ascorbic-acid and nitroxyl

Disruption of the redox balance in vivo is closely involved in the development of various diseases associated with oxidative stress. Therefore, methods for the in vivo analysis of antioxidants and free radicals are essential to elucidate the pathogenic mechanisms of such diseases. Although profluorescent nitroxide probes can be used to evaluate redox molecules with high sensitivity, these probes have low selectivity. Recently, we developed two profluorescent nitroxide probes, 15-((9-(ethylimino)-10-methyl-9Hbenzo[a]phenoxazin-5-yl)amino)-3,11-dioxa-7-azadispiro-hexadecan-7-yloxyl (Nile-DiPy) and 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen), which had high sensitivity and selectivity toward ascorbic acid and lipid-derived radicals, respectively. These probes can react sensitively and selectively to each target molecule and can be used in animal experiments. In this paper, we review the design strategies and application of these profluorescent nitroxide probes.

Topics: 4-Chloro-7-nitrobenzofurazan; Animals; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Cyclic N-Oxides; Fluorescent Dyes; Free Radicals; Mass Spectrometry; Nitrogen Oxides; Oxidation-Reduction; Oxidative Stress; Rats, Wistar

Nitroxides are stable, kinetically-persistent free radicals which have been successfully used in the study and intervention of oxidative stress, a critical issue pertaining to cellular health which results from an imbalance in the levels of damaging free radicals and redox-active species in the cellular environment. This review gives an overview of some of the biological processes that produce radicals and other reactive oxygen species with relevance to oxidative stress, and then discusses interactions of nitroxides with these species in terms of the use of nitroxides as redox-sensitive probes and redox-active therapeutic agents.

Topics: Animals; Antioxidants; Ascorbic Acid; Biopterins; Electron Spin Resonance Spectroscopy; Humans; Melatonin; Molecular Probes; Nitrogen Oxides; Oxidation-Reduction; Oxidative Stress; Spin Labels; Ubiquinone; Vitamin E

2,2,6,6-Tetramethylpiperidin-N-oxyl (TEMPO)-type nitroxides are susceptible to bioreduction, leading to a loss of radical properties. Although it has been reported that the steric and electronic environments around the N-O moiety of nitroxides affect the reduction, how the relative configuration of nitroxide derivatives alters it is unclear. In this study, we investigated the effect of diastereomers on the radical properties of C2- and C4-disubstituted TEMPO-type nitroxides. We succeeded in isolating the diastereomers of the studied nitroxides for the first time. In addition, we compared the reactivities of nitroxide derivatives with different substituents at the C2 and C4 positions toward ascorbate reduction. We found that the bulky substituents at both C2 and C4 and the electronic effect of C4 affected the reduction of the isomers. C2- and C4-disubstituted nitroxides were administered to mice for electron spin resonance imaging to assess bioreduction in the brain. Similar to the reactivity to reduction in vitro, a difference in the bioreduction of diastereomers was observed in brain tissues. Our research strongly indicates that bioreduction can be controlled by changing the relative configuration, which can be used in the design of nitroxide derivatives for biological applications.

Topics: Animals; Ascorbic Acid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Mice; Nitrogen Oxides; Oxidation-Reduction; Spin Labels

Nitroxide compounds have been used as redox-sensitive imaging probes by electron paramagnetic resonance (EPR) for assessing oxidative stress

Topics: Animals; Ascorbic Acid; Brain; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Mice; Nitrogen Oxides; Oxidation-Reduction

With a view to developing a theranostic nanomedicine for targeted drug delivery systems visible by magnetic resonance (MR) imaging, robust metal-free magnetic nanoemulsions (mean particle size less than 20 nm) consisting of a biocompatible surfactant and hydrophobic, low molecular weight 2,2,5-trimethyl-5-(4-alkoxy)phenylpyrrolidine-N-oxyl radicals were prepared in pH 7.4 phosphate-buffered saline (PBS). The structure of the nanoemulsions was characterized by electron paramagnetic resonance spectroscopy, and dynamic light scattering and small-angle neutron-scattering measurements. The nanoemulsions showed high colloidal stability, low cytotoxicity, enough reduction resistance to excess ascorbic acid, and sufficient contrast enhancement in the proton longitudinal relaxation time (T

Topics: Animals; Antineoplastic Agents, Phytogenic; Ascorbic Acid; Brain; Cell Proliferation; Drug Carriers; Dynamic Light Scattering; Electron Spin Resonance Spectroscopy; HeLa Cells; Humans; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Imaging; Magnetite Nanoparticles; Mice; Molecular Weight; Neutron Diffraction; Nitrogen Oxides; Oxidation-Reduction; Paclitaxel; Particle Size; Scattering, Small Angle

