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Dimitrios Tsikas – One of the best experts on this subject based on the ideXlab platform.
What we-authors, reviewers and editors of scientific work-can learn from the analytical history of biological 3-Nitrotyrosine.Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2017Co-Authors: Dimitrios TsikasAbstract:
Tyrosine and tyrosine residues in proteins are attacked by the reactive oxygen and nitrogen species peroxynitrite (O=N-OO-) to generate 3-Nitrotyrosine (3-NT) and 3-Nitrotyrosine-proteins (3-NTProt), respectively. 3-NT and 3-NTProt are widely accepted as biomarkers of nitr(os)ative stress. Over the years many different analytical methods have been reported for 3-NT and 3-NTProt. Reported concentrations often differ by more than three orders of magnitude, indicative of serious analytical problems. Strategies to overcome pre-analytical and analytical shortcomings and pitfalls have been proposed. The present review investigated whether recently published work on the quantitative measurement of biological 3-Nitrotyrosine did adequately consider the analytical past of this biomolecule. 3-Nitrotyrosine was taken as a representative of biomolecules that occur in biological samples in the pM-to-nM concentration range. This examination revealed that in many cases the main protagonists involved in the publication of scientific work, i.e., authors, reviewers and editors, failed to do so. Learning from the analytical history of 3-Nitrotyrosine means advancing analytical and biological science and implies the following key issues. (1) Choosing the most reliable analytical approach in terms of sensitivity and accuracy; presently this is best feasible by stable-isotope dilution tandem mass spectrometry coupled with gas chromatography (GC-MS/MS) or liquid chromatography (LC-MS/MS). (2) Minimizing artificial formation of 3-Nitrotyrosine during sample work up, a major pitfall in 3-Nitrotyrosine analysis. (3) Validating adequately the final method in the intendent biological matrix and the established concentration range. (4) Inviting experts in the field for critical evaluation of the novelty and reliability of the proposed analytical method, placing special emphasis on the compliance of the analytical outcome with 3-Nitrotyrosine concentrations obtained by validated GC-MS/MS and LC-MS/MS methods.
Homoarginine and 3-Nitrotyrosine in patients with takotsubo cardiomyopathyInternational journal of cardiology, 2014Co-Authors: Arslan Arinc Kayacelebi, Thanh H. Nguyen, Christopher Neil, John D. Horowitz, Jens Jordan, Dimitrios TsikasAbstract:
In recent years, homoargininewas shown to be a cardiovascular risk factor , and to herald a poor prognosis in heart failure patients . Yet, the underlying mechanism is elusive. Human and animal studies suggest that the enzyme responsible for the biosynthesis of homoarginine is arginine:glycine amidinotransferase (AGAT) [3–5]. Previously, excessive myocardial AGATgene expressionwas observed in heart failure; the authors implicated AGAT in cardiac creatine synthesis . This finding suggests that homoarginine synthesis in the myocardiummay be elevated in heart failure. Thus far, there is no information about the homoarginine homeostasis in takotsubo cardiomyopathy (TTC) and which potential role this quite neglected non-proteinogenic amino acid may play in the development and recovery of TTC. In TTC patients we recently observed that the plasma concentration of asymmetric dimethylarginine (ADMA), another arginine homologue, is lower than the control, whereas the responsiveness to nitric oxide (NO) is substantially greater compared to healthy females . This is of particular interest, because both L-arginine and L-homoarginine serve as substrates for NO synthases (NOS), while ADMA inhibits NOScatalyzed production of NO from these substrates . The aim of the present study was to measure plasma homoarginine concentration inTTC patients and healthy controls of a previous study  and to determine its relationship to 3-Nitrotyrosine, a biomarker of NO-related oxidative stress. Plasma homoarginine and 3nitrotyrosine were measured by validated, previously reported gas chromatography-tandem mass spectrometry (GC-MS/MS) methodologies [9,10]. Written informed consent was provided by all subjects included in the study, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the local Ethics Committee of the Central Northern Adelaide Health Service: the Queen Elizabeth Hospital and Lyell McEwin Hospital (protocol number, 009014). The plasma concentration of homoarginine was significantly reduced in TTC patients compared to healthy controls (mean ± SEM; 1298 ± 112 nmol/L vs. 2094 ± 321 nmol/L; median 1403 nmol/L vs. 1634 nmol/L) (Fig. 1A). 