Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

Abstract

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 approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) 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 approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-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 NO(X) exposure such as inflammation and smoking.

Figure 1

Conversion of NTyr to AcATyr

Figure 2

Treatment of rat plasma with peroxynitrite. Concentration-dependent formation of protein-bound NTyr. Rat plasma (200 μl) was treated with peroxynitrite (•) and processed according to Materials and Methods. Rat plasma was also treated with peroxynitrite that was allowed to decompose at pH 7.2 for 15 min before addition (○). Values represent the average of three determinations ± SD. ∗, P < 0.05; ∗∗, P < 0.01, one-tailed Students t test.

Figure 3

Representative HPLC chromatograms. AcATyr (A, EC) and AcTyr (B, UV) were monitored in processed hydrolyzates from RAW 264.7 cultures: spike = 300 fmol authentic AcATyr.