Ascorbic acid (AsA), also known as vitamin C, is a vital small-molecule antioxidant with multiple functions in vivo. It's the major natural antioxidant found in plants and is also an essential component of human nutrition. AsA plays a key role in many diseases-related biological metabolism. Therefore, sensitive and selective detection of AsA is greatly important in pharmaceutical, clinical and food industry. Here a sensitive and selective sensor for ascorbic acid detection based on the recovered fluorescence of NAPS-NO (N-propyl-triethoxysilane-4-(4-ylamino-1-oxy-2,2,6,6-tetramethylpiperdine)- naphthalimide) probe is described. The fluorescence of the naphthalimide moiety of NAPS-NO is inhibited by the nitroxide group, which is covalently linked to the fluorophore. Then, ascorbic acid reacts rapidly with the nitroxide moiety of NAPS-NO to form hydroxylamine, and the fluorescence properties of the naphthalimide moiety are recovered and the ESR signal decayed. Over a wide range from 80 nM to 50 μM, a good linear relationship between the fluorescence intensity and the concentration of ascorbic acid was found and the detection limit was estimated to be as low as 20 nM. To confirm the practical usefulness of the fluorophore-nitroxide probe, we demonstrated the use of NAPS-NO for the measurement of AsA in human blood serum and also successfully determined the concentration of AsA in HEK 293 cell lysate. Results from confocal laser scanning microscopy experiments demonstrated that this chemosensor is cell permeable and can be used as a fluorescent probe for monitoring ascorbic acid in living cells.

Topics: Ascorbic Acid; Biological Transport; Cell Extracts; Fluorescence; HEK293 Cells; Humans; Limit of Detection; Molecular Probes; Naphthalimides; Nitrogen Oxides; Piperidines; Spectrometry, Fluorescence

The role of NO in biology is well established. However, an increasing body of evidence suggests that azanone (HNO), could also be involved in biological processes, some of which are attributed to NO. In this context, one of the most important and yet unanswered questions is whether and how HNO is produced in vivo. A possible route concerns the chemical or enzymatic reduction of NO. In the present work, we have taken advantage of a selective HNO sensing method, to show that NO is reduced to HNO by biologically relevant alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent decomposition of the so-produced radical to yield HNO and an alkoxyl radical.

Topics: Alcohols; Animals; Ascorbic Acid; Cattle; Endothelial Cells; Nitric Oxide; Nitrogen Oxides; Oxidation-Reduction; Tyrosine

Much evidence supports the idea that oxidative stress is involved in the pathogenesis of epilepsy, and therapeutic interventions with antioxidants are expected as adjunct antiepileptic therapy. The aims of this study were to non-invasively obtain spatially resolved redox data from control and pentylenetetrazole (PTZ)-induced kindled mouse brains by electron paramagnetic resonance (EPR) imaging and to visualize the brain regions that are sensitive to oxidative damage. After infusion of the redox-sensitive imaging probe 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), a series of EPR images of PTZ-induced mouse heads were measured. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of redox status in vivo and mapped as a redox map. The obtained redox map showed heterogeneity in the redox status in PTZ-induced mouse brains compared with control. The co-registered image of the redox map and magnetic resonance imaging (MRI) for both control and PTZ-induced mice showed a clear change in the redox status around the hippocampus after PTZ. To examine the role of antioxidants on the brain redox status, the levels of antioxidants were measured in brain tissues of control and PTZ-induced mice. Significantly lower concentrations of glutathione in the hippocampus of PTZ-kindled mice were detected compared with control. From the results of both EPR imaging and the biochemical assay, the hippocampus was found to be susceptible to oxidative damage in the PTZ-induced animal model of epilepsy.

Topics: Animals; Ascorbic Acid; Brain; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Epilepsy; Glutathione; Hippocampus; Kindling, Neurologic; Male; Mice, Inbred C57BL; Nitrogen Oxides; Oxidation-Reduction; Pentylenetetrazole

Stimuli-responsive multimodality imaging agents have broad potential in medical diagnostics. Herein, we report the development of a new class of branched-bottlebrush polymer dual-modality organic radical contrast agents--ORCAFluors--for combined magnetic resonance and near-infrared fluorescence imaging in vivo. These nitroxide radical-based nanostructures have longitudinal and transverse relaxation times that are on par with commonly used heavy-metal-based magnetic resonance imaging (MRI) contrast agents. Furthermore, these materials display a unique compensatory redox response: fluorescence is partially quenched by surrounding nitroxides in the native state; exposure to ascorbate or ascorbate/glutathione leads to nitroxide reduction and a concomitant 2- to 3.5-fold increase in fluorescence emission. This behaviour enables correlation of MRI contrast, fluorescence intensity and spin concentration with tissues known to possess high concentrations of ascorbate in mice. Our in vitro and in vivo results, along with our modular synthetic approach, make ORCAFluors a promising new platform for multimodality molecular imaging.

