Protein-bound 3-nitrotyrosine Research Articles

High glucose leads to redistribution of the proteasomal system.

The impact of high glucose on the cellular redox state, causing both induction of antioxidative systems and also enhanced protein oxidation is discussed for a long time. It is established that elevated glucose levels are disrupting the cellular proteostasis and influencing the proteasomal system. However, it is still unresolved whether this is due to a reaction of the cellular proteasomal system towards the high glucose or whether this is a secondary reaction to inflammatory stimuli. Therefore, we used a dermal fibroblast cell line exposed to high glucose in order to reveal whether a response of the proteasomal system takes place. We investigated the α4 and the inducible iβ5 subunits of the 20S proteasome, as well as the Rpn1-subunit of the 19S proteasomal regulator complex, measured activity of the 20S, 20S1, and 26S proteasome and detected as well changes in expression as a redistribution into the nucleus. Interestingly, while the activity of the proteasomal forms rather decreased under high glucose treatment; higher expression levels of components of the proteasomal system and higher concentrations of protein-bound 3-nitrotyrosine and Nrf2 (nuclear factor [erythroid-derived 2]-like 2) were detected. However, no change in the cytosol-nucleus distribution could be detected for most of the quantified parameters. We concluded that high glucose alone, without additional inflammatory stimuli, provokes a regulatory response on the ubiquitin-proteasomal system.

Identification and relative quantification of 3-nitrotyrosine residues in fibrinogen nitrated in vitro and fibrinogen from ischemic stroke patient plasma using LC-MS/MS

Ischemic stroke is one of the leading causes of death and disability worldwide. This acute vascular event interferes with blood supply to the brain and induces a burst of free radicals such as nitric oxide and superoxide, producing peroxynitrite, a precursor of strong nitrating agents. Fibrinogen is one of the most abundant plasma proteins; it plays a role in the hemostatic system, mediating clot formation, which can be affected by nitrotyrosine formation. We hypothesized that nitration of fibrinogen by ONOOH and ONOOCO2− radical products could be one of the early events of the ischemic stroke, and protein-bound 3-nitrotyrosine could be a potential biomarker for diagnosis and/or prognosis of this condition. A targeted mass spectrometry approach was developed to analyze the nitration of fibrinogen and its association with ischemic stroke. First, a comprehensive mapping of 3-nitrotyrosine locations and their relative quantification was performed by LC-MS/MS, using in vitro nitrated fibrinogen samples. Twenty different 3-nitrotyrosine residues were identified on fibrinogen nitrated in vitro, varying with the peroxynitrite tofibrinogen molar ratio used. Nine tyrosine residues that were consistently modified at different treatment ratios were chosen to perform a targeted LC-MS/MS analysis in clinical samples. Enriched fibrinogen fractions from clinical samples from 24 ischemic stroke and 12 patients with non-inflammatory conditions were analysed with this method. Three of the nine tyrosine residues analysed (βY452, βY475 and γY380) showed a significant difference between the ischemic stroke and non-inflammatory disease groups. ROC curve analysis suggested an association of these residues either individually or in combination with ischemic stroke. Different tyrosine nitration patterns were also observed in fibrinogen modified in vitro and in vivo, suggesting differences in the nitration process in these situations. This is the first study showing a putative association between the nitration profile of specific tyrosine residues in human fibrinogen and ischemic stroke.

Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.

Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•–) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected.

Detection of protein-bound 3-nitrotyrosine in the plasma of pediatric patients with severe ARDS and avian influenza virus infection

Detection of protein-bound 3-nitrotyrosine in the plasma of pediatric patients with severe ARDS and avian influenza virus infection

Denitrase activity of Debaryomyces hansenii reduces the oxidized compound 3-nitrotyrosine in mice liver with colitis.

