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.

no / Vascular inflammation is implicated in the pathogenesis of atherosclerosis and abdominal aortic aneurysm (AAA), and is thought to involve reactive species such as the nitric oxide-derived oxidant peroxynitrite. In the present study nitrotyrosine was measured as a stable marker of peroxynitrite production in vivo. Perioperative blood samples were obtained from patients undergoing elective open or endovascular repair of an AAA and from patients with intermittent claudication, smoking aged-matched controls, non-smoking aged-matched controls and non-smoking young healthy controls. Plasma nitrotyrosine was measured by an enzyme-linked immunosorbent assay. The median plasma nitrotyrosine concentration in patients with an AAA (0·46 nmol nitrated bovine serum albumin equivalents per mg protein) was significantly higher than that in patients with intermittent claudication (0·35 nmol; P = 0·002), smoking controls (0·36 nmol; P = 0·001), non-smoking controls (0·35 nmol; P = 0·002) and young healthy controls (0·27 nmol; P < 0·001). Nitrotyrosine concentrations increased during early reperfusion in open AAA repair, but not during endovascular repair. AAA exclusion from the circulation reduced levels to control values (P = 0·001). Patients with an AAA had raised levels of circulating nitrated proteins compared with patients with claudication and controls, suggesting a greater degree of ongoing inflammation that was not related to smoking. Copyright

Abstract The aim of the study was to evaluate pulmonary endogenous and inhaled nitric oxide (NO) in neonates with severe respiratory failure. Infant autopsy documents were reviewed for fulminant early-onset bacterial pneumonia. 12 infants with the onset at &lt; 72 h of age and three control groups were identified. Immunohistochemistry revealed that 11 of the infants with early-onset pneumonia (92%) had no or faint inducible nitric oxide synthase (NOS2) staining in their alveolar macrophages (AM). All control infants, regardless of their postnatal age, had NOS2-positive AM. The marker of NO-toxicity, nitrotyrosine, was low in all specimens. To confirm this finding, airway specimens of 21 newborns requiring mechanical ventilation were examined. Seven of them had fulminant early-onset pneumonia with maternal ascending intra-uterine infection (IUI). The controls had no infection at birth despite IUI or neither infection nor IUI. In early-onset pneumonia, NOS2 and nitrotyrosine immunoreactivity were low at birth and increased during the recovery phase (p &lt; 0.05). Analyses of interleukin-1 and surfactant protein A showed the same pattern of age-dependent change. Of the autopsied infants, 12 had received inhaled NO (iNO) before death. Each case was paired with a matched control. Additional five infants without respiratory failure prior to death were also studied. The iNO-treated ones tended to have more intensive NOS2 staining in the bronchiolar epithelium and adjacent tissue than the controls. No differences in other NOS isoforms or nitrotyrosine were detected. A novel method for exhaled NO measurements of intubated infants was developed. Six preterm and six term newborns were prospectively recruited for expired and nasal NO measurements. During the first week of life, the preterm infants showed a different pattern of exhaled NO excretion compared to the term infants. For the pilot intervention study on very early iNO, the eligible patients had a birth weight &lt; 1500 g and progressive, therapy-resistant respiratory failure before five hours of age. Five infants received iNO, showed immediately improved oxygenation and survived without deleterious side effects. Deficient production of NO in small premature infants is associated with severe infection and respiratory failure. Very early iNO therapy may be exceptionally effective in a select group of infants, and did not appear to cause oxidation lung injury.

No description available.

