Dual-function of thiocyanate on nitrite-induced formation of reactive nitrogen oxide species in human oral cavity: Inhibition under neutral and enhancement under acidic conditions

Salivary nitrate is reduced to nitric oxide (NO) via nitrite in the human oral cavity. The nitrite and NO formed can be transformed to reactive nitrogen oxide species (RNOS). In this investigation, RNOS formed in mixed whole saliva and its fractions were detected by the oxidation of aminophenyl fluorescein (APF) and the transformation of 3-amino-4-monomethylamino-2′,7′-difluorofluorecein (DAF-FM) to its triazol form (DAF-FMT). Nitrite-induced oxidation of APF and formation of DAF-FMT increased as pH was decreased from 7 to 5 and SCN^? inhibited the oxidation of APF and the formation of DAF-FMT around neutral pH and enhanced at pH about 5. The SCN^?-dependent inhibition was due to the suppression of salivary peroxidase and the enhancement was due to the formation of NOSCN from HNO₂ and SCN^?. It is deduced that the increase in the concentrations of nitrite and H⁺ in the oral cavity may result in the enhanced formation of RNOS.     

Effects of pH on nitrite-induced formation of reactive nitrogen oxide species and their scavenging by phenolic antioxidants in human oral cavity

Nitrite-induced transformation of 3-amino-4-monomethylamino-2′,7′-difluorofluorecein (DAF-FM) to the triazol form (DAF-FMT) was studied using bacterial fraction of mixed whole saliva. The transformation at pH 7 was inhibited by SCN^?, suggesting that nitrosative stresses were small in the oral cavity at the pH value as SCN^? was a normal component of saliva. DAF-FMT formation was much faster at pH 5.2 than 7 and ONSCN generated by the reaction of HNO₂ with SCN^? mainly contributed to its formation at pH 5.2. Coffee and phenolic antioxidants inhibited the DAF-FMT formation less effectively at pH 5.2 than pH 7. The less effectiveness was discussed to be due to slow scavenging of ONSCN, which contributed mainly to the formation of DAF-FMT at pH 5.2, by coffee and phenolics. Since nitrite-induced formation of ONSCN should become faster as the pH decreases, it was suggested that nitrosative stresses to oral tissues became serious under acidic conditions.     

Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes

Human saliva, which contains nitrite, is normally mixed with gastric juice, which contains ascorbic acid (AA). When saliva was mixed with an acidic buffer in the presence of 0.1 mM AA, rapid nitric oxide formation and oxygen uptake were observed. The oxygen uptake was due to the oxidation of nitric oxide, which was formed by AA-dependent reduction of nitrite under acidic conditions, by molecular oxygen. A salivary component SCN⁻ enhanced the nitric oxide formation and oxygen uptake by the AA/nitrite system. The oxygen uptake by the AA/nitrite/SCN⁻ system was also observed in an acidic buffer solution. These results suggest that oxygen is normally taken up in the stomach when saliva and gastric juice are mixed.     

Increase in gastric pH reduces hypotensive effect of oral sodium nitrite in rats

The new pathway nitrate-nitrite-nitric oxide (NO) has emerged as a physiological alternative to the classical enzymatic pathway for NO formation from l-arginine. Nitrate is converted to nitrite by commensal bacteria in the oral cavity and the nitrite formed is then swallowed and reduced to NO under the acidic conditions of the stomach. In this study, we tested the hypothesis that increases in gastric pH caused by omeprazole could decrease the hypotensive effect of oral sodium nitrite. We assessed the effects of omeprazole treatment on the acute hypotensive effects produced by sodium nitrite in normotensive and L-NAME-hypertensive free-moving rats. In addition, we assessed the changes in gastric pH and plasma levels of nitrite, NO(x) (nitrate+nitrite), and S-nitrosothiols caused by treatments. We found that the increases in gastric pH induced by omeprazole significantly reduced the hypotensive effects of sodium nitrite in both normotensive and L-NAME-hypertensive rats. This effect of omeprazole was associated with no significant differences in plasma nitrite, NO(x), or S-nitrosothiol levels. Our results suggest that part of the hypotensive effects of oral sodium nitrite may be due to its conversion to NO in the acidified environment of the stomach. The increase in gastric pH induced by treatment with omeprazole blunts part of the beneficial cardiovascular effects of dietary nitrate and nitrite.     

