ascorbic-acid and peroxynitric-acid

Atrial fibrillation (AF), the most common chronic arrhythmia, increases the risk of stroke and is an independent predictor of mortality. Available pharmacological treatments have limited efficacy. Once initiated, AF tends to self-perpetuate, owing in part to electrophysiological remodeling in the atria; however, the fundamental mechanisms underlying this process are still unclear. We have recently demonstrated that chronic human AF is associated with increased atrial oxidative stress and peroxynitrite formation; we have now tested the hypothesis that these events participate in both pacing-induced atrial electrophysiological remodeling and in the occurrence of AF following cardiac surgery. In chronically instrumented dogs, we found that rapid (400 min(-1)) atrial pacing was associated with attenuation of the atrial effective refractory period (ERP). Treatment with ascorbate, an antioxidant and peroxynitrite decomposition catalyst, did not directly modify the ERP, but attenuated the pacing-induced atrial ERP shortening following 24 to 48 hours of pacing. Biochemical studies revealed that pacing was associated with decreased tissue ascorbate levels and increased protein nitration (a biomarker of peroxynitrite formation). Oral ascorbate supplementation attenuated both of these changes. To evaluate the clinical significance of these observations, supplemental ascorbate was given to 43 patients before, and for 5 days following, cardiac bypass graft surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared with 34.9% in control subjects. In combination, these studies suggest that oxidative stress underlies early atrial electrophysiological remodeling and offer novel insight into the etiology and potential treatment of an enigmatic and difficult to control arrhythmia. The full text of this article is available at http://www.circresaha.org.

Topics: Aged; Animals; Antioxidants; Ascorbic Acid; Atrial Fibrillation; Cardiac Pacing, Artificial; Coronary Artery Bypass; Dogs; Electrophysiology; Female; Heart Atria; Humans; Male; Middle Aged; Multivariate Analysis; Nitrates; Time Factors; Treatment Outcome; Tyrosine

Peroxynitric acid (O2NOOH) nitrates L-tyrosine and related compounds at pH 2-5. During reaction with O2(15)NOOH in the probe of a 15N NMR spectrometer, the NMR signals of the nitration products of L-tyrosine, N-acetyl-L-tyrosine, 4-fluorophenol and 4-methoxyphenylacetic acid appear in emission indicating a nitration via free radicals. Nuclear polarizations are built up in radical pairs [15NO2* , PhO*]F or [15NO2* , ArH*+]F formed by diffusive encounters of 15NO2 with phenoxyl-type radicals PhO or with aromatic radical cations ArH*+. Quantitative 15N CIDNP investigations with N-acetyl-L-tyrosine and 4-fluorophenol show that the radical-dependent nitration is the only reaction pathway. During the nitration reaction, the 15N NMR signal of 15NO3- also appears in emission. This is explained by singlet-triplet transitions in radical pairs [15NO2* , 15NO3*]S generated by electron transfer between O2(15)NOOH and H15NO2 formed as a reaction intermediate. During reaction of peroxynitric acid with ascorbic acid, 15N CIDNP is again observed in the 15N NMR signal of 15NO3- showing that ascorbic acid is oxidized by free radicals. In contrast to this, O2(15)NOOH reacts with glutathione and cysteine without the appearance of 15N CIDNP, indicating a direct oxidation without participation of free radicals.

Topics: Ascorbic Acid; Cysteine; Glutathione; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Nitrates; Nitrogen; Nitrogen Isotopes; Phenols; Solvents; Temperature; Tyrosine

With the immersion of corn into dilute sulfur oxide during starch-manufacturing processes, corn steep liquor (CSL) remains as leftover material. CSL is often used for fermentation, but its components are not fully understood. To determine the properties of CSL, 12 p-coumaric acid-related compounds were isolated from an ethyl acetate extract of CSL with the guidance of antioxidative activity on the rabbit erythrocyte membrane ghost system. The activity of these compounds was compared against oxidative damages, and it was elucidated that the activity of p-coumaric acid derivatives was mainly affected by their functional groups at the 3-position and less by the conjugated side chain. Moreover, p-coumaric acid derivatives exhibited inhibitory activity stronger than that of tocopherols and ascorbic acid on peroxynitrite-mediated lipoprotein nitration. These findings that p-coumaric acid derivatives, which might play a beneficial role against oxidative damage, exist in CSL suggest this byproduct might be a useful resource of phenolic antioxidants.

Topics: Acetates; Animals; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Coumaric Acids; Erythrocyte Membrane; Fermentation; Lipoproteins; Magnetic Resonance Spectroscopy; Nitrates; Phenols; Rabbits; Tyrosine; Vitamin E; Zea mays

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1-15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca(2+)-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca(2+) changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H2O2-induced changes in Ca(2+) homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.

Topics: Ascorbic Acid; Calcium; Catalase; Chelating Agents; Comet Assay; Data Interpretation, Statistical; DNA Damage; DNA, Single-Stranded; Dose-Response Relationship, Drug; Egtazic Acid; Flow Cytometry; Humans; Kinetics; Lymphocytes; Microscopy, Ultraviolet; Molsidomine; Nitrates; Nitric Oxide Donors; Superoxide Dismutase

Using EPR spectroscopy, we show that the water-soluble mononitrosyl iron complexes with N-methyl-D-glucamine dithiocarbamate (MNIC-MGD) ligands can easily react with superoxide and with peroxynitrite. The reaction with superoxide transforms the paramagnetic MNIC-MGD complex into an EPR silent complex with a reaction rate of 3 x 10(7) (M.s)(-1). Suppletion of ascorbate partially restores the complexes to their original paramagnetic state. We propose that the reaction of MNIC-MGD with either superoxide or peroxynitrite leads to identical EPR silent complexes. Our results have important implications for the technique of NO trapping in biosystems with Fe-dithiocarbamate complexes, where mononitrosyl-iron complexes (hydrophilic as well as hydrophobic) are formed as adducts in the trapping reaction. This principle is illustrated by NO trapping experiments on viable cultured endothelial cells. We find that MNIC-MGD acts as a very potent and water-soluble antioxidant with an efficiency exceeding most SOD mimics. Moreover, by accounting for the EPR silent fraction of iron complexes, the sensitivity of NO trapping can be enhanced considerably. The method was demonstrated for hydrophobic iron-dithiocarbamate complexes in endothelial cell cultures, where sensitivity for NO detection was enhanced by a factor of 5.

