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Dimitrios Tsikas - One of the best experts on this subject based on the ideXlab platform.
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Homoarginine and 3-Nitrotyrosine in patients with takotsubo cardiomyopathy
International 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 [1], and to herald a poor prognosis in heart failure patients [2]. 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 [6]. 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 [7]. 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 [8]. The aim of the present study was to measure plasma homoarginine concentration inTTC patients and healthy controls of a previous study [7] 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 [11]. 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 [11], in our TTC patients there was a negative correlation
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Analytical methods for 3-Nitrotyrosine quantification in biological samples: the unique role of tandem mass spectrometry
Amino 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.
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Determination of 3-Nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2005Co-Authors: Dimitrios Tsikas, Anja Mitschke, Maria-theresia Suchy, Frank-mathias Gutzki, Dirk O. StichtenothAbstract:Abstract 3-Nitrotyrosine (NO2Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-Nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)–tandem mass spectrometry (MS) for circulating 3-Nitrotyrosine. The present article describes the extension of this method to urinary 3-Nitrotyrosine. The method involves separation of urinary 3-Nitrotyrosine from nitrite, nitrate and l -tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-Nitrotyrosine and the internal standard 3-nitro- l -[2H3]tyrosine, and GC–tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5 ± 7.2) 3-Nitrotyrosine levels were determined to be 8.4 ± 10.4 nM (range, 1.6–33.2 nM) or 0.46 ± 0.49 nmol/mmol creatinine (range, 0.05–1.30 nmol/mmol creatinine). The present GC–tandem MS method provides accurate values of 3-Nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro- l -tyrosine by a healthy volunteer (27.6 μg/kg body weight) 3-nitro- l -tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro- l -tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-Nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.
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Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System
Circulation research, 2002Co-Authors: Dimitrios Tsikas, Edzard Schwedhelm, Jürgen C. FrölichAbstract:To the Editor: Reactive nitrogen species (RNS; eg, •NO, •NO2, ONOO−, NO2Cl) react preferably with tyrosine (Tyr) and protein-associated tyrosine (TyrProt) to form 3-Nitrotyrosine, ie NO2Tyr and NO2TyrProt, respectively.1 Therefore, detection of NO2Tyr and/or NO2TyrProt provides evidence for generation of RNS rather than specifically peroxynitrite (ONOO−).1 Besides this difficulty, Tarpey and Fridovich2 have recently discussed, in an article published in Circulation Research , the problematic measurement of NO2Tyr and NO2TyrProt, giving special attention to artifactual formation of NO2Tyr and NO2TyrProt by acidification of biological samples. This methodological pitfall is very important and well-recognized,3–10⇓⇓⇓⇓⇓⇓⇓ but it is not the sole methodological problem in 3-Nitrotyrosine detection. Tarpey and Fridovich2 restricted their discussion exclusively to artifactual formation of 3-Nitrotyrosine referring to Yi et al3 and Frost et …
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Gas chromatographic-tandem mass spectrometric quantification of free 3-Nitrotyrosine in human plasma at the basal state.
Analytical biochemistry, 1999Co-Authors: Edzard Schwedhelm, Dimitrios Tsikas, Frank-mathias Gutzki, Jürgen C. FrölichAbstract:A fully validated gas chromatographic–tandem mass spectrometric (GC–tandem MS) method for the accurate and precise quantification of free 3-Nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4–4.2 nM) for free 3-Nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-Nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-Nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-Nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-Nitrotyrosine and 3-nitro-l-[2H3]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-μl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester–pentafluoropropionyl amide–trimethylsilyl ether derivatives, and quantified by GC–tandem MS. Overall recovery was determined as 50 ± 5% using 3-nitro-l-[14C9]tyrosine. The limit of detection of the method was 4 amol of 3-Nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[14C9]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of ≥80% and a precision of ≥94%. The method should be useful to investigate the utility of plasma free 3-Nitrotyrosine as an indicator of nitric oxide (•NO)-associated oxidative stress in vivo in humans.