Topics: Animals; Ascorbic Acid; Contrast Media; Female; Fluorescence; HeLa Cells; Humans; Magnetic Resonance Imaging; Mice; Mice, Inbred BALB C; Molecular Imaging; Nitrogen Oxides; Oxidation-Reduction; Polymers

A study was conducted to determine the effect of carbon monoxide (CO), nitroxide (NOx) and sulfur dioxide (SO2) on ROS production, photosynthesis and ascorbate-glutathione pathway in strawberry plants. The results showed that both singlet oxygen (O2(-1)) and hydrogen peroxide (H2O2) content increased in CO, NOx and SO2 treated strawberry leaves. A drastic reduction of primary metabolism of plants (photosynthesis), with the closure of stomata, resulted in a reduction of protein, carbohydrate and sucrose content due to production of reactive oxygen species (ROS) under prolonged exposure of gas stress. The resulting antioxidant enzymes were increased under a low dose of gas stress, whereas they were decreased due to a high dose of gas stress. Our results indicate that increased ROS may act as a signal to induce defense responses to CO, NOx and SO2 gas stress. The increased level of antioxidant enzymes plays a significant role in plant protection due to which strawberry plants can be used as a hyperaccumulator to maintain environmental pollution, however, the defense capacity cannot sufficiently alleviate oxidative damage under prolonged exposure of CO, NOx and SO2 stress.

Topics: Ascorbic Acid; Carbon Monoxide; Fragaria; Glutathione; Nitrogen Oxides; Photosynthesis; Reactive Oxygen Species; Sulfur Dioxide

A series of sterically shielded pyrrolidine nitroxides were synthesized, and their reduction by ascorbate (vitamin C) indicate that nitroxide 3, a tetraethyl derivative of 3-carboxy-PROXYL, is reduced at the slowest rate among known nitroxides, i.e., at a 60-fold slower rate than that for 3-carboxy-PROXYL.

Topics: Ascorbic Acid; Kinetics; Models, Molecular; Molecular Structure; Nitrogen Oxides; Oxidation-Reduction; Pyrrolidines

Nitroxyl radicals (nitroxide) have great potential advantages as spin probes, antioxidants, contrast agents, and radiation-protecting agents. However, they are readily reduced by reductants in cells and lose their paramagnetic nature. Recently, tetraethyl-substituted nitroxyl radicals have been reported to have high stability toward reduction by ascorbic acid (AsA). We report the general considerations of tetraethyl nitroxyl radicals for in vivo application. The reason for the low reactivity to AsA reduction was the positive value of Gibbs energy between the tetraethyl nitroxyl radical and AsA. Further, these compounds had an inhibitory effect on lipid peroxidation despite having AsA resistance. They had low antiproliferative effects in HepG2 cells and HUVECs and did not have a lowering effect on blood pressure in animals. Further, after intravenous injection, the ESR signal intensities of tetraethyl-substituted piperidine nitroxyl radicals were very stable in mice over 20 min. These results suggest that tetraethyl-substituted nitroxyl radicals have stability against bioreduction with reductants such as AsA and confer onto them features as antioxidants and paramagnetic tracers/contrast agents. Hence, they will be useful in identifying the foci of oxidative stress in vivo using redox-based imaging approaches.

Topics: Animals; Antioxidants; Ascorbic Acid; Blood Pressure; Cells, Cultured; Contrast Media; Drug Stability; Electron Spin Resonance Spectroscopy; Hep G2 Cells; Humans; Lipid Peroxidation; Male; Mice; Mice, Inbred ICR; Nitrogen Oxides; Oxidation-Reduction; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley

We have attempted to explore the neuroprotective effectiveness of levetiracetam (LEV) by measuring its in vivo antioxidant effect in the hippocampus of rats in a freely moving state. Male Wistar rats were used for the estimation of the in vivo antioxidant effect of LEV through microdialysis combined with electron spin resonance spectroscopy. The antioxidant effect was examined using the principle by which a systemically administered blood-brain barrier-permeable nitroxide radical (PCAM) decreases in an exponential decay manner that is correlated with the amount of antioxidant in the brain. The PCAM decay ratio during perfusion with normal Ringer's solution was compared with that during 32 microM and 100 microM LEV co-perfusion. The in vivo antioxidant effect was examined. In addition, the expressions of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS) protein related to redox regulation were measured in the hippocampus of rats after 14 days of administration of LEV at a dose of 54 mg/day i.p. The half-life of PCAM was statistically shortened after LEV perfusion compared with the results of the control experiment. While the expression of the pro-oxidant protein iNOS was decreased, that of the antioxidant protein xCT was statistically increased by the administration of LEV. The role of xCT is to transport cystine, the internal material of glutathione, into the cell. The shortened half-life of the nitroxide radical by co-perfusion of LEV with increased xCT and decreased iNOS expression revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. The results of this study suggest that LEV synergistically enhances the basal endogenous antioxidant effect in the hippocampus with ascorbic acid and alpha-tocopherol. Our findings further suggest that LEV exerts a neuroprotective role by 1) modifying the expression of xCT and iNOS in connection with lipid peroxidation, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by up-regulation of the intake of cystine, an internal material of GSH.

