Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins

doi:10.1016/0278-6915(90)90173-K

Food and Chemical Toxicology

Volume 28, Issue 9, 1990, Pages 647-652

https://doi.org/10.1016/0278-6915(90)90173-K Get rights and content

Abstract

3-Nitrotyrosine (NTYR) in tissue or blood proteins was evaluated as a possible exposure marker for exogenous and endogenous nitrosating or nitrating agents. A sensitive and selective method for analysing NTYR by gas chromatography with a thermal energy analyser (GC-TEA) was developed. Using this method, a number of kinetic studies were carried out. It was found that free and protein-bound tyrosine residues easily react with nitrating/nitrosating agents to yield NTYR. NTYR formation in vivo showed a dose-dependent increase in NTYR in both plasma proteins and haemoglobin obtained from rats 24 hr after ip injection of various doses (0.5–2.5 μmol/rat) of tetranitromethane. Major urinary metabolites of NTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid (NHPL). About 44% and 5% of the oral dose of NTYR (100 μg/rat) was excreted as NHPA and NHPL, respectively. Eleven 24-hr human urine samples were analysed for NHPA by GC-TEA after ethyl acetate extraction and HPLC purification: quantities ranging from 0 to 7.9 μg/24 hr, mean ± SD 2.8 ± 2.3 (n = 11) were detected (detection limit 0.2 μg/litre). NTYR in proteins or its metabolites in urine can be readily analysed by GC-TEA as a new/additional marker for endogenous nitrosation and nitration.

References (21)

B.P. Dunn et al.
Determination of free and protein-bound N-nitrosoproline in nitrite-cured meat products
Fd Chem. Toxic.
(1984)
R.W. Giese et al.
Nitrotyrosine internal standard for amino acid analysis
Analyt. Biochem.
(1975)
O.H. Lowry et al.
Protein measurement with the Folin phenol reagent
J. biol. Chem.
(1951)
W.A. Prutz et al.
Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins
Archs Biochem. Biophys.
(1985)
H. Bartsch et al.
Human exposure to endogenous N-nitroso compounds: quantitative estimates in subjects at high risk for cancer of the oral cavity, esophagus, stomach and urinary bladder
Cancer Surveys
(1989)
R. Bonnet et al.
Nitrite and the environment
S. Calmels et al.
Screening of microorganisms for nitrosation catalysis at pH 7 and kinetic studies on nitrosamine formation from secondary amines by E. coli strains
Carcinogenesis
(1985)
S.G. Carmella et al.
Formation of hemoglobin adducts upon treatment of F344 rats with the tobacco specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N′-nitrosonornicotine
Cancer Res.
(1987)
C.-C. Cheng et al.
Estimation of nitrotyrosine derivatives in nitrosated foods
J. Formosan Med. Ass.
(1979)
M. Helman et al.
Isolation of nitrotyrosine-containing peptides by using an insoluble-antibody column
Biochem. J.
(1971)