Jay W Heinecke - One of the best experts on this subject based on the ideXlab platform.
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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.
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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 m
Analytical Biochemistry, 2002Co-Authors: Joseph P Gaut, Hung D. Tran, Jaeman Byun, 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.
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Isotope Dilution Mass Spectrometric Quantification of 3-Nitrotyrosine in Proteins and Tissues Is Facilitated by Reduction to 3-Aminotyrosine
Analytical biochemistry, 1998Co-Authors: Jan R. Crowley, Kevin E. Yarasheski, Christiaan Leeuwenburgh, John Turk, Jay W HeineckeAbstract:Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previousin vitrostudies have suggested that 3-Nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indicator of tissue injury by nitrogen speciesin vivo.Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-Nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-Nitrotyrosine by ion exchange chromatography, and reduction of 3-Nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to itsn-propyl, per-heptafluorobutyryl derivative and quantified by isotope dilution gas chromatography negative-ion chemical ionization mass spectrometry. Attomole levels of 3-Nitrotyrosine can be reproducibly measured in this manner. Quantifying 3-Nitrotyrosine levels of tissues by stable isotope dilution gas chromatography/mass spectrometry should provide a powerful tool for exploring the impact of reactive nitrogen species on oxidative reactionsin vivo.
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reactive nitrogen intermediates promote low density lipoprotein oxidation in human atherosclerotic intima
Journal of Biological Chemistry, 1997Co-Authors: Christiaan Leeuwenburgh, Stanley L. Hazen, Medora M Hardy, Peter Wagner, Shuji Ohishi, Urs P Steinbrecher, Jay W HeineckeAbstract:Oxidized low density lipoprotein (LDL) may be of central importance in triggering atherosclerosis. One potential pathway involves the production of nitric oxide (NO) by vascular wall endothelial cells and macrophages. NO reacts with superoxide to form peroxynitrite (ONOO-), a potent agent of LDL oxidation in vitro. ONOO- nitrates the aromatic ring of free tyrosine to produce 3-Nitrotyrosine, a stable product. To explore the role of reactive nitrogen species such as ONOO- in the pathogenesis of vascular disease, we developed a highly sensitive and specific method involving gas chromatography and mass spectrometry to quantify 3-Nitrotyrosine levels in proteins. In vitro studies demonstrated that 3-Nitrotyrosine was a highly specific marker for LDL oxidized by ONOO-. LDL isolated from the plasma of healthy subjects had very low levels of 3-Nitrotyrosine (9 +/- 7 micromol/mol of tyrosine). In striking contrast, LDL isolated from aortic atherosclerotic intima had 90-fold higher levels (840 +/- 140 micromol/mol of tyrosine). These observations strongly support the hypothesis that reactive nitrogen species such as ONOO- form in the human artery wall and provide direct evidence for a specific reaction pathway that promotes LDL oxidation in vivo. The detection of 3-Nitrotyrosine in LDL isolated from vascular lesions raises the possibility that NO, by virtue of its ability to form reactive nitrogen intermediates, may promote atherogenesis, counteracting the well-established anti-atherogenic effects of NO.
Harry Ischiropoulos - One of the best experts on this subject based on the ideXlab platform.
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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.
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Metabolism of 3-Nitrotyrosine Induces Apoptotic Death in Dopaminergic Cells
The 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.
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Oxygen Tension and Inhaled Nitric Oxide Modulate Pulmonary Levels of S-Nitrosocysteine and 3-Nitrotyrosine in Rats
Pediatric 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.