Oxidative Modulation of the Glutathione-redox Couple Enhances Lipopolysaccharide-induced Interleukin 12 P40 Production by a Mouse Macrophage Cell Line,J774A.1

Abstract

Reactions of salivary nitrite with components of wine were studied using an acidic mixture of saliva and wine. The formation of nitric oxide (NO) in the stomach after drinking wine was observed. The formation of NO was also observed in the mixture (pH 3.6) of saliva and wine, which was prepared by washing the oral cavity with wine. A part of the NO formation in the stomach and the oral cavity was due to the reduction of salivary nitrite by caffeic and ferulic acids present in wine. Ethyl nitrite produced by the reaction of salivary nitrite and ethyl alcohol in wine also contributed to the formation of NO. In addition to the above reactions, caffeic acid in wine could be transformed to the oxathiolone derivative, which might have pharmacological functions. The results obtained in this study may help in understanding the effects of drinking wine on human health.     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.     

Altered nitric oxide production in mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin or methamphetamine

Several studies have demonstrated the involvement of reactive nitrogen and oxygen species (RNOS) in the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridin (MPTP) and methamphetamine (METH), so the contribution of altered nitric oxide synthase (NOS) enzyme function can be suspected. In this study, about 50% increase in nitric oxide (NO) production in the mouse striatum was found between 4 and 12 h after a single MPTP injection, allowing an increased peroxynitrite (ONOO⁻) formation in the target brain region. However, METH injection induced a rapid decrease of NO formation both in mouse striatum and hippocampus, reaching its minimum level at 2 h, and restored to the control value after 6 h in the striatum and 12 h in the hippocampus. The uncoupled function of NOS with increased superoxide (O₂⁻) production after METH injection is suggested.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Characterisation of the major autoxidation products of 3-hydroxykynurenine under physiological conditions

3-Hydroxykynurenine (3-OHKyn) is a tryptophan metabolite that is readily autoxidised to products that may be involved in protein modification and cytotoxicity. The oxidation of 3-OHKyn has been studied here with a view to characterising the major products as well as determining their relative rates of formation and the role that H₂O₂ and hydroxyl radical (HO^·) may play in modifying the autoxidation process. Oxidation of 3-OHKyn generated several compounds. Xanthommatin (Xan), formed by the oxidative dimerisation of 3-OHKyn, was the major product formed initially. It was, however, found to be unstable, particularly in the presence of H₂O₂, and degraded to other products including the p-quinone, 4,6-dihydroxyquinolinequinonecarboxylic acid (DHQCA). A compound that has a structure consistent with that of hydroxy-xanthommatin (OHXan) was also formed in addition to at least two minor species that we were unable to identify. Hydrogen peroxide was formed rapidly upon oxidation of 3-OHKyn, and significantly influenced the relative abundance of the different autoxidation species. Increasing either pH (from pH 6 to 8) or temperature (from 25°C to 35°C) accelerated the rate of autoxidation but had little impact on the relative abundance of the autoxidation species. Using electron paramagnetic resonance (EPR) spectroscopy, a clear phenoxyl radical signal was observed during 3-OHKyn autoxidation and this was attributed to xanthommatin radical (Xan^·). Hydroxyl radicals were also produced during 3-OHKyn autoxidation. The HO^· EPR signal disappeared and the Xan^· EPR signal increased when catalase was added to the autoxidation mixture. The HO^· did not appear to play a role in the formation of the autoxidation products as evidenced using HO^· traps/scavengers. We propose that the cytotoxicity of 3-OHKyn may be explained by both the generation of H₂O₂ and by the formation of reactive 3-OHKyn autoxidation products such as the Xan^· and DHQCA.     

Altered Circadian Relationship Between Serum Nitric Oxide, Carbon Dioxide, and Uric Acid in Multiple Sclerosis

The free radical nitric oxide (NO^·) is involved in a variety of diverse biological processes from acting as a vasodilator in the cardiovascular system to being the rate-limiting component in the production of peroxynitrite (ONOO^-), a contributor to neurodegenerative disorders such as multiple sclerosis (MS). Uric acid (UA), the end product of purine metabolism in humans and a selective inhibitor of toxic reactions attributed to radicals formed by the interaction of ONOO^- and CO₂, is generally low in MS patients. We investigated the relationship between serum ONOO^-, CO₂, and UA in MS patients and normal controls by comparing the circadian characteristics of the NO^· metabolites nitrite/nitrate (NO), CO₂, and UA. In this preliminary study, we found the functional relationship ascribed to the circadian timing of the peak and trough levels of NO, CO₂, and UA in healthy subjects to be clearly altered in MS patients. These findings suggest that alterations in the temporal relationship between the 24 h pattern in serum ONOO^- formation and UA may either contribute to or reflect the disease processes in MS.     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Quinone-enhanced ascorbate reduction of nitric oxide: role of quinone redox potential

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, E₇¹. Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.     