Topics: Air; Antioxidants; Ascorbic Acid; Cell Line; Cerium; Dimerization; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Ferrous Compounds; Kinetics; Ligands; Nitrates; Nitric Oxide; Oxidants; Sorbitol; Spin Labels; Spin Trapping; Superoxide Dismutase; Superoxides; Thiocarbamates

A well-established protocol to increase the intracellular content of ascorbic acid was used to investigate the effects of the vitamin on DNA single-strand breakage and toxicity mediated by authentic peroxynitrite (ONOO(-)) in U937 cells. This protocol involved exposure for 60 min to 100 microM dehydroascorbic acid, which was taken up by the cells and converted into ascorbic acid via a GSH-independent mechanism. At the time of exposure to ONOO(-), which was performed in fresh saline immediately after loading with dehydroascorbic acid, the vitamin present in the cells was all in its reduced form. It was found that, in cells that are otherwise ascorbate-deficient, an increase in their ascorbic acid content does not prevent, but rather enhances, the DNA-damaging and lethal responses mediated by exogenous ONOO(-). These results therefore suggest that acute supplementation of ascorbic acid can be detrimental for individuals with pathologies associated with a decrease in ascorbic acid and in which ONOO(-) is known to promote deleterious effects.

Topics: Ascorbic Acid; Dehydroascorbic Acid; DNA Damage; Ferricyanides; Free Radical Scavengers; Humans; Iron Chelating Agents; Nitrates; Oxidation-Reduction; U937 Cells

Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2).

Topics: Animals; Antioxidants; Ascorbic Acid; Cattle; Free Radicals; NAD; Nitrates; Oxidation-Reduction; Rhodamines

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.

Topics: Ascorbic Acid; Carbon Dioxide; Dipeptides; Erythrocytes; Free Radical Scavengers; Globins; Humans; Immunoelectrophoresis; Mass Spectrometry; Nitrates; Oxyhemoglobins; Tyrosine

Peroxynitrite, a reactive cytotoxic species generated by the reaction of superoxide with nitric oxide, rapidly oxidizes phenylaminoethyl selenide (PAESe) and its para-substituted derivatives with second-order rate constants ranging from 900 to 3000 M(-1) s(-1) at neutral pH (pH 7.0) and 25 degrees C. These values are approximately 3 x 10(4) times greater than the corresponding rate constants for the reactions of selenides with hydrogen peroxide. The peroxynitrite reaction was also studied at alkaline pH. HPLC analysis confirms that both the peroxynitrite and hydrogen peroxide reactions produced the corresponding phenylaminoethyl selenoxide (PAESeO) as the sole selenium-containing product, with a stoichiometry of 1 mol of PAESe oxidized per 1 mol of PAESeO formed per 1 mol of oxidant reacted. The influence of para-substituents on the rate constants was investigated using Hammett plots; in both cases the data are consistent with an S(N)2-type mechanism, wherein the selenium atom acts as the nucleophile. Our results provide further evidence that organoselenium compounds may play a protective role in the defense against the many reactive oxidizing species produced in cellular metabolism.

Topics: Antihypertensive Agents; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Ethylamines; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Molecular Structure; Nitrates; Organoselenium Compounds; Oxidants; Oxidation-Reduction; Oxides; Oxidoreductases; Phenethylamines; Reactive Oxygen Species; Sulfur Compounds; Thermodynamics

Superoxide radical (O2-) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M(-1) s(-1)) of ONOO- with NADH (233 +/- 27), ubiquinol-0 (485 +/- 54) and GSH (183 +/- 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2-) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants.

Topics: Animals; Ascorbic Acid; Blotting, Western; Carbon Dioxide; Glutathione; Inhibitory Concentration 50; Kinetics; Mice; Mitochondria, Liver; NAD; Nitrates; Oxidation-Reduction; Spectrometry, Fluorescence; Superoxides; Tyrosine; Ubiquinone

To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe2+/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.. At 10 microM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe2+/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 microM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Ascorbic Acid; Chlorine; Diclofenac; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; In Vitro Techniques; Indicators and Reagents; Indomethacin; Iron; Kinetics; Lipid Peroxidation; Luminescent Measurements; Neutrophil Activation; Neutrophils; Nitrates; Reactive Oxygen Species; Sodium Hypochlorite; Spectrophotometry, Ultraviolet; Sulfonamides; Tetradecanoylphorbol Acetate