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Plasma 3-Nitrotyrosine and outcome in neonates with severe bronchopulmonary dysplasia after inhaled nitric oxide
Free radical biology & medicine, 2003Co-Authors: Scott A. Lorch, Harry Ischiropoulos, Beverly A Banks, Jason D. Christie, Jeffrey D. Merrill, John S. Althaus, Keri Schmidt, Philip L. Ballard, Roberta A. BallardAbstract:Plasma protein levels of 3-Nitrotyrosine and 3-chlorotyrosine were measured by LC-MS/MS at 0 and 72 h after the initiation of inhaled nitric oxide (INO) at 20 ppm in 22 prematurely born infants with clinically documented bronchopulmonary dysplasia. Infants were classified at the time of hospital discharge as either "off mechanical ventilation," "on mechanical ventilation," or "expired/organ failure." These outcomes were tested for association with changes in plasma levels of 3-Nitrotyrosine and 3-chlorotyrosine and selected clinical risk factors. Infants whose 3-Nitrotyrosine levels decreased over the 72 h period were more likely to wean off of mechanical ventilation (p =.03). There was no significant association between changes in 3-chlorotyrosne levels and outcome. After controlling for other variables, an odds ratio of 8.3 (95% CI: 1.3-54.4) for improved outcomes was observed if the 3-Nitrotyrosine levels decreased. These data suggest that nitrative and oxidative stress may be related to the severity of lung disease and, consequentially, the overall outcome in this select group of infants with severe bronchopulmonary dysplasia.
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Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-Nitrotyrosine.
Free Radical Biology and Medicine, 2000Co-Authors: Richard Lightfoot, David Shuman, Harry IschiropoulosAbstract:Abstract The oxygen-insensitive nitroreductases nfsA and nfsB are known to reduce para-nitrated aromatic compounds. We tested the hypothesis that these nitroreductases are capable of reducing 3-Nitrotyrosine in proteins and peptides, as well as in free amino acids using wild-type and nfsA nfsB mutant strains of Escherichia coli. E. coli homogenates were incubated with nitrated proteins and the level of 3-Nitrotyrosine immunoreactivity was assayed by Western blotting. Assay conditions that allow the nitroreductases to rapidly reduce nitrofurantoin did not result in the modification of 3-Nitrotyrosine in protein, peptide, or free amino acid. Stimulation of nfsA nfsB activity with paraquat had no effect on 3-Nitrotyrosine reduction. Nonlethal exposure of E. coli to peroxynitrite/CO2 resulted in the reproducible nitration of tyrosine residues in endogenous proteins. The degree of 3-Nitrotyrosine immunoreactivity over the 2-h postexposure period did not differ between mutant and wild-type strains. These results indicate that the nfsA and nfsB enzymes do not reduce 3-Nitrotyrosine.
Paul J. Thornalley - One of the best experts on this subject based on the ideXlab platform.
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Assay of 3-Nitrotyrosine in tissues and body fluids by liquid chromatography with tandem mass spectrometric detection.
Methods in enzymology, 2008Co-Authors: Naila Rabbani, Paul J. ThornalleyAbstract:3-Nitrotyrosine (3-NT) is a marker of protein nitration in physiological systems. It is present as 3-Nitrotyrosine residues in proteins of tissue, extracellular matrix, plasma, and other body fluids and food. It is also present in body fluids and some beverages as free Nitrotyrosine and is excreted in urine with the major urinary metabolite 3-nitro-4-hydroxyphenylacetic acid. Quantitation of 3-Nitrotyrosine requires tandem mass spectrometry for specific detection. The method developed to determine 3-Nitrotyrosine (along with protein glycation and oxidation adducts in a quantitative screening assay) by liquid chromatography with tandem mass spectrometric detection is described. The 3-NT residue contents of plasma protein, hemoglobin, lipoproteins, and cerebrospinal fluid protein and the concentrations of free 3-Nitrotyrosine in plasma, urine, and cerebrospinal fluid are given. Changes of 3-Nitrotyrosine residue and free 3-Nitrotyrosine in diabetes, cirrhosis, acute and chronic renal failure, and neurological disorders, including Alzheimer's disease, are presented and compared with independent estimates.
