLET SECOND-DERIVATIVE SPECTROPHOTOMETRIC DETERMINATION OF NITRITE AND NITRATE IN PREPARATIONS OF PEROXYNITRITE
Methods in Enzymology, 2004Co-Authors: Rachel M. Bolzan, Rafael Cueto, Giuseppe L Squadrito, Rao M. Uppu, William A PryorAbstract:Publisher Summary This chapter discusses the direct and simultaneous ultraviolet second-derivative spectrophotometric determination of nitrite and nitrate in preparations of peroxynitrite. This chapter discusses three methods for synthesizing peroxynitrite that are most commonly used for investigation of the NO 2 - and NO 3 - either present as impurities or produced during peroxynitrite decomposition: (1) ozonation of azide (2) reaction of isoamyl nitrite with hydrogen peroxide, and (3) reaction of hydrogen peroxide with nitrous acid. Commonly used methods for the detection of NO 2 - and NO 3 - involve the reduction of nitrate to nitrite. The initial concentrations of nitrite and nitrate for three different methods of synthesizing peroxynitrite have been determined using an ultraviolet second-derivative spectroscopy method.
-
Distinguishing reactivities of peroxynitrite and hydroxyl radical.
Methods in Enzymology, 2004Co-Authors: Giuseppe L Squadrito, Rao M. Uppu, William A PryorAbstract:Publisher Summary This chapter discusses the distinguishing reactivities of peroxynitrite and hydroxyl radical. The oxidations using peroxynitrite prepared by independent methods afford the same yields of ethylene and methionine sulfoxide from methionine. The presence of metal ion chelators such as ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) does not affect these yields, suggesting that trace amounts of metal ions are not involved in these processes when the peroxynitrite is treated with MnO2 to destroy the hydrogen peroxide, or when a hydrogen peroxide free preparation is selected. Ethylene is formed from both the reactions of methionine with peroxynitrite and the hydroxyl radical. The yield of ethylene from the reaction of methionine with peroxynitrite is not affected by hydroxyl radical scavengers that do not react with ground-state peroxynitrous acid (HOONO). These data suggest that peroxynitrite oxidations involve ground-state HOONO and/or excited-state peroxynitrous acid (HOONO*) but not hydroxyl radicals. The effects of metal ion chelators that either exacerbate or inhibit Fenton-type reactions on oxidations by peroxynitrite are also reviewed in the chapter.
-
Proton affinities of N—O anions and their protonated forms
Journal of Physical Organic Chemistry, 2002Co-Authors: Leo Klasinc, Saša Kazazić, Sean P. Mcglynn, William A PryorAbstract:The proton affinities (PA) of several N-O anions, namely the singly charged anions nitrite, peroxynitrite, nitrate and Peroxynitrate and the doubly charged anions cis- and trans-hyponitrite and oxyhyponitrite, were calculated to be 1413.8, 1440.5, 1348.5, 1414.7, 2065.2, 1987.4 and 1977.9 kJ mol(-1), respectively. The PA of the corresponding conjugate acids were 780.3, 749.8, 746.0, 724.7, 1394.9, 1448.9 and 1340.7 kJ mol(-1) respectively. The PA of cis- and trans-hyponitric and oxyhyponitric acid were 906.7, 723.8 and 728.2 kJ mol(-1) respectively. Comparison with the available experimental values for the acids indicates that the calculated values are good estimates of these important quantities
-
trolox inhibits peroxynitrite mediated oxidative stress and apoptosis in rat thymocytes
Archives of Biochemistry and Biophysics, 1996Co-Authors: Maria G Salgo, William A PryorAbstract:Abstract Peroxynitrite is a strong oxidant that reacts with a variety of biomolecules in vivo and in vitro. When rat thymocytes in phosphate buffer are exposed to 25 μ M peroxynitrite for 10 min, DNA single strand breaks (SSB) can be detected. These SSB are repaired if the cells are incubated in fresh media at 37°C for 120 min. In addition, DNA protein cross-links and apoptosis are observed 1 and 6 h, respectively, after peroxynitrite exposure. Peroxynitrite mediates the formation of thiobarbituric acid-reactive substances (TBARS) that may be responsible for the DNA–protein crosslinks (DPXL). Both TBARS and DPXL formation are lowered by posttreating the cells immediately after the 10-min exposure to peroxynitrite with Trolox, a water-soluble vitamin E analog. These results suggest that oxidative stress mediated by peroxynitrite can trigger a critical sequence of events ending in programmed cell death and that intracellular oxidation is a component of the apoptosis of thymocytes, since both oxidative processes and apoptosis can be prevented by Trolox. In addition to Trolox, we obtained partial data on three other phenolic antioxidants (3- tert -butyl-4-hydroxyanisole, butylated hydroxytoluene, and 2,6-diisopropylphenol). We find that Trolox and these three phenols similarly protect rat thymocytes from apoptosis mediated by peroxynitrite.