We have extended the application of our previously reported total oxidant scavenging capacity (TOSC) assay (Winston et al., Free Radical Biol. Med. 24, 480-493, 1998) to permit facile quantification of the absorbance capacity of antioxidants toward three potent oxidants, i.e., hydroxyl radicals, peroxyl radicals, and peroxynitrite. Respectively, these oxidants were generated by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (ABAP), and 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Each of these oxidants reacts with alpha-keto-gamma-methiolbutyric acid (KMBA), which is oxidized and yields ethylene. The antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene formation relative to a control reaction. Assay conditions were established in which control reactions give comparable yields of ethylene with each of the oxidants studied. Thus, the relative efficiency of various antioxidants could be compared under conditions of quantitatively similar KMBA oxidizing capability by the three oxidants. Reduced glutathione was an efficient scavenger of peroxyl radicals, but scavenged peroxynitrite and hydroxyl radicals relatively poorly. Uric acid, Trolox, and ascorbic acid were comparable scavengers of peroxynitrite and peroxyl radicals. Uric acid and Trolox were approximately an order of magnitude less efficient as scavengers of hydroxyl radicals. The classical hydroxyl radical scavenging agents mannitol, dimethyl sulfoxide, and benzoic acid had much higher TOSC values with hydroxyl than with peroxyl radicals or peroxynitrite. The very different chemical reactivity toward KMBA by the SIN-1- and iron-ascorbate-generated oxidants indicates that hydroxyl radical is not a major oxidant produced by the SIN-1 system. The data show that the TOSC assay is useful and robust in distinguishing the reactivity of various oxidants and the relative capacities of antioxidants to scavenge these oxidants.

Topics: Amidines; Antioxidants; Ascorbic Acid; Chromatography, Gas; Free Radical Scavengers; Free Radicals; Hydroxyl Radical; Methionine; Molsidomine; Nitrates; Nitric Oxide Donors; Oxidants; Oxidation-Reduction; Peroxides

Three isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin (MnTMPyP) were evaluated for their reaction with peroxynitrite. The Mn(III) complexes reacted with peroxynitrite anion with rate constants of 1.85 x 10(7), 3.82 x 10(6), and 4.33 x 10(6) M(-1) s(-1) at 37 degrees C for MnTM-2-PyP, MnTM-3-PyP, and MnTM-4-PyP, respectively, to yield the corresponding oxo-Mn(IV) complexes. Throughout the pH range from 5 to 8.5, MnTM-2-PyP reacted 5-fold faster than the other two isomers. The oxo-Mn(IV) complexes could in turn be reduced by glutathione, ascorbate, urate, or oxidize tyrosine. The rate constants for the reduction of the oxo-Mn(IV) complexes ranged from >10(7) M(-1) s(-1) for ascorbate to 10(3)-10(4) M(-1) s(-1) for tyrosine and glutathione. Cyclic voltammetry experiments show that there is no significant difference in the E1/2 of the Mn(IV)/Mn(III) couple; thus, the differential reactivity of the three isomeric complexes is interpreted in terms of electrostatic and steric effects. Micromolar concentrations of MnTM-2-PyP compete well with millimolar CO2 at reacting with ONOO-, and it can even scavenge a fraction of the ONOOCO2- that is formed. By being rapidly oxidized by ONOO- and ONOOCO2- and reduced by antioxidants such as ascorbate, urate, and glutathione, these manganese porphyrins, and especially MnTM-2-PyP, can redirect the oxidative potential of peroxynitrite toward natural antioxidants, thus protecting more critical targets such as proteins and nucleic acids.

Topics: Ascorbic Acid; Catalysis; Free Radical Scavengers; Glutathione; Hydrogen-Ion Concentration; Isomerism; Kinetics; Metalloporphyrins; Nitrates; Oxidants; Oxidation-Reduction; Porphyrins; Reducing Agents; Tyrosine; Uric Acid

Twelve substituted metalloporphyrins (MPs), some of which have been previously characterized with respect to superoxide dismutase and peroxynitrite decomposing activities, were evaluated for their ability to scavenge peroxynitrite in vitro at 37 degrees C. Because the overall effectiveness of MPs as catalytic peroxynitrite scavengers is a function of (1) how fast they react with peroxynitrite, (2) how fast they cycle back to the starting compound, and (3) how well they contain or quench the reactive intermediates generated, all of these properties were evaluated and compared directly under the same conditions. Of the various MPs tested, only the iron and manganese porphyrins showed significant reactivity with peroxynitrite. The Mn(IV) intermediates resulting from oxidation by peroxynitrite were relatively stable and rereduction to the Mn(III) forms was rate-limiting to catalytic decomposition of peroxynitrite. However, in the presence of oxidizeable substrates like phenolics, rereduction of Mn(IV) forms occurred very rapidly and both the Mn- and Fe-porphyrins catalyzed nitration and oxidation by peroxynitrite. Mn- and Fe-porphyrins enhanced the yield of nitrated phenolics by peroxynitrite as much as 5-fold at pH 7.4 and up to 12-fold at pH 9. 1, while total oxidative yield was more than doubled. Nitration enhancement by MPs was effectively inhibited by ascorbate, glutathione, or serum, although much higher concentrations of ascorbate were required to inhibit nitration catalyzed by either Mn or Fe tetramethylpyridyl porphyrin. Catalysis of peroxynitrite nitration by MPs appears to proceed via a radical-mediated reaction mechanism whereby the phenolic substrate rapidly reduces Mn(IV) = O or Fe[IV] = O to the +3 state to yield phenoxyl radical which then combines with the other primary product, nitrogen dioxide. Based on the rate constants and the proposed reaction mechanism, it is reasonable to suggest that Mn and Fe porphyrins could detoxify peroxynitrite in vivo by efficiently trapping the relatively unreactive peroxynitrite anion and, in effect, channeling it into a single reaction pathway which could then be more effectively scavenged by cellular reductants like ascorbate.