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protein glycation oxidation and nitration adduct residues and free adducts of cerebrospinal fluid in alzheimer s disease and link to cognitive impairment
Journal of Neurochemistry, 2005Co-Authors: Naila Ahmed, Paul J. Thornalley, Usman Ahmed, Klaus Hager, Gerd Fleischer, Gerald MunchAbstract:Increased damage to proteins by glycation, oxidation and nitration has been implicated in neuronal cell death leading to Alzheimer's disease (AD). Protein glycation, oxidation and nitration adducts are consequently formed. Quantitative screening of these adducts in CSF may provide a biochemical indicator for the diagnosis of AD. To assess this, we measured 11 glycation adducts, three oxidation adducts and a nitration adduct, determining both protein adduct residues and free adducts, in CSF samples of age-matched normal healthy subjects (n = 18) and subjects with Alzheimer's disease (n = 32). In CSF protein, the concentrations of 3-Nitrotyrosine, Nɛ-carboxymethyl-lysine, 3-deoxyglucosone-derived hydroimidazolone and N-formylkynurenine residues were increased in subjects with Alzheimer's disease. In CSF ultrafiltrate, the concentrations of 3-Nitrotyrosine, methylglyoxal-derived hydroimidazolone and glyoxal-derived hydroimidazolone free adducts were also increased. The Mini-Mental State Examination (MMSE) score correlated negatively with 3-Nitrotyrosine residue concentration (p < 0.05), and the negative correlation with fructosyl-lysine residues just failed to reach significance (p = 0.052). Multiple linear regression gave a regression model of the MMSE score on 3-Nitrotyrosine, fructosyl-lysine and Nɛ-carboxyethyl-lysine residues with p-values of 0.021, 0.031 and 0.052, respectively. These findings indicate that protein glycation, oxidation and nitration adduct residues and free adducts were increased in the CSF of subjects with Alzheimer's disease. A combination of nitration and glycation adduct estimates of CSF may provide an indicator for the diagnosis of Alzheimer's disease.
Jürgen C. Frölich - One of the best experts on this subject based on the ideXlab platform.
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Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System
Circulation research, 2002Co-Authors: Dimitrios Tsikas, Edzard Schwedhelm, Jürgen C. FrölichAbstract:To the Editor: Reactive nitrogen species (RNS; eg, •NO, •NO2, ONOO−, NO2Cl) react preferably with tyrosine (Tyr) and protein-associated tyrosine (TyrProt) to form 3-Nitrotyrosine, ie NO2Tyr and NO2TyrProt, respectively.1 Therefore, detection of NO2Tyr and/or NO2TyrProt provides evidence for generation of RNS rather than specifically peroxynitrite (ONOO−).1 Besides this difficulty, Tarpey and Fridovich2 have recently discussed, in an article published in Circulation Research , the problematic measurement of NO2Tyr and NO2TyrProt, giving special attention to artifactual formation of NO2Tyr and NO2TyrProt by acidification of biological samples. This methodological pitfall is very important and well-recognized,3–10⇓⇓⇓⇓⇓⇓⇓ but it is not the sole methodological problem in 3-Nitrotyrosine detection. Tarpey and Fridovich2 restricted their discussion exclusively to artifactual formation of 3-Nitrotyrosine referring to Yi et al3 and Frost et …
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Gas chromatographic-tandem mass spectrometric quantification of free 3-Nitrotyrosine in human plasma at the basal state.
Analytical biochemistry, 1999Co-Authors: Edzard Schwedhelm, Dimitrios Tsikas, Frank-mathias Gutzki, Jürgen C. FrölichAbstract:A fully validated gas chromatographic–tandem mass spectrometric (GC–tandem MS) method for the accurate and precise quantification of free 3-Nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4–4.2 nM) for free 3-Nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-Nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-Nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-Nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-Nitrotyrosine and 3-nitro-l-[2H3]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-μl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester–pentafluoropropionyl amide–trimethylsilyl ether derivatives, and quantified by GC–tandem MS. Overall recovery was determined as 50 ± 5% using 3-nitro-l-[14C9]tyrosine. The limit of detection of the method was 4 amol of 3-Nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[14C9]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of ≥80% and a precision of ≥94%. The method should be useful to investigate the utility of plasma free 3-Nitrotyrosine as an indicator of nitric oxide (•NO)-associated oxidative stress in vivo in humans.