3-Nitrotyrosine plasma concentrations were similar in TTC patients and in healthy controls (mean ± SEM; 2355 ± 217 pmol/L vs. 2227 ± 146 pmol/L; median 1915 pmol/L vs. 2170 pmol/L) (Fig. 1B). Pearson correlation between homoarginine and 3-Nitrotyrosine concentrations revealed a significant negative relationship in TTC patients (Fig. 2A). In contrast, a positive relationship was observed in the control group (Fig. 2B). No relationship was obtained when all homoarginine and 3-Nitrotyrosine data from TTC patients and controls were correlated (not shown). In the TTC patients, plasma homoarginine concentration correlated inversely with systolic blood pressure (SBP) (Fig. 2C). It is worth mentioning that plasma homoarginine concentrationwas found to correlate positively with SBP in an elderly population (50–87 years) non-suffering from takotsubo cardiomyopathy . In contrast, plasma 3-Nitrotyrosine concentration did not correlate with SBP (Fig. 2C). Our study indicates that plasma homoarginine concentrations are reduced in TTC patients. They are considerably lower than those measured by us and others in healthy subjects [1–5]. With the exception of four TTC patients, the plasma concentrations of 3nitrotyrosine measured in the other TTC patients and in the control subjects are comparable with those reported in the literature for healthy and ill subjects. The limited number of TTC patients and healthy controls investigated in our study limits the power of our findings. Nevertheless, the results of the present study in TTC supports recent studies indicating homoarginine as a novel marker of cardiovascular disease [1–5]. In contrast to elderly males and females with normal or impaired glucose metabolism or with type 2 diabetes mellitus but without TTC , in our TTC patients there was a negative correlation
Analytical methods for 3-Nitrotyrosine quantification in biological samples: the unique role of tandem mass spectrometryAmino Acids, 2012Co-Authors: Dimitrios TsikasAbstract:
Reactive-nitrogen species, such as peroxynitrite (ONOO^−) and nitryl chloride (NO_2Cl), react with the aromatic ring of tyrosine in soluble amino acids and in proteins to form 3-Nitrotyrosine. The extent of nitration can be quantified by measuring 3-Nitrotyrosine in biological matrices, such as blood, urine, and tissue. This article reviews and discusses current analytical methodologies for the quantitative determination of 3-Nitrotyrosine in their soluble and protein-associated forms, with the special focus being on free 3-Nitrotyrosine. Special emphasis is given to analytical approaches based on the tandem mass spectrometry methodology. Pitfalls and solutions to overcome current methodological problems are emphasized and requirements for quantitative analytical approaches are recommended. The reliability of current analytical methods and the suitability of 3-Nitrotyrosine as a biomarker of nitrative stress are thoroughly examined.
Harry Ischiropoulos – One of the best experts on this subject based on the ideXlab platform.
Identification of immunoglobulins that recognize 3-Nitrotyrosine in patients with acute lung injury after major trauma.American journal of respiratory cell and molecular biology, 2006Co-Authors: Leonor Thomson, Stanley L. Hazen, Jason D. Christie, Caryn Vadseth, Paul N. Lanken, Harry IschiropoulosAbstract:
Tyrosine nitration is a nitric oxide–derived post-translational modification of proteins. Elevated levels of specific plasma proteins modified by tyrosine nitration have been detected during acute and chronic inflammatory conditions, including acute lung injury (ALI). In the present study we examined whether circulating immunoglobulins against nitrated proteins are present in the plasma of subjects with clinically documented ALI. Affinity chromatography using covalently linked 3-Nitrotyrosine was employed to identify plasma proteins that bind to this unusual amino acid. Western blotting and liquid chromatography-tandem mass spectrometry of in-gel digested protein bands revealed that the major proteins eluted from the affinity column were IgM and IgG. An enzyme-linked immunosorbent assay (ELISA) based on competition of horseradish peroxidase–derivatized 3-Nitrotyrosine binding to plasma with unlabeled 3-Nitrotyrosine was developed and validated. Using this ELISA, the levels of immunoglobulins that recognize 3-Nitrotyrosine were significantly higher in the plasma of subjects with ALI compared with both normal control subjects and subjects with major trauma who did not develop ALI (0.36± 0.14 versus 0.03 ± 0.05, and 0.25 ± 0.15; P < 0.001 and P = 0.006, respectively). These data indicate that tyrosine-nitrated proteins induce the production of specific immunoglobulins during acute phase response and inflammation.
