Detection of Blood Nitric Oxide Congeners and Nitrated Proteins in Human Health and Anaemia

Ingested inorganic nitrate (NO3⁻) has multiple physiological effects in the human body, including vasodilation, lowering blood pressure, inhibiting platelet aggregation and improving skeletal muscle function. Ingested NO3⁻ enters the enterosalivary circulation, resulting in increased NO3⁻ and nitrite (NO2⁻) concentrations in plasma. NO3⁻ metabolism also involves S nitrosothiol (RSNO) formation, and it has been suggested that this is dependent on the presence of an intracellular iron pool. 3 Nitrotyrosine (Tyr-NO2) is formed by the action of nitrating species on Tyr residues of proteins, and haemoglobin (Hb) is susceptible to nitration. Protein Tyr-NO2 formation has been implicated in Alzheimer’s disease (AD). The main aim of this thesis was to investigate whether reactive nitrogen species (RNS), i.e. S nitrosothiol concentrations ([RSNO]), [NO2⁻] and [NO3⁻], in the blood compartments (whole blood, plasma, red blood cells) of healthy volunteers and in the plasma of anaemic patients, were increased after ingestion of NO3⁻-rich beetroot juice (BR). The above RNS were measured by ozone-based chemiluminescence. A new protocol for collecting and treating the RBCs and whole blood samples was developed to improve the sensitivity of RSNO measurements in healthy volunteers. The effect of ingested BR on the levels of nitrated Hb, in red blood cells (RBCs) of healthy volunteers and plasma of anaemic patients, was determined by semi-quantitative western blotting. To assess whether the levels of nitrated Hb were increased in the RBCs of AD patients compared to non-dementia (ND) control volunteers, the levels of nitrated Hb were also determined by western blotting. All data are reported as medians with the interquartile ranges unless otherwise indicated. BR ingestion elevated the median RBC [RSNO] in healthy subjects (n = 5) almost five-fold to 110 (93–125) nM, whilst the median plasma [RSNO] in healthy subjects (n = 14) increased almost ten-fold to 104 (58–151) nM compared to before administration of NO3⁻ rich beetroot juice at baseline (BL-BR) and/or PL (Mann-Whitney test, p<0.01). The median [NO2⁻] and [NO3⁻] in plasma, whole blood and RBCs of healthy volunteers (n = 7) were increased after ingesting BR compared to BL-BR and PL (p<0.05, BR vs. BL-BR or PL in all three biofluids). The median levels of nitrated Hb in RBCs from healthy volunteers, did not show a statistically significant difference between BR ingestion and PL. The median plasma [RSNO] in anaemic patients (n = 12) was significantly increased to 118 (53-205) nM after ingesting BR juice compared to BL BR and PL, which were 12 (8-15) nM and 13 (10-18) nM, respectively (p<0.01). RBC samples were not available from the anaemia patient cohort but, as assessed by western blotting, BR ingestion did not cause a significant change in the median plasma nitrated Hb level (intensity of the anti-Tyr-NO2 staining of the Hb band, normalised to the intensity of the anti-Hb staining of the Hb band) compared to PL in anaemic patients. The median level of nitrated Hb was significantly increased in the RBCs of AD patients, to a value of 19.5 (16.2 – 20.9) arbitrary units (intensity of the anti-Tyr-NO2 staining of the Hb band, normalised to the intensity of the anti-Hb staining of the Hb band), compared to RBCs from non-dementia (ND) volunteers which had a median value of 14.2 (13.5 – 14.4) arbitrary units (p