Inflammation and NOx-induced nitration: Assay for 3-nitrotyrosine by HPLC with electrochemical detection

AUTOR(ES)
FONTE

The National Academy of Sciences of the USA

RESUMO

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-l-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be ≈0–1 residues per 106 Tyr with a detection limit of 0.5 per 107 Tyr when ≥100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an ≈6-fold higher level of protein-bound NTyr compared with control cells and cells treated with NG-monomethyl-l-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to ≈13 residues per 106 Tyr, an ≈40-fold elevation compared with plasma protein of untreated rats; cotreatment with NG-monomethyl-l-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NOx exposure such as inflammation and smoking.

ACESSO AO ARTIGO

Documentos Relacionados