Metabolism of 3-Nitrotyrosine Induces Apoptotic Death in Dopaminergic CellsThe Journal of neuroscience : the official journal of the Society for Neuroscience, 2006Co-Authors: Beatrice Blanchard-fillion, Delphine Prou, Manuela Polydoro, David Spielberg, Elpida Tsika, Zeneng Wang, Stanley L. Hazen, Michael Koval, Serge Przedborski, Harry IschiropoulosAbstract:
Intrastriatal injection of 3-Nitrotyrosine, which is a biomarker for nitrating oxidants, provokes dopaminergic neuronal death in rats by unknown mechanisms. Herein, we show that extracellular 3-Nitrotyrosine is transported via the l-aromatic amino acid transporter in nondopaminergic NT2 cells, whereas in dopaminergic PC12 cells, it is transported by both the l-aromatic amino acid and the dopamine transporters. In both cell lines, 3-Nitrotyrosine is a substrate for tyrosine tubulin ligase, resulting in its incorporation into the C terminus of α-tubulin. In NT2 cells, incorporation of 3-Nitrotyrosine into α-tubulin induces a progressive, reversible reorganization of the microtubule architecture. In PC12 cells, 3-Nitrotyrosine decreases intracellular dopamine levels and is metabolized by the concerted action of the aromatic amino acid decarboxylase and monoamine oxidase. Intracellular levels of 133 μmol of 3-Nitrotyrosine per mole of tyrosine did not alter NT2 viability but induced PC12 apoptosis. The cell death was reversed by caspases and aromatic amino acid decarboxylase and monoamine oxidase inhibitors. 3-Nitrotyrosine induced loss of tyrosine hydroxylase-positive primary rat neurons, which was also prevented by an aromatic amino acid decarboxylase inhibitor. These findings provide a novel mechanism by which products generated by reactive nitrogen species induce dopaminergic neuron death and thus may contribute to the selective neurodegeneration in Parkinson9s disease.
Oxygen Tension and Inhaled Nitric Oxide Modulate Pulmonary Levels of S-Nitrosocysteine and 3-Nitrotyrosine in RatsPediatric Research, 2004Co-Authors: Scott A. Lorch, David Munson, Richard T Lightfoot, Harry IschiropoulosAbstract:
The oxidative environment within the lung generated upon administration of oxygen may be a critical regulator for the efficacy of inhaled nitric oxide therapy, possibly as a consequence of changes in nitrosative and nitrative chemistry. Changes in S-nitrosocysteine and 3-Nitrotyrosine adducts were therefore evaluated after exposure of rats to 80% or >95% oxygen for 24 or 48 h with and without 20 ppm inhaled nitric oxide. Exposure to 80% oxygen led to increased formation of S-nitrosocysteine and 3-Nitrotyrosine adducts in lung tissue that were also associated with increased expression of iNOS. The addition of inhaled nitric oxide in 80% oxygen exposure did not alter any of these adducts in the lung or in the bronchoalveolar lavage (BAL). Exposure to >95% oxygen led to a significant decrease in S-nitrosocysteine and an increase in 3-Nitrotyrosine adducts in the lung. Co-administration of inhaled nitric oxide with >95% oxygen prevented the decrease in S-nitrosocysteine levels. The levels of S-nitrosocysteine and 3-Nitrotyrosine returned to baseline in a time-dependent fashion after termination of exposure to >95% oxygen and inhaled nitric oxide. These data suggest the formation of S-nitrosating and tyrosine-nitrating species is regulated by oxygen tensions and co-administration of inhaled nitric oxide restores the nitrosative chemistry without a significant impact upon the nitrative pathway.
Jay W Heinecke – One of the best experts on this subject based on the ideXlab platform.