Topics: Ascorbic Acid; Catalysis; Free Radical Scavengers; Iron; Kinetics; Manganese; Metalloporphyrins; Models, Chemical; Molecular Conformation; Molecular Structure; Nitrates; Oxidants

Recent studies have shown that peroxynitrite oxidizes thiol groups through competing one- and two-electron pathways. The two-electron pathway is mediated by the peroxynitrite anion and prevails quantitatively over the one-electron pathway, which is mediated by peroxynitrous acid or a reactive species derived from it. In CO2-containing fluids the oxidation of thiols might follow a different mechanism owing to the rapid formation of a different oxidant, the nitrosoperoxycarbonate anion (ONOOCO2(-)). Here we present evidence that in blood plasma peroxynitrite induces the formation of a disulphide cross-linked protein identified by immunological (anti-albumin antibodies) and biochemical criteria (peptide mapping) as a dimer of serum albumin. The albumin dimer did not form in plasma devoid of CO2 and its formation was enhanced by ascorbate. However, analysis of thiol groups showed that reconstituting dialysed plasma with NaHCO3 protected protein thiols against the oxidation mediated by peroxynitrite and that the simultaneouspresence of ascorbate provided further protection. Ascorbate alone did not protect thiol groups from peroxynitrite-mediated oxidation. ESR spin-trapping studies with N-t-butyl-alpha-phenylnitrone (PBN) revealed that peroxynitrite induced the formation of protein thiyl radicals and their intensity was markedly decreased by plasma dialysis and restored by reconstitution with NaHCO3. PBN completely inhibited the formation of albumin dimer. Moreover, the addition of iron-diethyldithiocarbamate to plasma demonstrated that peroxynitrite induced the formation of protein S-nitrosothiols and/or S-nitrothiols. Our results are consistent with the hypothesis that NaHCO3 favours the one-electron oxidation of thiols by peroxynitrite with formation of thiyl radicals, ;NO2, and RSNOx. Thiyl radicals, in turn, are involved in chain reactions by which thiols are oxidized to disulphides.

Topics: Ascorbic Acid; Blood Proteins; Chromatography, Gel; Chromatography, High Pressure Liquid; Dimerization; Electron Spin Resonance Spectroscopy; Electrons; Free Radicals; Humans; Nitrates; Oxidation-Reduction; Serum Albumin; Sodium Bicarbonate; Sulfhydryl Compounds

Apoptosis induced by peroxynitrite in cultured cerebellar granule cells was confirmed morphologically by chromatin condensation and biochemically by DNA laddering. A 30 min exposure to peroxynitrite (10 microM) initiated oxidative stress, which caused the formation of thiobarbituric acid-reactive substances (TBARS) and the alteration of cell membrane fluidity. Peroxynitrite treatment also caused ATP decrease and thus activated the apoptotic program. Pre-treating cells with antioxidant EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H -1- benzopyran-6-yl-hydrogen phosphate] potasium salt), a new water-soluble derivative of vitamin C and vitamin E, attenuated oxidative injury and prevents cells from apoptosis. The results suggest that EPC-K1 might be used as a potential therapeutic agent for diseases associated with NO/ONOO(-)-mediated neuronal injury.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Cell Survival; Cells, Cultured; Cerebellum; DNA Fragmentation; Lipid Peroxidation; Membrane Fluidity; Neurons; Nitrates; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances; Vitamin E

The peroxynitrite anion and the nitrogen dioxide (radical) are important toxic species which can arise in vivo from nitric oxide. Both in vivo and in vitro cell protection is demonstrated for beta-carotene in the presence of vitamin E and vitamin C. A synergistic protection is observed compared to the individual anti-oxidants and this is explained in terms of an electron transfer reaction in which the beta-carotene radical is repaired by vitamin C.

Topics: Antioxidants; Ascorbic Acid; beta Carotene; Cell Survival; Drug Synergism; Free Radicals; Humans; Jurkat Cells; Lymphocytes; Nitrates; Nitrogen Dioxide; Oxidants; Vitamin E

Peroxynitrite (ONOO-), a potent oxidant formed by reaction of nitric oxide (NO.) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO. Reaction of ONOO- with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO. Kinetic analysis of NO. production after reaction of ONOO- with GSH established that NO. originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO- does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2. or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO- with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO-. Our results indicate that ONOO- may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO- may represent an important and often overlooked component of NO. biochemistry.

Topics: Ascorbic Acid; Cells, Cultured; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Glutathione; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Luminescent Measurements; Nitrates; Nitric Oxide

The free radicals nitric oxide and superoxide react to form peroxynitrite (ONOO-), a potent cytotoxic oxidant. This study was designed to evaluate whether addition of L-Ascorbic acid (AsC) into the culture medium decreases peroxynitrite-induced apoptosis in human intestinal epithelial (T84) and murine macrophage (RAW 264.7) cell lines. In Experiment 1, T84 and RAW 264.7 cells were divided in two protocols: (1) treated with 100-300 microM ONOO- and incubated for 4 h, and (2) treated with 10-100 microM ONOO- and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 microM ONOO- and 500 microM AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 microM AsC then exposed to 300 microM ONOO- for 4 h. Cell viability (necrosis) was assessed by trypan blue dye exclusion. Apoptosis was quantified with a cell death detection ELISA assay. In the 4 h protocol, ONOO- induced apoptosis in T84 and RAW 264.7 cells, at levels of 100-300 microM. Concentrations of ONOO- greater than 300 microM caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO- induced apoptosis at lower concentrations (50;-75 microM), with concentrations > 75 microM resulting in necrosis. AsC administered to the media or with preincubation plus washout, decreased peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells. These results indicate that ONOO- may contribute to the pathophysiology of gut inflammation by promoting cell death and ascorbic acid may protect against peroxynitrie-induced damage.

