This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Reactive nitrogen species (RNS) are small, highly reactive molecules that are arise from the synthesis of nitric oxide (NO) within biological tissues. These species are present at low concentrations in healthy tissues where they mediate a variety of signaling events, and at higher concentrations in inflammed tissues where they are overproduced. One of the most successful methods for obtaining insights into the chemical actions of RNS (i.e. formation of nitrite, nitroso, and nitrosyl species) is the widely known 'NO chemiluminescence assay'. This technique is based on one-electron chemical assays that release NO from target substances, which is subsequently quantified via chemiluminescence detection. One of the limitations of this technique is the inability of its detection to track isotope-labelled RNS and thus to obtain essential information on the formation, consumption, and recycling of RNS. The goal of this project is to initiate a new collaboration with Matthew Grisham, a Professor of Physiology at LSUHSC in Shreveport, aimed at modifying NO chemiluminescence assays that will enable them to discriminate between 14N- and 15N-labelled NO, as well as other NO-related species. This new approach will be implemented with mass spectrometry with the aid of sample enrichment. Specifically, the immediate focus of this project will be to develop a sample enrichment technique that reduces the volume occupied by NO purged from reduction assays, from 10 milliliters to 100 microliters. This level of enrichment should be sufficient to allow NO reduction assays to detect NO and related species under inflammatory conditions and possibly even from healthy tissues. Preliminary experiments using nitric oxide and chemiluminescence detection suggest that the method can achieve this level of enrichment.
The specific aims of this proposal are: (1) to implement the above level of enrichment for RNS-containing samples and to couple the enriched sample into a mass spectrometer, (2) to obtain sufficient preliminary data that demonstrate the viability of the technique for tracing isotope-labelled RNS under inflammatory conditions, and (3) to use this preliminary data in proposals to NIH and NSF to achieve the ultimate goal of elucidating the role of RNS in inflammation.
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