Copper-mediated intra-ligand oxygen transfer in gas-phase complexes with 3-Nitrotyrosine.Journal of mass spectrometry : JMS, 2005Co-Authors: Tomas Vaisar, Jay W Heinecke, Jennifer L. Seymour, František TurečekAbstract:
Gas-phase ternary complexes with Cu(II) and 2,2′-bipyridine (bpy) of tyrosine, 3-aminotyrosine, 3-Nitrotyrosine and 3-Nitrotyrosine methyl ether are formed readily upon electrospraying aqueous methanol solutions containing the components. In contrast to Cu(bpy) complexes of tyrosine, 3-aminotyrosine and other aromatic amino acids, the complexes of 3-Nitrotyrosine and its methyl ether undergo unusual collisionally activated dissociations (CADs) that involve Cu-mediated transfer of an oxygen atom from the nitro group. With 3-Nitrotyrosine this results in an expulsion of carbonic acid, H2CO3, whereas with 3-Nitrotyrosine methyl ether an OH migration forms Cu(OH)bpy+ as the predominant product. To the best of our knowledge, this is the first case of an intra-ligand redox reaction in a gas-phase organometallic complex. The reaction mechanism of this unusual dissociation was elucidated by a combination of isotope labeling, accurate mass measurements, energy-resolved CAD mass spectra and density functional theory calculations of ion structures and relative energies. Copyright © 2005 John Wiley & Sons, Ltd.
Myeloperoxidase produces nitrating oxidants in vivo.Journal of Clinical Investigation, 2002Co-Authors: Joseph P Gaut, Jaeman Byun, Hung D. Tran, Wendy M. Lauber, James A Carroll, Richard S. Hotchkiss, Abderrazzaq Belaaouaj, Jay W HeineckeAbstract:
Despite intense interest in pathways that generate reactive nitrogen species, the physiologically relevant mechanisms for inflammatory tissue injury remain poorly understood. One possible mediator is myeloperoxidase, a major constituent of neutrophils, monocytes, and some populations of macrophages. The enzyme uses hydrogen peroxide and nitrite to generate 3-Nitrotyrosine in vitro. To determine whether myeloperoxidase produces nitrating intermediates in vivo, we used isotope dilution gas chromatography/mass spectrometry to quantify 3-Nitrotyrosine in two models of peritoneal inflammation: mice infected with Klebsiella pneumoniae and mice subjected to cecal ligation and puncture. Both models developed an intense neutrophil inflammatory response, and the inflammatory fluid contained markedly elevated levels of 3-chlorotyrosine, a marker of myeloperoxidase action. In striking contrast, 3-Nitrotyrosine levels rose only in the mice infected with K. pneumoniae. Levels of total nitrite and nitrate were 20-fold higher in mice injected with K. pneumoniae than in mice subjected to cecal ligation and puncture. Levels of 3-Nitrotyrosine failed to increase in mice infected with K. pneumoniae that lacked functional myeloperoxidase. Our observations provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available.
artifact free quantification of free 3 chlorotyrosine 3 bromotyrosine and 3 nitrotyrosine in human plasma by electron capture negative chemical ionization gas chromatography mass spectrometry and liquid chromatography electrospray ionization tandem mAnalytical Biochemistry, 2002Co-Authors: Joseph P Gaut, Jaeman Byun, Hung D. Tran, Jay W HeineckeAbstract:
Halogenation and nitration of biomolecules have been proposed as key mechanisms of host defense against bacteria, fungi, and viruses. Reactive oxidants also have the potential to damage host tissue, and they have been implicated in disease. In the current studies, we describe specific, sensitive, and quantitative methods for detecting three stable markers of oxidative damage: 3-chlorotyrosine, 3-bromotyrosine, and 3-Nitrotyrosine. Our results indicate that electron capture–negative chemical ionization–gas chromatography/mass spectrometry (EC–NCI GC/MS) is 100-fold more sensitive than liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-MS/MS) for analyzing authentic 3-chlorotyrosine, 3-bromotyrosine, and 3-Nitrotyrosine. Using an isotopomer of tyrosine to evaluate artifactual production of the analytes during sample preparation and analysis, we found that artifact generation was negligible with either technique. However, LC-MS/MS proved cumbersome for analyzing multiple samples because it required 1.5 h of run and equilibration time per analysis. In contrast, EC-NCI GC/MS required only 5 min of run time per analysis. Using EC-NCI GC/MS, we were able to detect and quantify attomole levels of free 3-chlorotyrosine, 3-bromotyrosine, and 3-Nitrotyrosine in human plasma. Our results indicate that EC-NCI GC/MS is a sensitive and specific method for quantifying free 3-chlorotyrosine, 3-bromotyrosine, and 3-Nitrotyrosine in biological fluids in a single, rapid analysis and that it avoids generating any of the analytes ex vivo.