Topics: Animals; Apoptosis; Ascorbic Acid; Cell Line; Cell Survival; Culture Media; Epithelium; Free Radical Scavengers; Humans; Intestines; Macrophages; Mice; Necrosis; Nitrates; Trypan Blue

Exposure of human blood plasma to peroxynitrite in the presence of 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) resulted in the trapping of a strongly immobilized nitroxide radical adduct. The adduct was due to protein-centred radicals derived not only from serum albumin but also from other major plasma proteins (fibrinogen, IgG, alpha1-antitrypsin and transferrin). Urate significantly protected plasma from the peroxynitrite-induced DBNBS-plasma protein adduct, whereas ascorbate and glutathione were protective at concentrations exceeding those usually found in plasma. Alkylation of plasma -SH groups did not affect the intensity of DBNBS-plasma protein adduct, whereas bicarbonate increased its formation, thus showing a pro-oxidant effect. The DBNBS-plasma protein adduct provided little structural information, but subsequent non-specific-protease treatment resulted in the detection of an isotropic three-line spectrum, indicating the trapping of radicals centred on a tertiary carbon. The nitrogen hyperfine coupling constant of this adduct and its superhyperfine structure were similar to those of DBNBS-tryptophan peptides with the alpha-amino group of tryptophan linked in the amide bond, consistent with a radical adduct formed at C-3 of the indole ring of tryptophan-containing peptides. DBNBS was unable to trap radicals derived from peroxynitrite-treated tyrosine or tyrosine-containing peptides. Methionine treated with peroxynitrite resulted in the trapping of at least two DBNBS-methionine adducts with hyperfine structures different from that of protease-treated DBNBS-plasma proteins. These results demonstrate that peroxynitrite induced in blood plasma the formation of protein radicals centred on tryptophan residues and underline the relevance of the one-electron oxidation pathway of peroxynitrite decomposition in biological fluids.

Topics: Ascorbic Acid; Benzenesulfonates; Bicarbonates; Blood Proteins; Chromatography, Gel; Electron Spin Resonance Spectroscopy; Electron Transport; Free Radicals; Glutathione; Humans; Methionine; Nitrates; Nitroso Compounds; Peptides; Pronase; Spin Trapping; Sulfhydryl Compounds; Tryptophan; Uric Acid

The reactions of new spin trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H) with superoxide radicals and peroxynitrite were studied. The rate constants were determined as 3.2 x 10(3) and 4.5 x 10(9) M-1s-1, respectively. It was found that 2mM of spin trap CP-H or 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H) provide almost the same spin trapping efficacy. In contrast to TEMPONE-H the reaction of CP-H with peroxynitrite was inhibited by 20 mM DMSO. This simplifies the quantification of peroxynitrite formation. During the reaction of CP-H and TEMPONE-H with superoxide radicals or peroxynitrite the stable nitroxide radicals 3-carboxy-proxyl (CP) and 2,2,6,6-tetramethyl-4-oxo-piperidinoxyl (TEMPONE) are formed. It was found that the rate of reduction of CP by glutathione or by smooth muscle cells was two-fold slower and the reduction of CP by ascorbate was 66-fold slower than corresponding rates of reduction of TEMPONE. Therefore quantification of the formation of superoxide radicals and of peroxynitrite by CP-H is much less hindered by a variety of biological reductants than in case of TEMPONE-H. Thus, CP-H is more suitable for spin trapping of superoxide radicals and peroxynitrite in biological systems than the TEMPONE-H.

Topics: Animals; Ascorbic Acid; Cysteine; Electron Spin Resonance Spectroscopy; Glutathione; Muscle, Smooth; Nitrates; Oxidation-Reduction; Piperidines; Pyrrolidines; Spin Trapping; Superoxides

beta-Carotene absorbed 2 equimolar amounts of NO2 accompanying the complete destruction of beta-carotene. Electron spin resonance study using 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl revealed that no significant amounts of NO were released by the interaction. Nitrogen atoms derived from NO2 were tightly bound to the beta-carotene molecules. Destruction of beta-carotene was inhibited little by alpha-tocopherol and polyunsaturated fatty esters, and slightly by ascorbyl palmitate, indicating that beta-carotene was a more effective scavenger of NO2. ONOOH/ONOO- and 3-morpholinosydononimine similarly destroyed beta-carotene. The results suggest that beta-carotene contributes to the prevention of cytotoxicity and genotoxicity of NO2 and ONOOH/ONOO- derived from NO.

Topics: Amiloride; Ascorbic Acid; beta Carotene; Docosahexaenoic Acids; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Nitrates; Nitrogen Dioxide; Vitamin E

The recent literature suggests that free radicals and reactive oxygen species may account for many pathologies, including those of the nervous system.. The influence of various reactive oxygen species on the rate of glutamate uptake by astrocytes was investigated on monolayers of primary cultures of mouse cortical astrocytes.. Hydrogen peroxide and peroxynitrite inhibited glutamate uptake in a concentration-dependent manner. Addition of copper ions and ascorbate increased the potency and the efficacy of the hydrogen peroxide effect, supporting the potential neurotoxicity of the hydroxyl radical. The free radical scavenger dimethylthiourea effectively eliminated the inhibitory potential of a mixture containing hydrogen peroxide, copper sulphate, and ascorbate on the rate of glutamate transport into astrocytes. The inhibitory effect of hydrogen peroxide on glutamate uptake was not altered by the inhibition of glutathione peroxidase, whereas the inhibition of catalase by sodium azide clearly potentiated this effect. Superoxide and nitric oxide had no effect by themselves on the rate of glutamate uptake by astrocytes. The absence of an effect of nitric oxide is not due to an inability of astrocytes to respond to this substance, since the same cultures did respond to nitric oxide with a sustained increase in cytoplasmic free calcium.. These results confirm that reactive oxygen species have a potential neurotoxicity by means of impairing glutamate transport into astrocytes, and they suggest that preventing the accumulation of hydrogen peroxide in the extracellular space of the brain, especially during conditions that favor hydroxyl radical formation, could be therapeutic.

