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The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00–1.34) and 0.97 (0.00–1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Plasma S-nitrosothiols (RSNOs) may act as a circulating form of nitric oxide that affects vascular function and platelet aggregation. Their role in liver ischemia/reperfusion (I/R) injury is largely unknown. The aim of the present study was to investigate the changes in plasma RSNOs following liver I/R injury. Two groups of New Zealand white rabbits were used (n=6, each): the I/R group underwent 60 min lobar liver ischemia and 7 h reperfusion, while the sham group underwent laparotomy but no liver ischemia. Serial RSNO levels were measured in plasma by electron paramagnetic resonance (EPR) spectrometry, nitrite/nitrates by capillary electrophoresis, hepatic microcirculation by laser Doppler flowmetry, redox state of hepatic cytochrome oxidase by near-infrared spectroscopy, liver iNOS mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) and the oxidation of dihydrorhodamine to rhodamine by fluorescence. The effect of the antioxidant N-acetylcysteine (NAC) on RSNOs formation and DHR oxidation was tested in a third group of animals (n=6) undergoing lobar liver I/R. Hepatic I/R was associated with a significant increase in plasma RSNOs, plasma nitrites, hepatic iNOS mRNA expression, impairment in hepatic microcirculation, decrease in the redox state of cytochrome oxidase, and significant production of rhodamine. The changes were more obvious during the late phase of reperfusion (>4 h). NAC administration decreased plasma RSNOs and oxidation of DHR to RH (P