Topics: alpha-Tocopherol; Amino Acid Transport System y+; Amino Acid Transport Systems, Acidic; Amino Acids; Animals; Antioxidants; Ascorbic Acid; Blotting, Western; Electron Spin Resonance Spectroscopy; Hippocampus; Levetiracetam; Male; Microdialysis; Neuroprotective Agents; Nitric Oxide Synthase Type II; Nitrogen Oxides; Piracetam; Rats; Rats, Wistar

Piperidine and pyrrolidine nitroxyl radicals (nitroxide) contain unpaired electrons and have been widely recognized as antioxidants, contrast agents, spin probes, radiation protective agents and polymerization mediators. Nitroxyl radicals can react with free radicals and reductants and their reactivities depend on the basic structure of the nitroxyl radicals themselves. However, reductants easily reduce these radicals and they lose their paramagnetic nature and function. Therefore, the aim of this study was to develop various functional nitroxyl radicals, particularly focusing on stability towards AsA through the improvement of the synthetic route for a series of 2,6-substituted nitroxyl radicals. Tetraethyl-substituted piperidine nitroxyl radical 8 exhibited resistance to AsA reduction and 2,6-dispiro-4',4''-dipyrane-piperidin-4-one-N-oxyl 5 had a second-order rate constant 10-times greater than those of hydroxyl-TEMPO and oxo-TEMPO. The 2,6-substituted compound offers various reactivities towards AsA and the possibility to be used as a new antioxidant, contrast agent and radical polymerizer.

Topics: Antioxidants; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Nitrogen Oxides

Although the ascorbate-dependent reduction of S-nitrosothiol to the corresponding thiol function is frequently used for analyzing S-nitrosocysteinyl residues in proteins, it proceeds with low yields. Our re-investigation of the ascorbate-S-nitrosothiol reaction demonstrates now the intermediacy of nitroxyl (HNO/3NO-) that is highly effective in oxidizing thiols. The occurrence of the HNO reporter molecule, i.e., N2O, during and after reaction was unequivocally demonstrated by 15N NMR. The yield of HNO from the S-nitrosoglutathione-ascorbate reaction was determined with the aid of a Mn(III)-complex to 60%, a value that was significantly higher than the one of nitric oxide formation (48%) at physiological pH. The same HNO yield was observed with a S-nitrosothiol bound to a protein (i.e., S-nitroso-papain). With the known chemistry of nitroxyl, it was possible to optimize the experimental conditions so that the GSNO-ascorbate reaction yielded stoichiometric amounts of glutathione after a reaction period of 1 min.

Topics: Ascorbic Acid; Magnetic Resonance Spectroscopy; Molecular Structure; Nitric Oxide; Nitrogen Oxides; S-Nitrosothiols

This technical note reports a continuous-wave electron paramagnetic resonance (CW-EPR) imager that can visualize the distribution of free radicals with a half-life of subminutes in three-dimensional (3D) space. A total of 46 EPR spectra under magnetic field gradients, called projections, were obtained for image reconstruction at an interval of 3.6 s. A shortened data-acquisition time was achieved with the use of analog signals that drove field gradient coils in the imager. 3D mapping of the half-lives of nitroxyl radicals (4-hydroxyl-2,2,6,6-tetramethyl-piperidinyl-1-oxyl) was demonstrated in their reduction reaction with ascorbic acid. Inhomogeneous half-lives were clearly mapped pixel-by-pixel in a sample tube.