Topics: Animals; Animals, Newborn; Antioxidants; Ascorbic Acid; Astrocytes; Catalase; Free Radical Scavengers; Glutamic Acid; Glutathione Peroxidase; Hydrogen Peroxide; Mice; Mice, Inbred C57BL; Nitrates; Nitric Oxide; Reactive Oxygen Species; Superoxides; Thiourea

Cholesterol and alpha-tocopherol oxidations were studied in brain subcellular fractions isolated from cerebral hemispheres of 4-month-old, male Fischer 344 rats. The fractions were suspended in buffered media (pH 7.4, 37 degrees C0 and oxidized by adding (i) ferrous iron (Fe2+) with or without ascorbate or (ii) peroxynitrite (an endogenous oxidant produced by the reaction of superoxide and nitric oxide). Treatment of subcellular fractions with Fe2+ in the presence or absence of ascorbate produced primarily 7-keto- and 7-hydroxy-cholesterols and small amounts of 5 alpha, 6 alpha-epoxycholesterol. Since brain contains high levels of ascorbate, and release of iron could result in oxysterol formation. Peroxynitrite oxidized alpha-tocopherol but not cholesterol. Hence, the toxicity of peroxynitrite or nitric oxide could not be due to cytotoxic oxysterols. When synaptosomes were incubated for 5 min in the presence of 0.5 to 2 microM Fe2+ and ascorbate, alpha-tocopherol was oxidized while cholesterol remained unchanged. Thus, alpha-tocopherol is functioning as an antioxidant, protecting cholesterol. Diethylenetriaminepentaacetic acid blocked production of oxysterols, whereas citrate, ADP and EDTA did not. A significant percentage of mitochondrial cholesterol was oxidized by treatment with Fe2+ and ascorbate. Hence, mitochondrial membrane properties dependent on cholesterol could be particularly susceptible to oxidation. The oxysterols formed were retained within the membranes of synaptosomes and mitochondria. The 7-oxysterols produced are known to be inhibitors of membrane enzymes and also can modify membrane permeability. Hence, oxysterols may plan an important role in brain tissue damage during oxidative stress.

Topics: Animals; Antioxidants; Ascorbic Acid; Borohydrides; Brain; Chelating Agents; Cholesterol; Chromatography, Gas; Ferrous Compounds; Male; Mitochondria; Nitrates; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Inbred F344; Synaptosomes; Vitamin E

Peroxynitrite, the reaction product of O2.- and .NO, is a toxic compound involved in several oxidative processes that modify proteins. The mechanisms of these oxidative reactions are not completely understood. In this study, using direct ESR at 37 degrees C, we observed that peroxynitrite induced in human blood plasma a long-lived singlet signal at g = 2.004 arising from proteins. This signal was not due to a specific plasma protein, because several purified proteins were able to form a peroxynitrite-induced g = 2.004 signal, but serum albumin and IgG showed the most intense signals. Hydroxyurea, a tyrosyl radical scavenger, strongly inhibited the signal, and horseradish peroxidase/H2O2, a radical-generating system known to induce tyrosyl radicals, induced a similar signal. Furthermore peptides containing a Tyr in the central portion of the molecule were able to form a stable peroxynitrite-dependent g = 2.004 signal, whereas peptides in which Tyr was substituted with Gly, Trp or Phe and peptides with Tyr at the N-terminus or near the C-terminus did not form radicals that were stable at 37 degrees C. We suggest that Tyr residues are at least the major radical sources of the peroxynitrite-dependent g = 2.004 signal at 37 degrees C in plasma or in isolated proteins. Although significantly enhanced by CO2/bicarbonate, the signal was detectable in whole plasma at relatively high peroxynitrite concentrations (>2 mM) but, after removal of ascorbate or urate or in dialysed plasma, it was detectable at lower concentrations (100-1000 microM). Our results suggest that the major role of ascorbate and urate is to reduce or 'repair' the radical(s) centred on Tyr residues and not to scavenge peroxynitrite (or nitrosoperoxycarbonate, the oxidant formed in CO2-containing fluids). This mechanism of inhibition by plasma antioxidants may be a means of preserving the physiological functions of peroxynitrite.

Topics: Amino Acid Sequence; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; Molecular Sequence Data; Nitrates; Sulfhydryl Compounds; Tyrosine; Uric Acid

Formation of peroxynitrite by the fast reaction between nitric oxide and superoxide anion may represent a critical control point in cells producing both species, leading to either down-regulation of the physiological effects of superoxide anion and nitric oxide by forming an inert product, nitrate, or to potentiation of their toxic effects by oxidation of nearby molecules by peroxynitrite. (The term peroxynitrite is used to refer to the sum of all possible forms of peroxynitrite anion and peroxynitrous acid unless otherwise specified.) In this report we demonstrate that, in spite of all the antioxidant defences present in human plasma, its interaction with peroxynitrite leads to generation of free radical intermediates such as (i) the ascorbyl radical, detected by direct EPR, (ii) the albumin-thiyl radical, detected by spin-trapping experiments with both N-tert-butyl-alpha-phenylnitrone and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and (iii) a uric acid-derived free radical, detected as the DMPO radical adduct in plasma whose thiol groups were previously blocked with 5,5-dithiobis-(2-nitrobenzoic acid). The identity of the latter adduct was confirmed by parallel experiments demonstrating that it is not detectable in plasma pretreated with uricase, whereas it is formed in incubations of peroxynitrite with uric acid. Peroxynitrite-mediated oxidations were also followed by oxygen consumption and ascorbate and plasma-thiol depletion. Our results support the view that peroxynitrite-mediated one-electron oxidation of biomolecules may be an important event in its cytotoxic mechanism. In addition, the data have methodological implications by providing support for the use of EPR methodologies for monitoring both free radical reactions and ascorbate concentrations in biological fluids.

