The Development of Analytical Methods for Investigations of Dynorphin A 1-17 Metabolism in the Central Nervous System and Peripheral Tissues and Transport at the Blood Brain Barrier

Dynorphin A 1-17 (Dyn A 1-17) is an endogenous neuropeptide that acts preferentially at the kappa opioid receptor. Elevated concentrations of Dyn A 1-17 have demonstrated neurotoxicity via a non-opioid mechanism, potentially mediated by NMDA (N-methyl-D-aspartate) receptors. This neurotoxicity has further been implicated in a variety of conditions including neuropathic pain, stress, depression, and neurological disorders including; Alzheimer's and Parkinson's disease. Unlike traditional small molecule neurotransmitters, peptides can undergo enzymatic degradation once released into the extracellular space and these metabolites can then go on to exhibit their own unique properties in vivo, often at sites far distal to their releasing cells. The investigation of dynorphin metabolism is especially crucial at blood brain barrier, where a peptide metabolite may exhibit substantially different transport properties. Traditional methods for the quantification of neuropeptides are immunoassays such as ELISA (enzyme linked immunosorbent assay) and RIA (radioimmunoassay). While these techniques have excellent limits of detection, cross-reactivity between related species is a significant issue. Therefore, it is essential to develop analytical methodologies capable of simultaneously determining the concentrations of both the parent peptide and its metabolites.;In this thesis, the metabolism of Dyn A 1-17 in both the central nervous system and peripheral tissues as well as with an in vitro cell culture model of the blood brain barrier (BBB) is investigated. Methods for the separation and detection of Dyn A 1-17 and four of its key metabolites; Dyn A 1-6, Dyn A 1-8, Dyn A 1-13, and Dyn A 2-17 were developed using capillary electrophoresis (CE) with copper complexation and UV detection and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Dyn A 1-6 was found to be a major metabolite in the above mentioned systems and therefore its permeability at the BBB was investigated using bovine brain microvessel endothelial cells (BBMECs). The effect of this peptide on the permeability of the low molecular weight, low permeability compound fluorescein was also explored.;In addition to CE and LC-MS/MS, microchip electrophoresis is also investigated. More specifically the development of an immunoaffinity microchip electrophoresis system was begun. Online immunoaffinity enables sample clean-up and preconcentration on the separation device. By combining immunoaffinity with highly efficient electrophoretic separations, an integrated device to investigate dynorphin metabolism is fabricated. Miniaturization decreases analysis time, significantly improving temporal resolution. Additionally preconcentration will improve limits of detection, making the detection of endogenous concentrations of dynorphin peptides from biological samples feasible. Towards this goal, antibody screening and some initial microchip electrophoresis studies with amperometric and laser-induced fluorescence detection were investigated. Future directions include the incorporation of this device with on-line microdialysis sampling and the development of an immunoaffinity microchip electrophoresis system for investigating neuropeptides implicated in neuropathic pain.