The Reaction of no With Superoxide

The rate constant for the reaction of NO with ·O₂^- was determined to be (6.7 ± 0.9) × 10⁹ 1 mol^-1 s^-1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O₂^- and the formation of the product ^-OONO. The decay rate of ^-OONO, in the presence of 0.1 moll^-1 formate, ranges from 1.2s^-1 at pH 5 to about 0.2s^-1 in strong base, the latter value probably reflecting catalysis by formate.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1–4?h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1-4 h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.     

Nitroxidation, nitration, and oxidation of a BODIPY fluorophore by RNOS and ROS.

BODIPY C11 581/591 (BODIPY11) represents a sensitive probe for quantification of relative antioxidant capacity. However, the mechanism of BODIPY11 fluorescence decay in the presence of reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) requires clarification. Azo-initiators provide a continuous source of peroxyl radicals that in simple, aerobic, homogeneous, buffered solution simulate lipid peroxyl radical formation. Inhibition of BODIPY11 fluorescence decay was assayed and quantified for several families of antioxidants, including phenols, NO donors, and thiols. Fluorescence decay of BODIPY11 in these systems demonstrated similar patterns of antioxidant activity to those observed in classical oxygen pressure measurements, and provided a readily applied quantification of antioxidant capacity and mechanistic information, which was analyzed by measurement of induction periods, initial rates, and net oxidation. LC/MS analysis confirmed that peroxyl radical-induced irreversible fluorescence decay of the BODIPY11 fluorophore is due to oxidative cleavage of the activated phenyldiene side chain. The behavior of BODIPY11 towards RNOS was more complex, even in these simple systems. Incubation of BODIPY11 with bolus peroxynitrite or a sydnonimine peroxynitrite source produced a variety of novel products, characterized by LC/MS, derived from oxidative cleavage, nitroxidation, and nitration reactions. The "NO scavenger" PTIO reinforced the antioxidant activity of NO, and inhibited BODIPY11 oxidation induced by the sydnonimine. These observations suggest that BODIPY11 is a well-behaved fluorescence probe for peroxidation and antioxidant studies, but that for study of RNOS even co-application of fluorescence decay with LC/MS measurements requires careful analysis and interpretation.     

Role of antioxidants in the nitric oxide-elicited inhibition of dopamine uptake in cultured mesencephalic neurons. Insights into potential mechanisms of nitric oxide-mediated neurotoxicity

Under aerobic conditions the addition of (C₂N₅)₂N(N[O]NO)⁻ · Na⁺(DEA/NO), S-nitroso-N-macetyl penicillamine and nitric oxide (NO)-saturated buffer, but not S-nitroso- -glutathione, to dopamine solutions resulted in dopamine o-semiquinone formation that was dependent on the formation of a NO/oxygen intermediate. High pressure liquid chromatography (HPLC) electrochemical analysis of dopamine demonstrated that the DEA/NO-induced oxidation of dopamine was abrogated in the presence of the antioxidants, ascorbate and glutathione. NO spontaneously released from DEA/NO decreased [³H]dopamine accumulation in primary cultures of mesencephalic neurons in a dose-dependent fashion. In contrast, [³H]γ-aminobutyric acid uptake by mesencephalic neurons tested under the same conditions was unchanged. When DEA/NO was added to incubation buffer that contained [³H]dopamine and the antioxidant, ascorbate or glutathione, [³H]dopamine uptake was also inhibited. These data excluded that oxidation of extracellular [³H]dopamine by the intermediates of the NO/O₂ reaction could have caused this decrease. Instead, NO may have acted directly on a not yet identified target operative in the regulation of dopamine storage and release. Analysis of the rate constants for the NO reaction with ascorbate, glutathione and dopamine revealed that dopamine quinone formation was delayed by the presence of antioxidants. Since the formation of NO as well as neurotransmitter release are activated during ischemia reperfusion injury, it is possible that prolonged NO exposure could deplete antioxidants and facilitate the oxidation of dopamine and thereby cause neurotoxicity.     

Suppression of nitric oxide oxidation to nitrite by curcumin is due to the sequestration of the reaction intermediate nitrogen dioxide, not nitric oxide

Curcumin, a phytochemical with antioxidant and other cytoprotective properties, has been reported to reduce nitrite formation during nitric oxide (NO) oxidation in solution. This decrease in nitrite production was attributed to the direct sequestration of NO by curcumin. In this report, we confirm that curcumin inhibits nitrite formation from DEA/NO-derived NO in a concentration-dependent manner. However, curcumin over a concentration range of 3-50 microM had no effect on the concentration of free NO (0.5 microM) in solution at 37 degrees C as assessed using an NO electrode. We conclude that the inhibitory effect of curcumin on the oxidation of NO to nitrite is due to its known sequestration of the reaction intermediate nitrogen dioxide (NO(2)). The ability of curcumin to sequester NO(2), but not NO, suggests that curcumin may be useful for separating the actions of NO(2) from those of NO in various biological systems.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.