Topics: Ascorbic Acid; Electron Spin Resonance Spectroscopy; Equipment Design; Half-Life; Imaging, Three-Dimensional; Nitrogen Oxides; Oxidation-Reduction; Sensitivity and Specificity; Time Factors

Low-molecular-weight S-nitrosothiols (RSNOs) are well known for their capability to transnitrosate cysteine residues of enzymes thereby altering their catalytic activity. It is unknown, however, whether N-nitrosomelatonin (NOMela) which is highly effective in transnitrosating low-molecular-weight thiols (RSHs) can also alter protein function. In the present study, we report on such a capability with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a target enzyme. Reaction of NOMela with GAPDH resulted in an increase of RSNOs at the expense of RSHs. Somewhat surprisingly, NOMela was about 10-fold more effective than S-nitrosocysteine in inhibiting GAPDH. Vitamin C and glutathione increased the NOMela-dependent inhibition of the enzyme by accelerating the intermediacy of nitroxyl which is also highly effective in nitrosating RSHs. The occurrence of this intermediate during the NOMela-vitamin C reaction was verified by using Mn(III)-tetrakis(1-methyl-4-pyridyl)porphorin pentachloride as nitroxyl scavenger. The NOMela-dependent inactivation of GAPDH was so effective that this reaction can be used to quantify NOMela with high sensitivity.

Topics: Ascorbic Acid; Cysteine; Free Radical Scavengers; Glutathione; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Maleimides; Melatonin; Metalloporphyrins; Nitrogen Oxides; Nitroso Compounds; S-Nitrosothiols; Sulfhydryl Compounds

Aminoglutethimide (AG) is a first-generation aromatase inhibitor used for estrogen-dependent breast cancer. Unfortunately, its use has also been associated with agranulocytosis. We have investigated the metabolism of AG by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that AG oxidation by MPO/H2O2 would produce an AG cation radical that, in the absence of a biochemical reductant, would lead to the oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms only by the reaction of DMPO with a protein free radical. We found that AG metabolism by MPO/H2O2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. Glutethimide, a congener of AG that lacks the aromatic amine, did not cause DMPO-MPO formation, indicating the necessity of oxidation of the aniline moiety in AG. When analyzed by electron spin resonance spectroscopy, we detected a phenyl radical adduct, derived from AG, which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with AG/H2O2 and was found to contain DMPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by one of two free radical drug metabolites of AG, one of which was characterized by spin trapping with 2-methyl-2-nitrosopropane. These results are the first demonstration of MPO free-radical detection by the anti-DMPO antibody that results from drug oxidation. We propose that drug-dependent free radical formation on MPO may play a role in the origin of agranulocytosis.

Topics: Adenosine Triphosphate; Agranulocytosis; Aminoglutethimide; Aniline Compounds; Aromatase Inhibitors; Ascorbic Acid; Blotting, Western; Chromatography, Affinity; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Enzyme-Linked Immunosorbent Assay; Free Radicals; Glucose; Glutethimide; HL-60 Cells; Humans; Hydrogen Peroxide; Nitrogen Oxides; Nitroso Compounds; Peroxidase; Spectrophotometry

To suppress enzymatic reduction of nitroxyl group of spin probes, this study designed two new nitroxyl probes, 4-hydroxy and 4-oxopiperidine-N-oxyls having 4'-hydroxyspirocyclohexyl groups at the 2- and 6-positions of the piperidine ring (hydroxy-DICPO and oxo-DICPO, respectively). The decay of the EPR signal of these probes in mouse liver homogenates was significantly suppressed compared with that of 4-hydroxy- and 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (hydroxy-TEMPO and oxo-TEMPO, respectively), although hydroxy-DICPO and oxo-DICPO showed no difference in the reactivities with ascorbic acid. While both hydroxy- and oxo-DICPO reacted with hydroxyl radicals, only hydoxy-DICPO lost its EPR signal by the reaction with superoxide anion radical in the presence of cysteine. This feature is similar to that observed for hydroxy- and oxo-TEMPO. These results suggest that the introduction of spirocyclohexyl groups to nitroxyl spin probes is effective for protecting the nitroxyl group against enzymatic reduction without changing the characteristics of the reaction with oxygen radicals.

Topics: Animals; Ascorbic Acid; Cyclic N-Oxides; Electrochemistry; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Liver; Mice; Models, Chemical; Nitrogen Oxides; Palmitic Acids; Reactive Oxygen Species; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Spiro Compounds

Nitrite may be a source for nitric oxide (*NO), particularly in highly acidic environments, such as the stomach. Diet products contribute also with reductants that dramatically increase the production of *NO from nitrite. Red wine has been attributed health promoting properties largely on basis of the reductive antioxidant properties of its polyphenolic fraction. We show in vitro that wine, wine anthocyanin fraction and wine catechol (caffeic acid) dose- and pH-dependently promote the formation of *NO when mixed with nitrite, as measured electrochemically. The production of *NO promoted by wine from nitrite was substantiated in vivo in healthy volunteers by measuring *NO in the air expelled from the stomach, following consumption of wine, as measured by chemiluminescence. Mechanistically, the reaction involves the univalent reduction of nitrite, as suggested by the formation of *NO and by the appearance of EPR spectra assigned to wine phenolic radicals. Ascorbic and caffeic acids cooperate in the reduction of nitrite to *NO. Moreover, reduction of nitrite is critically dependent on the phenolic structure and nitro-derivatives of phenols are also formed, as suggested by caffeic acid UV spectral modifications. The reduction of nitrite may reveal previously unrecognized physiologic effects of red wine in connection with *NO bioactivity.

