Document Detail


Artifactual nitration controlled measurement of protein-bound 3-nitro-L-tyrosine in biological fluids and tissues by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.
MedLine Citation:
PMID:  12387616     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A sensitive and selective method is presented to accurately determine the level of protein-bound 3-nitro-L-tyrosine (NTyr) in rat plasma and kidney samples. This assay is based on isotope dilution liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation entails protein precipitation, acid hydrolysis with 6 N HCl, and solid-phase extraction (using reverse and aminopropyl phase cartridges) prior to the determinative step. For kidney samples, NTyr is converted into its butyl ester to improve sensitivity. The potential formation of artifactual NTyr during the acid hydrolysis step was carefully followed and determined by supplementation of the samples with (13)C-labeled L-tyrosine (Tyr) prior to protein digestion. Hence, the concomitant measurement of formation of (13)C-enriched NTyr enabled the accurate determination of artifactual NTyr. This approach was employed to measure the basal level of protein-bound NTyr in rat plasma and kidney samples, revealing levels in the range of 4-18 micromol/mol of Tyr and 50-68 micromol/mol of Tyr, respectively. No artifactual nitration of Tyr was observed in kidney proteins, whereas in the case of plasma the contribution of the artifactual response ranged from 16 to 40%. This method allows the analysis of protein-bound NTyr with a full control of the artifactual nitration of tyrosine during the proteolysis and/or sample preparation. Reliable detection of NTyr in proteins may allow insight into the role of nitric oxide-derived oxidants under various pathological conditions.
Authors:
Thierry Delatour; Janique Richoz; Jacques Vuichoud; Richard H Stadler
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Chemical research in toxicology     Volume:  15     ISSN:  0893-228X     ISO Abbreviation:  Chem. Res. Toxicol.     Publication Date:  2002 Oct 
Date Detail:
Created Date:  2002-10-21     Completed Date:  2003-04-17     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8807448     Medline TA:  Chem Res Toxicol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1209-17     Citation Subset:  IM    
Affiliation:
Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland. thierry.delatour@rdls.nestle.com
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MeSH Terms
Descriptor/Qualifier:
Animals
Artifacts
Chemical Precipitation
Chromatography, Liquid / methods
Female
Hydrolysis
Kidney / chemistry
Protein Binding
Rats
Rats, Inbred F344
Sensitivity and Specificity
Spectrometry, Mass, Electrospray Ionization / methods
Tyrosine / analogs & derivatives*,  analysis*,  chemistry*
Chemical
Reg. No./Substance:
3604-79-3/3-nitrotyrosine; 55520-40-6/Tyrosine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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