INFLAMMATION AND NOX-INDUCED NITRATION - ASSAY FOR 3-NITROTYROSINE BYHPLC WITH ELECTROCHEMICAL DETECTION

The identification of N-15-labeled 3-nitrotyrosine (NTyr) by gas chrom atography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with N-15-L-arginine confirms that nitric oxide synthase (NOS) is involved In the nitration of protein-bound ty rosine (Tyr), An assay is presented for NTyr that employs HPLC with ta ndem electrochemical and UV detection. The assay involves enzymatic hy drolysis 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 esti mate the level of protein-bound NTyr in normal rat plasma to be approx imate to 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when greater than or equal to 100 nmol of Tyr is analyzed an d when precautions are taken to limit nitration artifacts, Zymosan-tre ated RAW 264.7 cells were shown to have an approximate to 6-fold highe r level of protein-bound NTyr compared with control cells and cells tr eated with N-G-monomethyl-L-arginine, an inhibitor of NOS, Intraperito neal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximate to 13 residues per 10(6) Tyr, an app roximate to 40-fold elevation compared with plasma protein of untreate d rats; cotreatment with N-G-monomethyl-L-arginine inhibited the forma tion of NTyr in plasma protein from blood and peritoneal exudate by 69 % and 53%, respectively. This assay offers a highly sensitive and quan titative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states a ssociated with NOx exposure such as inflammation and smoking.