Topics: Antioxidants; Ascorbic Acid; Caffeic Acids; Diet; Electron Spin Resonance Spectroscopy; Flavonoids; Gastric Mucosa; Humans; Nitric Oxide; Nitrites; Nitrogen Oxides; Phenols; Polyphenols; Spectrophotometry, Ultraviolet; Wine

The quinones 1,4-naphthoquinone (NQ), methyl-1,4-naphthoquinone (MNQ), trimethyl-1,4-benzoquinone (TMQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ-0) enhance the rate of nitric oxide (NO) reduction by ascorbate in nitrogen-saturated phosphate buffer (pH 7.4). The observed rate constants for this reaction were determined to be 16+/-2,215+/-6,290+/-14 and 462+/-18 M-1 s-1, for MNQ, TMQ, NQ and UQ-0, respectively. These rate constants increase with an increase in quinone one-electron redox potential at neutral pH, E1(7). Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.

Topics: Ascorbic Acid; Benzoquinones; Naphthoquinones; Nitric Oxide; Nitrogen Oxides; Nitrous Oxide; Oxidation-Reduction

Nitroxyl radicals are known to act as radical scavenging antioxidants. In the present study, a lipophilic nitroxyl radical, cyclohexane-1-spiro-2'-(4'-oxyimidazolidine-1'-oxyl)-5'-spiro-1"-cyclohexane (nitroxyl radical I) was synthesized and its antioxidant capacity was assessed in comparison with a hydrophilic nitroxyl radical, 4-hydroxy-2,2,6,6-tetra-methylpiperidine-N-oxyl (Tempol). Both nitroxyl radical I and Tempol inhibited methyl linoleate oxidation induced by free radicals, and the efficacy increased with decreasing partial pressure of oxygen, the effect being more pronounced for nitroxyl radical I than Tempol. Their hydroxylamines inhibited lipid peroxidation more effectively than their corresponding parent nitroxyl radicals. In liposomal membranes, a synergistic effect was observed in the combination of nitroxyl radical I with ascorbic acid, whereas only an additive effect was observed between Tempol and ascorbic acid. The present study suggests that nitroxyl radical I and its hydroxylamine may act as potent antioxidants, especially in combination with ascorbic acid under hypoxic conditions.

Topics: Air; Antioxidants; Ascorbic Acid; Benzhydryl Compounds; Biochemistry; Cell Hypoxia; Cyclohexanes; Drug Synergism; Free Radicals; Hydroxylamine; Imidazolidines; Linoleic Acids; Lipid Peroxidation; Nitrogen; Nitrogen Oxides; Oxidation-Reduction; Oxygen; Pressure; Spiro Compounds

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.

Topics: Ascorbic Acid; Cyclic N-Oxides; Membranes, Artificial; Models, Chemical; Nitrogen Oxides; Solutions; Water

1. This study made use of a nitric oxide-sensitive electrode to examine possible means of generating nitric oxide from nitroxyl anion (NO(-)) released upon the decomposition of Angeli's salt. 2. Our results show that copper ions (from CuSO(4)) catalyze the rapid and efficient oxidation of nitroxyl to nitric oxide. Indeed, the concentrations of copper required to do so (0.1 - 100 microM) are roughly 100-times lower than those required to generate equivalent amounts of nitric oxide from S-nitroso-N-acetyl-D,L-penicillamine (SNAP). 3. Experiments with ascorbate (1 mM), which reduces Cu(2+) ions to Cu(+), and with the Cu(2+) chelators, EDTA and cuprizone, and the Cu(+) chelator, neocuproine, each at 1 mM, suggest that the oxidation is catalyzed by copper ions in both valency states. 4. Some compounds containing other transition metals, i.e. methaemoglobin, ferricytochrome c and Mn(III)TMPyP, were much less efficient than CuSO(4) in catalyzing the formation of nitric oxide from nitroxyl, while FeSO(4), FeCl(3), MnCl(2), and ZnSO(4) were inactive. 5. Of the copper containing enzymes examined, Cu-Zn superoxide dismutase and ceruloplasmin were weak generators of nitric oxide from nitroxyl, even at concentrations (2500 and 30 u ml(-1), respectively) vastly greater than are present endogenously. Two others, ascorbate oxidase (10 u ml(-1)) and tyrosinase (250 u ml(-1)) were inactive. 6. Our findings suggest that a copper-containing enzyme may be responsible for the rapid oxidation of nitroxyl to nitric oxide by cells, but the identity of such an enzyme remains elusive.