Topics: Ascorbic Acid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; Kinetics; Nitrates; Oxygen Consumption; Serum Albumin; Spin Labels; Time Factors; Uric Acid

Peroxynitrite is formed by the reaction of superoxide with nitric oxide, an important neurotransmitter. Incubation of rat brain synaptosomes with peroxynitrite resulted in the consumption of antioxidant substances such as alpha-tocopherol, ascorbate, and thiols. Membrane cholesterol was not oxidized under the same conditions. alpha-Tocopherol and ascorbate in synaptosomes were oxidized very rapidly by peroxynitrite. In contrast, previous reports in the literature have shown that peroxynitrite treatment did not oxidize tocopherol in human plasma. Peroxynitrite in sufficient concentrations oxidized all of the tocopherol and ascorbate in synaptosomes. Thus, the oxidant is able to diffuse to the different membranes in synaptosomes and oxidize tocopherol in all of them. alpha-Tocopherol is converted quantitatively to tocopherolquinone during the oxidation. Significant amounts of thiols (at least 30% of the total thiols) do not seem to be accessible to oxidation by peroxynitrite. However, the concentration of thiols is much higher than those of tocopherol and ascorbate. Addition of the hydroxyl radical quenchers benzoate or mannitol or the enzymes superoxide dismutase or catalase (alone or together) did not affect the oxidation of tocopherol and ascorbate by peroxynitrite, whereas cysteine and glutathione blocked the oxidation. Therefore, reactive oxygen species may not be directly involved as intermediates in oxidations induced by peroxynitrite. The latter is a potent oxidizing agent that can oxidize substances such as tocopherols, ascorbate, and thiols in the immediate vicinity of its formation. The antioxidant nutrients ascorbate and tocopherol could play important roles in protecting brain from oxidative damage induced by peroxynitrite.

Topics: Animals; Ascorbic Acid; Brain; Dose-Response Relationship, Drug; Humans; Hydrogen-Ion Concentration; Nitrates; Rats; Sulfhydryl Compounds; Synaptosomes; Vitamin E

Peroxynitrite, formed by reaction of superoxide and nitric oxide, appears to be an important tissue-damaging species generated at sites of inflammation. In this paper, we compare the abilities of several biological antioxidants to protect against peroxynitrite-dependent inactivation of alpha 1-antiproteinase, and to inhibit tyrosine nitration upon addition of peroxynitrite. GSH and ascorbate protected efficiently in both systems. Uric acid inhibited tyrosine nitration but not alpha 1-antiproteinase inactivation. The possibility that ascorbic acid is an important scavenger of reactive nitrogen species in vivo is discussed.

Topics: alpha 1-Antitrypsin; Antioxidants; Arthritis, Rheumatoid; Ascorbic Acid; Humans; Lipid Peroxidation; Nitrates; Oxidation-Reduction; Oxidative Stress; Pancreatic Elastase; Synovial Fluid; Tyrosine

We previously reported that mesencephalic dopaminergic neurons are resistant to cytotoxicity induced by nitric oxide (NO). This study investigated the intracellular mechanism that protects dopaminergic neurons against NO toxicity in rat mesencephalic cultures. Peroxynitrite anion, an active metabolite of NO, caused significant cytotoxic effects against dopaminergic and nondopaminergic neurons, but NO caused cytotoxic effects restricted to nondopaminergic neurons. In addition, we studied the effects of ascorbate, an anti-oxidant, on NO-induced neurotoxicity against dopaminergic neurons and found that coadministration of ascorbate failed to affect resistance against NO-induced neurotoxicity. These findings suggest that the protecting mechanism from NO neurotoxicity in dopaminergic neurons is based on inhibition of conversion of NO to peroxynitrite anion, is independent of the NO redox state, and is possibly due to suppression of superoxide anion production. Furthermore, we investigated NO-induced neurotoxicity with or without pretreatment with sublethal doses of methylphenylpyridium ion (MPP+). Following pretreatment with 1 microM MPP+, which did not show significant cytotoxic effects against dopaminergic neurons, NO demonstrated significant cytotoxicity. Therefore, MPP+ may inhibit the protecting systems from NO neurotoxicity in dopaminergic neurons.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Antioxidants; Ascorbic Acid; Cell Survival; Cells, Cultured; Dopamine; Drug Resistance; Mesencephalon; Nerve Tissue Proteins; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Parkinson Disease; Rats; Tyrosine 3-Monooxygenase

The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.

Topics: Adenosine Diphosphate; Animals; Ascorbic Acid; Blotting, Western; Cattle; Ferric Compounds; Kinetics; Lipid Peroxides; NAD; NADP; NADP Transhydrogenases; Nitrates; Oxidation-Reduction; Peptide Mapping; Stress, Physiological; Submitochondrial Particles; Tyrosine; Ubiquinone

A new mechanism is presented for the oxidation of ascorbate by peroxynitrite. Our mechanism involves the reaction of ascorbate both with ground-state peroxynitrous acid (HOONO) and with a reactive intermediate (HOONO*); the reactive intermediate is postulated to be formed in the decay of HOONO to form nitrate. At physiological pH, the ascorbate monoanion (AH-) is the predominant ascorbate species. The plot of the observed rate constant for peroxynitrite decay (kobs) vs AH- for the reaction of peroxynitrite with AH- shows two regions, one linear and one curved. In the linear region, which involves high AH- concentrations, the reaction is dominated by the bimolecular reaction between HOONO and AH-. At lower AH- concentrations, this bimolecular reaction slows and reactions with both HOONO and HOONO* produce the observed curvature. Analysis of the data leads to the estimation of the ratio of rate constants for the reaction of AH- with HOONO* (k2*) and the decay of HOONO to nitrate (kN), giving the value of k2*/kN = 3158 +/- 505 M-1; and of the rate constant (k2) for the reaction between AH- and HOONO, k2 = 236 +/- 14 M-1 s-1. Ascorbate displays higher selectivity for HOONO* than does methionine or 2-keto-4-thiomethylbutanoic acid, two substrates whose reactivity toward HOONO and HOONO* has previously been reported. The biological relevance of the reaction of ascorbate with peroxynitrite is discussed in terms of the rate constants and the concentrations of AH- typically found in biological systems; ascorbate may react with HOONO*, although the reaction with ground-state HOONO probably is too slow to occur in vivo.

