Measurement of nitric oxide metabolites and protein nitration in healthy and inflammatory human tissues and bio-fluids

Abstract

The central thesis of this project is that damage caused by reactive nitrogen species, e.g. 3-nitrotyrosine (Tyr-NO2), constitutes a marker of disease progression/severity. A new sensitive electrochemiluminescence ELISA was optimised and validated for Tyr-NO2 measurement, giving a lower limit of quantification of 0.04 nM BSA-NO2, intra- and inter-assay CVs of 6.5% and 11.3%, an average recovery of 106 ± 3% and average linearity 0.998 ± 0.001. Nitrative stress, carbonyl stress and C-reactive protein (CRP) concentrations were measured before and after major elective surgery. CRP measurements confirmed the induction of an inflammatory response. Median serum Tyr-NO2 levels increased post-surgery to a median (inter-quartile range) value of 0.97 (0 – 1.7) fmol nitrated BSA (BSA-NO2) equivalents/mg protein compared with a pre-surgery level of 0.59 (0 – 1.3) fmol BSA-NO2 equivalents/mg protein (p<0.05). Oxidative damage was confirmed by serum protein carbonyl levels (p<0.05). In a second pre-/post- surgery study, patients who developed sepsis postoperatively had significantly higher serum Tyr-NO2 levels one day prior to diagnosis (median (IQR) 4.5 (1.65 – 8.21) fmol BSA-NO2 equivalents/mg protein) compared to patients without sepsis (1.2 (0.74 – 5.97) fmol BSA-NO2 equivalents/mg protein; p<0.05). Tyr-NO2 levels have not previously been measured before clinical diagnosis. However, Tyr-NO2 did not improve upon CRP as a diagnostic marker (area under the curve: Tyr-NO2 0.69 versus CRP 0.88). Nitrate (NO3¯) supplementation in healthy smokers was also studied. Plasma Tyr-NO2 levels were unaltered by supplementation or smoking status. Salivary nitration was unaffected by smoking and decreased with NO3¯ supplementation: the median (IQR) pre-supplementation was 0.67 (0.31-1.14) and post-supplementation was 0.43 (0.12-0.61) pmol BSA-NO2 equivalents/mg protein. Ozone-based chemiluminescence was utilised for nitrite (NO2¯) and NO3¯ measurement as indicators of ˙NO production. Plasma and salivary NO2¯ and NO3¯ concentrations increased significantly with NO3¯ supplementation (p<0.05). In contrast to published studies, brain frontal lobe Tyr-NO2 levels were not higher in dementia: the median (IQR) levels in dementia were 0.29 (0.19-0.57) and in non-dementia controls were 0.3 (0.22-0.55) pmol BSA-NO2 equivalents/mg protein. However, the median brain tissue NO2¯ concentration was significantly higher in the Alzheimer’s disease group (p<0.05). Western blotting revealed that nitration was predominantly in a few select proteins, with TOF-MS/MS analysis suggesting haemoglobin is one of these proteins. Measurement of nitrative stress using ozone-based chemiluminescence and an electrochemiluminescence-based-ELISA overcomes earlier methodological flaws, such as low sensitivity. Detection of total Tyr-NO2 in different inflammatory states indicates that its measurement could have potential as a marker of disease, but measurement of nitration in specific proteins may be more informative than total Tyr-NO2.