Topics: Analysis of Variance; Ascorbic Acid; Chelating Agents; Copper; Enzymes; Iron Compounds; Manganese Compounds; Nitric Oxide; Nitrites; Nitrogen Oxides; Oxidation-Reduction; Zinc Compounds

Previously, we reported that X-irradiation enhanced the signal decay of a spin probe injected into whole mice measured by in vivo ESR, and that the observed enhancement was suppressed by the pre-administration of cysteamine, a radioprotector [Miura, Y., Anzai, K., Urano, S. and Ozawa, T. (1997) Free Rad. Biol. Med. 23: 533-540]. In the present study, the suppression activity of the X-ray-induced increase in the ESR signal decay rate (termed suppression index, SI) was measured for several radioprotectors: 5-hydroxytryptamine (5-HT), S-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR-2721), 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPOL), cimetidine, interleukin-1 beta (IL-1 beta) and stem cell factor (SCF). The enhancement of the ESR signal decay of carbamoyl-PROXYL due to X-irradiation was suppressed by a treatment with all of the radioprotectors examined, showing positive SI values. However, a dose-dependency of 5-HT or WR-2721 was not observed, suggesting that several mechanisms exist for radioprotection and a modification of the signal decay rate. Although the in vivo ESR system cannot be used in place of the 30-day survival method for the assessment of radioprotectors, this system might be applicable to in vivo, non-invasive screening prior to using the 30-day survival method.

Topics: Amifostine; Animals; Ascorbic Acid; Chromans; Cimetidine; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Interleukin-1; Mice; Nitrogen Oxides; Oxidation-Reduction; Oxidative Stress; Pyrrolidines; Radiation-Protective Agents; Recombinant Proteins; Serotonin; Spin Labels; Stem Cell Factor; Vitamin E; Whole-Body Irradiation

Reduction-resistant nitroxides are particularly interesting for biomedical applications. beta-Phosphorylated pyrrolidinyl nitroxides, a new series of stable pyrrolidinoxyl radicals prepared in our laboratory, were tested toward ascorbate reduction in phosphate buffer at pH 7.4. The kinetics of decay were monitored by ESR and compared to those of two reference nitroxides, PCA and Proxyl. The introduction of a beta-phosphoryl group on a pyrrolidinoxyl structure resulted in a moderate increase of the reduction rate constant. However, inside the phosphorylated series, slight structural modifications can induce significant changes in the rate constants.

Topics: Ascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; In Vitro Techniques; Kinetics; Nitrogen Oxides; Oxidation-Reduction; Phosphorylation; Pyrrolidines

The interaction of nitric oxide (.NO) with Rose Bengal (RB) in the presence of electron donors was investigated. Upon illumination of a mixture of RB and .NO with visible light, an enhancement in the rate of .NO consumption was observed that increased with increasing RB concentration. In the presence of electron donors (NADH, glutathione, or ascorbate), the rates of .NO depletion increased further. NADH enhanced .NO depletion to a greater extent than either glutathione or ascorbate. Photoactivated RB under anaerobic conditions reacts with NADH to form the RB anion radical (RB.-), which has a characteristic visible absorption band centered at 418 nm. Rose Bengal anion radical disporportionates to give RB and a colorless reduced form of RB, RBH-. The net result of this process is the photobleaching of RB. The presence of .NO during irradiation of RB and NADH introduced a lag time into the kinetics of RB photobleaching. The length of this lag time was proportional to the concentration of .NO. A similar lag time, which was also dependent on the .NO concentration, was observed in the kinetics of formation of RB.-. The three-line electron spin resonance (ESR) spectrum of RB.-, with an intensity ratio 1:2:1, was obtained during irradiation of RB and NADH under anaerobic conditions. .NO introduced a concentration-dependent lag time into the kinetics of the appearance of this ESR signal. We propose that .NO oxidizes RB.- to regenerate RB and thus inhibit photobleaching until .NO is consumed. This reaction predicts the formation of NO-, the one-electron reduced form of .NO. Nitrous oxide, a characteristic dimerization product of NO-, was detected by gas chromatography. This evidence indicates the occurrence of a Type I mechanism between photoactivated RB and .NO.

