This proposal is aimed at identifying pathways involved in oxidative damage in asthma. The work will entail studying the mechanisms of oxidative damage in asthma with the emphasis on studies of eosinophil peroxidase, an enzyme released by activated eosinophils. Peroxidase secretion by leukocytes during asthmatic episodes is implicated in the production of reactive oxidant species. These reactive intermediates participate in the modification of biological markets, such as proteins. The cellular mechanisms controlling protein modification during asthma are not fully understood. Here it is proposed to enhance our understanding of the pathways involved in oxidative damage in vivo and the mechanism through which eosinophils contribute to these processes. The studies will initially examine mechanisms of protein modification by activated leukocytes using a mass spectrometry to look at levels of specific modified amino acids produced in proteins. This work will then be extended to in vivo samples to search for these distinct markers of oxidation pathway of tissue damage. In vivo protein damage by these distinct oxidation pathways will be identified and quantified by using stable markers of tyrosine oxidation (i.e. chlorination,. bromination, nitration, tyroslyation and free-metal dependent hydroxylation) in clinical specimens of segmental antigen challenge, blood plasma and urine samples.