The oxidation of tyrosine to 3-nitrotyrosine is irreversible, and due to this characteristic, 3-nitrotyrosine is used as a marker for oxidative stress in a range of diverse chronic and degenerative diseases. It has been established that the yeast Debaryomyces hansenii (D. hansenii) can assimilate free 3-nitrotyrosine as unique source of nitrogen, and during saline stress, has a high denitrase activity to detoxify this compound in a reaction that involves the liberation of nitrogen dioxide from 3-nitrotyrosine. However, until now it has not been determined whether D. hansenii can detoxify protein-bound 3-nitrotyrosine such as nitrated proteins present in different chronic illnesses. TThe aim of the present study was to evaluate the denitrase activity of D. hansenii to reduce 3-nitrotyrosine from liver proteins of mice with colitis. Firstly, the levels of reactive oxygen species of liver tissue of colitic and control mice were measured by the reaction with the 2'7'-dichlorofluorescein diacetate. Denitrase activity of D. hansenii was evaluated by incubating cell extracts of the yeast with protein extracts from livers of mice with colitis. Following incubation, 3-nitrotyrosine was measured, and to corroborate that denitrase reaction had occurred, the production of nitrites was measured. In samples of liver tissue from mice with colitis, the maximum levels of reactive oxygen species were up to two times higher compared with the control livers. Following the incubation of colitic liver samples with cell extracts of D. hansenii, it was observed that 3-nitrotyrosine decreased to the basal concentration of control liver samples, and that the concentration of nitrites was increased. These results indicate that denitrase of D. hansenii extracts can effectively detoxify 3-nitrotyrosine bound to proteins and that the extracts could be used to decrease protein oxidation damage in chronic degenerative diseases.

The role of charge in the toxicity of polymer-coated cerium oxide nanomaterials to Caenorhabditis elegans

This study examined the impact of surface functionalization and charge on ceria nanomaterial toxicity to Caenorhabditis elegans. The examined endpoints included mortality, reproduction, protein expression, and protein oxidation profiles. Caenorhabditis elegans were exposed to identical 2–5nm ceria nanomaterial cores which were coated with cationic (diethylaminoethyl dextran; DEAE), anionic (carboxymethyl dextran; CM), and non-ionic (dextran; DEX) polymers. Mortality and reproductive toxicity of DEAE-CeO2 was approximately two orders of magnitude higher than for CM-CeO2 or DEX-CeO2. Two-dimensional gel electrophoresis with orbitrap mass spectrometry identification revealed changes in the expression profiles of several mitochondrial-related proteins and proteins that are expressed in the C. elegans intestine. However, each type of CeO2 material exhibited a distinct protein expression profile. Increases in protein carbonyls and protein-bound 3-nitrotyrosine were also observed for some proteins, indicating oxidative and nitrosative damage. Taken together the results indicate that the magnitude of toxicity and toxicity pathways vary greatly due to surface functionalization of CeO2 nanomaterials.

Neuroprotective Efficacy of Mitochondrial Antioxidant MitoQ in Suppressing Peroxynitrite-Mediated Mitochondrial Dysfunction Inflicted by Lead Toxicity in the Rat Brain.

Lead (Pb) is one of the most pollutant metals that accumulate in the brain mitochondria disrupting mitochondrial structure and function. Though oxidative stress mediated by reactive oxygen species remains the most accepted mechanism of Pb neurotoxicity, some reports suggest the involvement of nitric oxide (•NO) and reactive nitrogen species in Pb-induced neurotoxicity. But the impact of Pb neurotoxicity on mitochondrial respiratory enzyme complexes remains unknown with no relevant report highlighting the involvement of peroxynitrite (ONOO-) in it. Herein, we investigated these effects in in vivo rat model by oral application of MitoQ, a known mitochondria-specific antioxidant with ONOO- scavenging activity. Interestingly, MitoQ efficiently alleviated ONOO--mediated mitochondrial complexes II, III and IV inhibition, increased mitochondrial ATP production and restored mitochondrial membrane potential. MitoQ lowered enhanced caspases 3 and 9 activities upon Pb exposure and also suppressed synaptosomal lipid peroxidation and protein oxidation accompanied by diminution of nitrite production and protein-bound 3-nitrotyrosine. To ascertain our in vivo findings on mitochondrial dysfunction, we carried out similar experiments in the presence of different antioxidants and free radical scavengers in the in vitro SHSY5Y cell line model. MitoQ provided better protection compared to mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase suggesting the predominant involvement of ONOO- compared to •NO and O2•-. However, dimethylsulphoxide and catalase failed to provide protection signifying the noninvolvement of •OH and H2O2 in the process. The better protection provided by MitoQ in SHSY5Y cells can be attributed to the fact that MitoQ targets mitochondria whereas mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase are known to target mainly cytoplasm and not mitochondria. Taken together the results from the present study clearly brings out the potential of MitoQ against ONOO--induced toxicity upon Pb exposure indicating its therapeutic potential in metal toxicity.