Topics: Ascorbic Acid; Electron Transport; Free Radicals; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Methionine; Models, Chemical; Nitrates; Oxidation-Reduction

Peroxynitrite [O = NOO-, oxoperoxonitrate(1-)bd is a strong oxidant that may be formed in vivo by the reaction of O2.- and NO(.). Oxoperoxonitrate(1-) reacts with molecules in aqueous acidic solutions via pathways that involve the highly reactive hydrogen oxoperonitrate either as an intermediate in a first-order reaction or as a reactive agent in a simple second-order reaction. ESR experiments show that hydrogen oxoperoxonitrate oxidizes monohydrogen L-ascorbate by one electron: when mixed at pH ca. 5 and passed through a flow cell within 0.1 s, the two-line ESR signal of the ascorbyl radical anion (aH = 0.18 T, g = 2.005) is observed. The overall stoichiometry of the reaction was 1 mol of ascorbate oxidized per mol of oxoperoxonitrate(1-) added. The kinetics of the reaction were studied over the pH range 4.0-7.5 by stopped-flow spectrometry. Hydrogen oxoperoxonitrate, observed between 300 and 350 nm, and the oxoperoxonitrate(1-) anion, at 302 nm, disappear faster than predicted for the first-order isomerization to NO3-. The rate increases from pH 4 to 5.8, and then decreases with increasing pH. The rate variation suggests a bimolecular reaction either between the oxoperoxonitrate(1-) anion and ascorbic acid or between hydrogen oxoperoxonitrate and the monohydrogen ascorbate anion. Although the two pathways are kinetically indistinguishable, the pKa values of ascorbic acid and hydrogen oxoperoxonitrate strongly suggest that the reacting species are hydrogen oxoperoxonitrate and monohydrogen ascorbate. The second-order rate constant for this reaction is 235 +/- 4 M-1s-1 at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Ascorbic Acid; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Kinetics; Mathematics; Nitrates; Oxidation-Reduction; Spectrophotometry; Temperature; Thermodynamics

Electron spin resonance (ESR) spin trapping was utilized to investigate the reaction of peroxynitrite with thiols and ascorbate at physiological pH. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The reaction of peroxynitrite with DMPO generated 5,5-dimethylpyrrolidone-(2)-oxy-(1) (DMPOX). Formate enhanced the peroxynitrite decomposition but did not generate any detectable amount of formate-derived free radicals. Thus, the spin trapping measurements provided no evidence for hydroxyl (.OH) radical generation in peroxynitrite decomposition at physiological pH. Thiols (glutathione, cysteine, and penicillamine) and ascorbate reacted with peroxynitrite to generate the corresponding thiyl and ascorbyl radicals. The one-electron oxidation of thiols by peroxynitrite may be one of the important mechanisms for peroxynitrite-induced toxicity and ascorbate may provide a detoxification pathway.

Topics: Ascorbic Acid; Cyclic N-Oxides; Dehydroascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Hydrogen-Ion Concentration; Models, Biological; Nitrates; Spin Labels; Sulfhydryl Compounds

Endothelial cells and activated phagocytes produce both nitric oxide (.NO) and superoxide (O2.-), which react to form peroxynitrite. Peroxynitrite has been suggested to be directly cytotoxic and also to decompose into other toxic species. In order to understand the consequences of peroxynitrite generation in vivo, we examined its reaction with human blood plasma. Peroxynitrite decreased the total peroxyl-trapping capacity of plasma. In terms of specific antioxidants, addition of peroxynitrite to plasma leads to rapid oxidation of ascorbic acid, uric acid and plasma SH groups. The oxidation of plasma SH groups was enhanced in dialysed plasma and returned to control levels by the addition of physiological levels of bicarbonate. Evidence was found for formation of nitro-adducts to aromatic side chains in plasma proteins by peroxynitrite. Peroxynitrite also leads to depletion of ubiquinol and formation of traces of lipid hydroperoxides in plasma, although alpha-tocopherol levels were only slightly decreased. Peroxynitrite formation in human body fluids is likely to cause antioxidant depletion and oxidative damage.

Topics: Antioxidants; Ascorbic Acid; Blood Proteins; Glutathione; Humans; Kinetics; Lipid Peroxides; Nitrates; Oxidants; Oxidation-Reduction; Serum Albumin; Vitamin E

The decomposition of peroxynitrite in the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) generated 5,5-dimethylpyrrolidone-(2)-oxy-(1) (DMPOX) without formation of DMPO/OH. Formate enhanced the peroxynitrite decomposition but did not generate any detectable amount of formate-derived free radicals. Glutathione, cysteine, penicillamine, and ascorbate reacted with peroxynitrite to generate the corresponding thiyl and ascorbyl radicals. The results show that the decomposition of peroxynitrite did not generate any significant amount of OH radicals, and one-electron reduction of peroxynitrite by ascorbate may be one of the important peroxynitrite detoxification pathways.

Topics: Ascorbic Acid; Cyclic N-Oxides; Cysteine; Electron Spin Resonance Spectroscopy; Formates; Free Radicals; Glutathione; Hydroxyl Radical; Nitrates; Penicillamine; Spin Labels