Topics: Anions; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Glutathione; Kinetics; Light; Models, Chemical; NAD; Nitric Oxide; Nitrogen Oxides; Nitrous Oxide; Oxidation-Reduction; Photochemistry; Rose Bengal; Spectrophotometry

Fatty acid nitroxide radicals in CO-gassed erythrocytes are reduced by intracellular components with a half-life of about 160 min. In this study, using reduction rate constants of fatty acid spin labels to determine the reduction quantitatively, we found that catalase, glutathione, glutathione peroxidase, superoxide dismutase, and vitamin E--as well as hemoglobin, individually or in concerted manner, contributed little in reducing membrane nitroxides. Ascorbic acid appeared to be the predominant component in the erythrocyte to reduce membrane nitroxides. However, ascorbic acid solutions at 0.1 mM or less, concentrations similar to those found in the erythrocyte, produced no observable reduction in spin labeled membranes during the 2 h monitoring period. Ascorbic acid solutions at about 1 mM were needed to exhibit rate constants similar to those observed in labeled erythrocyte samples. It was also found that beta-nicotinamide adenine dinucleotide, beta-nicotinamide adenine dinucleotide phosphate, and heat-sensitive components in the erythrocyte enhanced the ability of ascorbic acid to scavenge nitroxide radicals in the erythrocyte membrane near the membrane surface.

Topics: Adult; Antioxidants; Ascorbic Acid; Carboxyhemoglobin; Catalase; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Erythrocytes; Free Radicals; Glutathione; Glutathione Peroxidase; Hemoglobins; Humans; Nitrogen Oxides; Superoxide Dismutase; Vitamin E

We have investigated the optimal nominal power settings for the electron paramagnetic resonance detection of typical nitroxides, nitroxide spin adducts, and the ascorbyl free radical. In room temperature aqueous solution, we find that, for all the nitroxides examined, saturation effects begin at approx. 25 mW nominal power with maximum signal intensity achieved at approx. 100 mW power when using a TM110 cavity. For the ascorbyl free radical, we find that saturation effects begin at approx. 16 mW nominal power and that maximum peak-to-peak signal amplitude is obtained at approx. 40 mW microwave power. For the ascorbyl free radical, we find that a modulation amplitude of approx. 0.65 G yields the maximum signal height for the doublet signal. This information will help researchers maximize the EPR signal height of minimally detectable free radicals such as encountered in biological systems.

Topics: Ascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Microwaves; Nitrogen Oxides; Spin Labels

Nitroxide free radicals interact with Hb/metHb, Mb/metMb and with peroxidases/phenols to induce a catalase-like conversion of H2O2 to O2 (catalatic activity), without being substantially consumed in the process. The mechanism of this reaction is postulated to involve a one-electron oxidation of the nitroxide to the immonium oxene, which then reacts further to release oxygen and the nitroxide. An involvement of the immonium oxene in the reaction mechanism is consistent with ferryl heme reduction by nitroxides and a detection of the reduced nitroxide when the reaction mixture is supplemented with the two-electron reductant sodium borohydride. The nitroxide-induced catalatic activity is completely inhibited when the reaction mixture is supplemented with glutathione. Nitroxides suppress free radical formation by hydroperoxide-activated heme proteins, as inferred from their inhibition of the spin-trapping of glutathionyl radicals. H2O2 decomposition and a suppression of reactive free radical formation by heme proteins appears to be an antioxidant activity of nitroxides, which is distinct from their previously reported superoxide dismutating activity and which may be a factor in their protective action in models of cardiac reperfusion injury.

Topics: Ascorbic Acid; Catalysis; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Glutathione; Hemoglobins; Hydrogen Peroxide; Myoglobin; Nitrogen Oxides; Oxidation-Reduction; Oxygen; Peroxidases; Structure-Activity Relationship

The reduction rates of five-membered pyrrolidine and pyrroline, and six-membered piperidine nitroxides (alternatively termed nitroxyls) containing various substituents were determined under homogeneous conditions using ascorbate, and electrochemically under heterogeneous conditions. The results were compared with data from the literature. It was shown that the increased rates of reduction of six-membered nitroxides, compared with those of the five-membered nitroxides, cannot be explained on the basis of differences in electrochemical potentials but, rather, can be ascribed to differences in the accessibility of the nitroxide group. A double bond in the five-membered nitroxyls increases the reduction rate. Within any ring system, the reduction rates of nitroxides using ascorbate can be correlated with the inductive substituent constants. The half-way potentials for electrochemical reduction within a series of nitroxides based on the same ring correlate with logarithms of the rates using ascorbate and with the inductive constants. The potentials for one-electron oxidation of the nitroxides were related to the inductive constants.

Topics: Ascorbic Acid; Carbon; Electrochemistry; Electrodes; Kinetics; Mercury; Nitrogen Oxides; Oxidation-Reduction; Piperidines; Pyrrolidines

Topics: Animals; Ascorbic Acid; Free Radicals; Mice; Mice, Nude; Nitrogen Oxides; Skin; Thioredoxin-Disulfide Reductase