Increased S-nitrosothiols are associated with spinal cord injury in multiple sclerosis

Multiple sclerosis (MS) is an immune-mediated disorder associated with inflammation, demyelination and axonal damage. In search of potential biomarkers of spinal cord lesions in MS related to nitric oxide metabolites, we measured total nitrite and nitrate levels, and protein-bound nitrotyrosine and S-nitrosothiol concentrations in the serum of MS patients at different stages of the disease. Sixty-eight patients and 36 healthy volunteers were included in the study. Total nitrite and nitrate levels were augmented in relapsing-remitting MS, while increased S-nitrosothiol concentrations were found both in relapsing-remitting and secondary-progressive MS. Further analysis demonstrated that S-nitrosothiol levels were selectively increased in patients with spinal cord injury. The data suggest that high S-nitrosothiol concentration may be a potential serum biomarker for spinal cord injury in MS.

Protective effects of Lycium barbarum polysaccharide on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway.

Oxidative stress plays an important role in Parkinson’s disease and other neurodegenerative disorders. Lycium barbarum polysaccharides (LBP), the main active ingredients extracted from the fruits of Lycium barbarum L., have been shown to be a potent antioxidant. In the present study, we investigated the protective effects, and the possible mechanism of action of LBP against 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells. Our data demonstrated that LBP significantly reversed the 6-OHDA-induced decrease in cell viability, prevented 6-OHDA-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. Furthermore, LBP also slowed the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), decreased the level of protein-bound 3-nitrotyrosine (3-NT) and intracellular free Ca2+, and inhibiting the overexpression of nuclear factor κB (NF-κB), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). These results demonstrate that LBP prevents 6-OHDA-induced apoptosis in PC12 cells, at least in part through the ROS-NO pathway.

Immunoglobulins Against Tyrosine-Nitrated Epitopes in Coronary Artery Disease

Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine-nitrated proteins, a footprint of oxygen- and nitrogen-derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of patients with coronary artery disease (CAD). However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of patients with CAD. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in patients with CAD (n=374) and subjects without CAD (non-CAD controls, n=313). A 10-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in patients with CAD (3.75±1.8 μg antibody Eq/mL plasma versus 0.36±0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. The results of this cross-sectional study suggest that posttranslational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neo-epitopes for the elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD.

Enzyme immunoassay for detection of protein-bound nitrotyrosine in brain tissue and cerebrospinal fluid: Methodological issues

Protein-bound nitrotyrosine is a standard marker of nitrosative stress in vivo, its levels being associated with severity of nitrosative stress in neurological diseases. Using two commercially available antibodies we have developed protocols for an indirect competitive enzyme-linked immunosorbent assay (ELISA) aimed at detecting protein-bound nitrotyrosine. The working concentration range of the assay is 0.094–4 μg/mL, the lowest limit of detection 0.063 μg/mL, IC50 0.538 μg/mL. In a pilot study protein-bound nitrotyrosine levels in cerebrospinal fluid of acute stroke patients and in brain tissue of rats after focal ischemia were evaluated.

Protective effect of tetrahydroxystilbene glucoside on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway.

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4′-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress.

