Abstract3-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.
|Date of Award||2009|
|Supervisor||Martin Cox (Supervisor) & John Heptinstall (Supervisor)|