3-nitrotyrosine (3NT), a stable end product arising from the interaction of proteins and reactive nitrogen species such as peroxynitrite, is produced during periods of oxidative stress. 3NT is, therefore, of interest as a potential biomarker in a variety of disease states where oxidative stress is known to be involved in the pathology, for example intermittent claudication. The aim of this thesis was to develop sensitive and specific immunoassays to assess the levels of 3NT in plasma samples from claudicants and to investigate the protein nitration profile. Clinical data and plasma samples were collected from claudicant (n=33) and control (n=6) subjects. Analysis of data confirmed the difficulty of using parameters such as ankle brachial index (ABI) in diagnosis, supporting the need for investigations into potential biomarkers. Development of indirect and competitive ELISAs using electrochemically nitrated bovine serum albumin as the standard revealed that the detection of 3NT was dependent on the antibody being able to access the 3NT-residues within the protein. Various denaturing conditions and different types of microtitre plate were utilised during development. Initially the presence of 3NT in claudicant or control whole plasma samples could only be detected using dot blot immunodetection. Affinity purification techniques for the fractionation of the plasma proteins were therefore applied. Subsequently, 3NT-containing plasma proteins were found to be present in all of the claudicant and control samples using the developed competitive ELISA. Proteomic analysis of the 3NT-affinity purified samples, using MALDI-MS and LC-ESI-MS/MS, confirmed the presence of human serum albumin, serotransferrin and apolipoprotein A1 and A2 precursors within those protein bands staining immunopositive for 3NT on SDS-PAGE gels. The identification of apolipoprotein A1 within 3NT-immunopositive bands confirms previous reports suggesting the oxidative modification of HDL may contribute to the link between inflammation and the pathology of atherosclerosis.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Damage to visual pathways can lead to irreversible blindness. Posterior visual pathways, located within a watershed area, are predisposed to hypoperfusion/reperfusion injury. In a novel rat model of bilateral common carotid artery occlusion (BCCAO), oxidative injury to the superior colliculus (SC), a major visual center within the watershed area was evaluated, in addition to its effects on retinal ganglion cells (RGCs). Nitrotyrosine, a footprint of peroxynitrite-mediated oxidative injury in the SC, and microtubule-associated protein 2, a dendrite marker in the retina, were assessed using immunofluorescence and confocal microscopy. Nitrotyrosine-immunoreactivity in the SC was increased 2 weeks after BCCAO compared to controls. Microtubule-associated protein 2-immunoreactivity in the central inner plexiform layer was reduced 3 weeks after BCCAO compared to controls. Global incomplete cerebral hypoperfusion/reperfusion induced oxidative injury in the SC and retrograde RGC dendritic changes. This suggests that cerebrovascular injury affecting the posterior visual pathways may contribute to vision loss in patients.

Nitric oxide (NO) has been proved to reduce parasite burden in vertebrates infected with Schistosoma, Fasciola, Brugia or Taenia. NO negatively influences parasite growth and development, which then leads to smaller parasite-caused damage to the liver during schistosomosis and stimulates healing processes in muscles infected with Toxocara canis. Peroxynitrite, formed from NO and superoxide, significantly reduces the viability of F. hepatica adults. In case of T. regenti, the neuropathogenic schistosome, the cells capable of NO production (macrophages, neutrophils, eosinophils, microglia and astrocytes) migrate to the site of the infection suggesting that NO might affect T. regenti infection as well. Therefore, the production of NO and its effect on the course of the infection was examined in vivo and the effect of peroxynitrite on T. regenti schistosomula was examined in vitro to assess the role of reactive nitrogen species during the infection. Our results from in vivo experiments demonstrate that although the infection did not significantly elevate nitrite/nitrate results in the sera, NO is locally produced in the early stages of the infection in both the skin and the spinal cord as shown by immunohistochemical detection of inducible NO synthase. Diminishing NO production by aminoguanidine...

The aim of the present study was to find out whether adaption to chronic hypoxia affects the expresion of TNF-α and IL-10 in rat myocardium. TNF-α is a proinflammatory cytokine, which amplifies inflammatory reaction, while IL-10 has opposite antiinflammatory effect. We also measured concentration of nitrotyrosine as a marker of nitrosative stress. We used male Wistar rats divided into four groups: 1) normoxic controls; 2) exposed to continous normobaric hypoxia (10% O2) for three days or 3) for three weeks and 4) exposed to intermittent normobaric hypoxia (10% O2) for three weeks with one hour daily reoxygenation. Cytosolic and membrane proteins (cytosolic and particulate fractions) were obtained from the left ventricle, right ventricle and interventricular septum. Concentrations of TNF-α and IL-10 in both fractions were measured by ELISA. Continous hypoxia increased TNF-α production in particulate fractions from all ventricular parts and decreased the ratio of IL-10/TNF-α in particulate and cytosolic fractions. Intermittent hypoxia redistributed TNF-α from cytosol into the particulate fraction and prevented the drop of IL-10/TNF-α ratio in the cytosolic fraction. The highest concentration of nitrotyrosine was found in the particulate fraction from the right ventricle after three days of hypoxia....

No description available.