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

Abstract

The identification of 15 N-labeled 3-nitrotyrosine (NTyr) by gas chromatography͞mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15 N-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 proteinbound NTyr in normal rat plasma to be Ϸ0-1 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 Ϸ6-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 Ϸ13 residues per 10 6 Tyr, an Ϸ40-fold elevation compared with plasma protein of untreated rats; cotreatment with N Gmonomethyl-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.

Nitrative thioredoxin inactivation as a cause of enhanced myocardial ischemia/reperfusion injury in the aging heart

“…To verify the specificity of the assay system, samples were pre-treated with sodium hydrosulfite as described by Ischiropoulos et al [19] to convert 3-nitrotyrosine to aminotyrosine [20][21][22]. In brief, ischemic/reperfused heart tissue (n=3 rats) was homogenized and the supernatant pH was adjusted to 9.0 by addition of sodium hydroxide.…”

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

“…Regardless of the mechanism of formation in vivo, protein 3-NT levels provide a useful measure of the severity of tissue exposure to reactive nitrogen species and a telling indicator of inflammatory disease severity and progression Brodsky et al, 2004;HalejcioDelophont et al, 2001;Skinner et al, 1997;Upmacis et al, 2007). Semiquantitative immunological methods (Ischiropoulos, 1998), as well as quantitative high-pressure liquid chromatography (HPLC)-based methods that utilize ultraviolet-visible (UV/VIS) absorption, electrochemistry, and mass spectrometry (MS) for detection (Frost et al, 2000;Maruyama et al, 1996;Schwedhelm et al, 1999;Shigenaga et al, 1997;Yi et al, 2000), have all been employed for the quantification of protein 3-NT residues; each method offers its own particular strengths and limitations. Importantly, these alternative 3-NT detection methods differ widely with regard to their relative sensitivity, specificity, throughput, and accessibility (because of differing requirements for expensive and/or specialized instrumentation).…”

Methodological Considerations on the Detection of 3-Nitrotyrosine in the Cardiovascular System

“…2-9 Avoidance of acidic conditions prevents artifactual formation of 3-nitrotyrosine. [3][4][5][6]9 Regarding NO 2 TyrProt, this means that plasma proteins must be hydrolyzed enzymatically under nonacidic conditions. 6,10 Especially in MS-based methods, in which artifactual formation of NO 2 Tyr from the necessary derivatization may occur, 3,4,9 separation of NO 2 Tyr from Tyr is absolutely required and can be easily achieved by HPLC.…”