Artifactual Nitration Controlled Measurement of Protein-Bound 3-Nitro-L-tyrosine in Biological Fluids and Tissues by Isotope Dilution Liquid Chromatography Electrospray Ionizati
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- 作者：Thierry Delatour ; Janique Richoz ; Jacques Vuichoud ; and Richard H. Stadler
- 刊名：Chemical Research in Toxicology
- 出版年：2002
- 出版时间：October 2002
- 年：2002
- 卷：15
- 期：10
- 页码：1209 - 1217
- 全文大小：163K
- 年卷期：v.15,no.10(October 2002)
- ISSN：1520-5010
文摘
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 onisotope dilution liquid chromatography coupled with electrospray ionization tandem massspectrometry (LC-ESI-MS/MS). The sample preparation entails protein precipitation, acidhydrolysis with 6 N HCl, and solid-phase extraction (using reverse and aminopropyl phasecartridges) prior to the determinative step. For kidney samples, NTyr is converted into itsbutyl ester to improve sensitivity. The potential formation of artifactual NTyr during the acidhydrolysis step was carefully followed and determined by supplementation of the samples with13C-labeled L-tyrosine (Tyr) prior to protein digestion. Hence, the concomitant measurementof formation of 13C-enriched NTyr enabled the accurate determination of artifactual NTyr.This approach was employed to measure the basal level of protein-bound NTyr in rat plasmaand kidney samples, revealing levels in the range of 4-18 
mol/mol of Tyr and 50-68 mol/mol of Tyr, respectively. No artifactual nitration of Tyr was observed in kidney proteins, whereasin the case of plasma the contribution of the artifactual response ranged from 16 to 40%. Thismethod allows the analysis of protein-bound NTyr with a full control of the artifactual nitrationof tyrosine during the proteolysis and/or sample preparation. Reliable detection of NTyr inproteins may allow insight into the role of nitric oxide-derived oxidants under variouspathological conditions.
mol/mol of Tyr and 50-68 mol/mol of Tyr, respectively. No artifactual nitration of Tyr was observed in kidney proteins, whereasin the case of plasma the contribution of the artifactual response ranged from 16 to 40%. Thismethod allows the analysis of protein-bound NTyr with a full control of the artifactual nitrationof tyrosine during the proteolysis and/or sample preparation. Reliable detection of NTyr inproteins may allow insight into the role of nitric oxide-derived oxidants under variouspathological conditions.