Antibody‐Free Detection of Protein Tyrosine Nitration in Tissue Sections

Inflamed proteins: Tyrosine nitration is a covalent post-translational protein modification that represents a biomarker for inflammatory diseases. We introduce a novel method to detect nitrotyrosine in biological samples by chemoselective conversion into a fluorophore. We describe the applicability of this methodology to detect protein-bound nitrotyrosine by fluorescence microscopy in tissue sections and on Western blot membranes.

Myocardial stunning is associated with impaired calcium uptake by sarcoplasmic reticulum

Myocardial stunning (temporary post-ischaemic contractile dysfunction) may be caused by oxidative stress and/or impaired myocyte calcium homeostasis. Regional myocardial stunning was induced in open-chest pigs (segment shortening reduced to 68.3 ± 4.7% of baseline) by repetitive brief circumflex coronary occlusion (I/R). Reduced glutathione was depleted in stunned myocardium (1.34 ± 0.06 vs. 1.77 ± 0.11 nmol/mg, p = 0.02 vs. remote myocardium) indicating regional oxidant stress, but no regional differences were observed in protein-bound 3-nitrotyrosine or S-nitrosothiol content. Repetitive I/R did not affect myocardial quantities of the sarcolemmal sodium–calcium exchanger, L-type channel, SR calcium ATPase and phospholamban, or the kinetics of ligand binding to L-type channels and SR calcium release channels. However, initial rates of oxalate-supported 45Ca uptake by SR were impaired in stunned myocardium (41.3 ± 13.5 vs. 73.0 ± 15.6 nmol/min/mg protein, p = 0.03). The ability of SR calcium ATPase to sequester cytosolic calcium is impaired in stunned myocardium. This is a potential mechanism underlying contractile dysfunction.

Biodistribution and oxidative stress effects of a systemically-introduced commercial ceria engineered nanomaterial

The objective was to characterize the biodistribution of nanoscale ceria from blood and its effects on oxidative stress endpoints. A commercial 5% crystalline ceria dispersion in water (average particle size ~31±4 nm) was infused intravenously into rats (0, 50, 250 and 750 mg/kg), which were terminated 1 or 20 h later. Biodistribution in rat tissues was assessed by microscopy and ICP-AES/MS. Oxidative stress effects were assessed by protein-bound 4-hydroxy 2-trans-nonenal (HNE), protein-bound 3-nitrotyrosine (3-NT), and protein carbonyls. Evans blue (EB)-albumin and Na fluorescein (Na2F) were given intravenously as blood-brain barrier integrity markers. The initial ceria t½ in blood was ~7 min. Brain EB and Na2F increased some at 20 h. Microscopy revealed peripheral organ ceria agglomerations but little in the brain. Spleen Ce concentration was >liver >blood >brain. Reticuloendothelial tissues cleared ceria. HNE was significantly increased in the hippocampus at 20 h. Protein carbonyl and 3-NT changes were small. The nanoparticle characterizations before and after biodistribution, linked with the physiological responses, provide a foundation for evaluating the effects of engineered nanomaterial physico-chemical properties on peripheral organ distribution, brain entry and resultant toxicity.

The role of nitric oxide on DNA damage induced by benzene metabolites

Benzene, a tobacco constituent, is a leukemogen in humans and a carcinogen in rodents. Several benzene metabolites generate superoxide anion (O(2)(.-)) and induce nitric oxide synthase in the bone marrow of mice. We hypothesized that the reaction of nitric oxide (*NO) with O(2)(.-) leads to the formation of peroxynitrite as an intermediate during benzene metabolism. This hypothesis was supported by demonstrating that the exposure of mice to benzene produced nitrated metabolites and enhanced the levels of protein-bound 3-nitrotyrosine in the bone marrow of mice in vivo. In the current study, we investigated the influence of nitric oxide, generated from sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, on DNA strand breaks induced by each single or binary benzene metabolite at different doses and compared the levels of the DNA damage induced by each benzene metabolite in the presence of nitric oxide with the levels of DNA strand breaks induced by peroxynitrite at similar doses in vitro. We found that among benzene metabolites only 1,2,4-trihydroxybenzene (BT) can induce significant DNA damage in the absence of nitric oxide. While 1,4-dihydroxybenzene (HQ), 1,4-benzoquinone (BQ) and 1,2-dihydroxybenzene (CAT) require .NO to induce DNA strand breaks, hydroquinone was the most potent DNA-damaging benzene metabolite in the presence of *NO. The order of DNA breaks by benzene metabolites in the presence of *NO is: Peroxynitrite = HQ > BT > BQ > CAT. The *NO and O(2)(.-) scavengers inhibited DNA damage induced by [HQ+*NO]. Benzene, trans,trans-muconaldehyde, and phenol, do not induce DNA strand breaks either in the absence or presence of *NO. However, adding phenol to [HQ+*NO] leads to greater DNA damage than [HQ+*NO] alone. Collectively, these results suggest that nitric oxide is an important factor in DNA damage induced by certain benzene metabolites, probably via the formation of the peroxynitrite intermediate. Phenol, the major benzene metabolite that does not induce DNA damage alone and is inactive in vivo, synergistically enhances DNA damage induced by potent benzene metabolite in the presence of nitric oxide.