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Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer
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Covalent amino acid modification significantly expands protein functional capability in regulating biological processes. Tyrosine residues can undergo phosphorylation, sulfation, adenylation, halogenation, and nitration. These posttranslational modifications (PTMs) result from the actions of specific enzymes: tyrosine kinases, tyrosyl-protein sulfotransferase(s), adenylate transferase(s), oxidoreductases, peroxidases, and metal-heme containing proteins. Whereas phosphorylation, sulfation, and adenylation modify the hydroxyl group of tyrosine, tyrosine halogenation and nitration target the adjacent carbon residues. Because aberrant tyrosine nitration has been associated with human disorders and with animal models of disease, we have created an updated and curated database of 908 human nitrated proteins. We have also analyzed this new resource to provide insight into the role of tyrosine nitration in cancer biology, an area that has not previously been considered in detail. Unexpectedly, we have found that 879 of the 1971 known sites of tyrosine nitration are also sites of phosphorylation suggesting an extensive role for nitration in cell signaling. Overall, the review offers several forward-looking opportunities for future research and new perspectives for understanding the role of tyrosine nitration in cancer biology.
Nitrite ion modifies tyrosine and lysine residues of extracellular matrix proteins
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Age-related macular degeneration (AMD) is a disease characterized by degenerative changes in the retinal pigment epithelium and Bruch's membrane. Inflammation is considered a major risk factor for the development and progression of AMD. Nitrite is a potent byproduct of inflammation and has been detected at elevated concentrations in AMD donor tissue. We hypothesize that nitrite chemically modifies the extracellular matrix (ECM) of Bruch's membrane as an initial step to degenerative changes observed in AMD. Non-enzymatically nitrated synthetic ECM peptides, fibronectin and laminin, were used as model systems for inflammation. Using LC/MS, we identified that nitration preferentially occurred on tyrosine and deamination of lysine under the studied conditions. At tyrosine residues, 3-nitrotyrosine was produced and shifted the total mass by the addition of 45 amu. Deamination of lysine occurred and resulted in the formation of either an alkene or alcohol group. The alkene group was observed with a loss of 17 amu. An addition of 1 amu was observed with alcohol formation. We hypothesize that these initial chemical modifications to the structure of ECM proteins may be the responsible for altering the structure and consequent function of Bruch's membrane.
Biomarkers of nitro-oxidation and oxidative stress
2018, Current Opinion in Toxicology
Macromolecule oxidation in response to reactive oxygen species (ROS) is associated with a variety of diseases. The recognition of NO as a key regulator of redox signalling has more recently led to the discovery that reactive nitrogen species (RNS) also elicit modifications of macromolecules which are also involved in pathophysiological processes. This article provides an overview of key biomarkers used to assess oxidation, peroxidation and nitration/nitrosation signalling, stressing the necessity to analyse multiple biomarkers to understand the redox mechanisms involved in biological responses.
SLE autoantibodies are well recognized by peroxynitrite-modified-HSA: Its implications in the pathogenesis of SLE
2018, International Journal of Biological Macromolecules
Systemic lupus erythematosus (SLE) is an autoimmune disorder where the role of inflammatory processes in the etiopathogenesis is well documented. Despite extensive research, the trigger for initiation of the disease has not been identified. Peroxynitrite, a strong nitrating/oxidizing agent has been reported in SLE and other autoimmune diseases. In this study, human serum albumin (HSA) was exposed to peroxynitrite for 30 min at 37 °C. The structure of HSA was grossly perturbed when examined by various physico-chemical techniques. Peroxynitrite mediated nitration of HSA was confirmed by LCMS/MS. Furthermore, increase in hydrodynamic radius of peroxynitrite-modified-HSA suggests the attachment of nitro group(s). Aggregation in peroxynitrite-modified-HSA was evident in a TEM scan. Nitration, oxidation, cross linking, aggregation etc conferred immunogenicity on peroxynitrite-modified-HSA. High titre antibodies were elicited in rabbits immunized with peroxynitrite-modified-HSA. Induced antibodies were highly specific for peroxynitrite-modified-HSA but showed considerable binding with other nitrated molecules. Direct binding/inhibition ELISA carried out with autoantibodies in SLE sera showed preferential binding with peroxynitrite-modified-HSA. Anti-nDNA positive IgG from SLE sera showed preference for peroxynitrite-modified-HSA when subjected to immunoassay (direct binding and inhibition) and mobility shift assay. Our results reinforce the role of augmented inflammation in SLE progression.
What we—authors, reviewers and editors of scientific work—can learn from the analytical history of biological 3-nitrotyrosine
2017, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
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.
Nitrated apolipoprotein AI/apolipoprotein AI ratio is increased in diabetic patients with coronary artery disease
2016, Atherosclerosis
Citation Excerpt :
Purified human HDL (MyBioSource, # MBS173147, total protein 40 mg/ml, standard curve range 1–12 ng/ml) was used as standard for the determination of plasma concentrations of apoAI. For NT-apoAI standard, purified human apoAI (Biomedical Technologies, # BT-927) was nitrated with sodium nitrite [23,24]. Briefly, purified apoAI (∼1 mg/ml) was adjusted to pH 3.5 and mixed with 200 mM sodium nitrite and then incubated at 37 °C for 24 h under low agitation.
Recent studies have suggested that determination of HDL function may be more informative than its concentration in predicting its protective role in coronary artery disease (CAD). Apolipoprotein AI (apoAI), the major protein of HDL, is nitrosylated in vivo to nitrated apoAI (NT-apoAI) that might cause dysfunction. We hypothesized that NT-apoAI/apoAI ratio might be associated with diabetes mellitus (DM) in CAD patients.
We measured plasma NT-apoAI and apoAI levels in 777 patients with coronary artery disease (CAD) by ELISA. Further, we measured plasma cholesterol efflux potential in subjects with similar apoAI but different NT-apoAI levels.
We found that median NT-apoAI/apoAI ratio was significantly higher in diabetes mellitus (DM) (n = 327) versus non-diabetic patients (n = 450). Further analysis indicated that DM, thiobarbituric acid-reactive substances and C-reactive protein levels were independent predictors of higher NT-apoAI/apoAI ratio. There was negative correlation between NT-apoAI/apoAI and use of anti-platelet and lipid lowering drugs. The cholesterol efflux capacity of plasma from 67 individuals with differing NT-apoAI but similar apoAI levels from macrophages in vitro was negatively correlated with NT-apoAI/apoAI ratio.
Higher NT-apoAI/apoAI ratio is significantly associated with DM in this relatively large German cohort with CAD and may contribute to associated complications by reducing cholesterol efflux capacity.

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Food and Chemical Toxicology

Abstract

References (21)

Determination of free and protein-bound N-nitrosoproline in nitrite-cured meat products

Fd Chem. Toxic.

Nitrotyrosine internal standard for amino acid analysis

Analyt. Biochem.

Protein measurement with the Folin phenol reagent

J. biol. Chem.

Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins

Archs Biochem. Biophys.

Human exposure to endogenous N-nitroso compounds: quantitative estimates in subjects at high risk for cancer of the oral cavity, esophagus, stomach and urinary bladder

Cancer Surveys

Nitrite and the environment

Screening of microorganisms for nitrosation catalysis at pH 7 and kinetic studies on nitrosamine formation from secondary amines by E. coli strains

Carcinogenesis

Formation of hemoglobin adducts upon treatment of F344 rats with the tobacco specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N′-nitrosonornicotine

Cancer Res.

Estimation of nitrotyrosine derivatives in nitrosated foods

J. Formosan Med. Ass.

Isolation of nitrotyrosine-containing peptides by using an insoluble-antibody column

Biochem. J.

Cited by (239)

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer

Nitrite ion modifies tyrosine and lysine residues of extracellular matrix proteins

Biomarkers of nitro-oxidation and oxidative stress

SLE autoantibodies are well recognized by peroxynitrite-modified-HSA: Its implications in the pathogenesis of SLE

What we—authors, reviewers and editors of scientific work—can learn from the analytical history of biological 3-nitrotyrosine

Nitrated apolipoprotein AI/apolipoprotein AI ratio is increased in diabetic patients with coronary artery disease