Atherosclerosis: A Link between Lipid Intake and Protein Tyrosine Nitration

Atherosclerosis, a disease characterized by plaque formation in the arterial wall that can lead to heart attack and stroke, is a principal cause of death in the world. Since the 1990's, protein nitrotyrosine formation has been known to occur in the atherosclerotic plaque. This potentially damaging reaction occurs as a result of tyrosine modification by reactive nitrogen species, such as nitrogen dioxide radical, which forms upon peroxynitrite decomposition or nitrite oxidation by hydrogen peroxide-activated peroxidase enzymes. The presence of protein-bound nitrotyrosine can be considered an indicator of a loss in the natural balance of oxidants and antioxidants, and as such, there is an emerging view that protein-bound nitrotyrosine may be a risk factor for cardiovascular disease. This review brings together evidence that the accumulation of protein nitrotyrosine during atherogenesis is more widespread than initially thought (as its presence can be detected not only in the lesion but also in the blood stream and other organs) and is closely linked to lipid intake.

Nitration of soluble proteins in organotypic culture models of Parkinson's disease

Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP +) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP + treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 μM MPP +, but significant DA depletions occurred at much lower concentrations of MPP + (1 μM). Chronic MPP + or rotenone treatments (3 weeks) caused maximum protein nitration by 1 μM (twofold) or 10 nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 μM) prevented protein nitration by MPP +, but did not protect against MPP +-induced DA depletion or inhibition of TH activity. Acute incubation with 100 μM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.

Distinct inhibitory mechanisms of isoquercitrin gallate and its aglycone on zymosan-induced peroxynitrite production in macrophages

Peroxynitrite, the coupling product of superoxide and nitric oxide (NO) radicals, plays as a pathogenic mediator in the oxidative stress-implicated diseases. Quercetin 3- O-β-(2″galloyl)-glucopyranoside (Q-32) is an isoquercitrin gallate. In this study, Q-32 was found to inhibit zymosan-induced production of protein-bound 3-nitrotyrosine, a stable metabolite of peroxynitrite, in macrophages RAW 264.7, and its inhibitory mechanism was also documented to be different from that of its aglycone, quercetin. Both Q-32 and quercetin inhibited not only zymosan- but also phorbol 12-myristate 13-acetate-induced superoxide productions in the macrophages. Q-32 did not affect NO production and inducible NO synthase (iNOS) expression in zymosan-stimulated macrophages RAW 2647. However, quercetin inhibited zymosan-induced NO production as well as down-regulated zymosan-induced iNOS expression at the transcription level. Further, quercetin inhibited zymosan-induced nuclear factor (NF)-κB transcriptional activity but also NF-κB-dependent iNOS promoter activity. Taken together, Q-32 and quercetin could provide invaluable tools to investigate zymosan-induced oxidative stress with distinct antioxidant mechanisms.

Disposition of Protein-bound 3-nitrotyrosine in Rat Plasma Analysed by a Novel Protocol for HPLC-ECD

3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 +/- 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 +/- 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 +/- 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo.