The use of precolumn derivatization and capillary electrophoresis with laser-induced fluorescence detection for the investigation of the transport and metabolism of substance P at the blood-brain barrier.

摘要

This dissertation describes the development and use of an analytical method for the investigation of the transport and metabolism of substance P (SP) at the blood-brain barrier (BBB). SP is a neuropeptide present throughout the body in the low-to-subpicomole per gram tissue level and in the cerebrospinal fluid and plasma at picomolar concentrations. The most common method for the determination of SP utilizes radioimmunoassay. However, this is a very time-consuming technique that typically requires sample volumes of more than 10 mL to quantitate endogenous levels of the peptide. In this dissertation, a method based on precolumn derivatization and capillary electrophoresis with laser-induced fluorescence detection is described. Although this method did not allow for determination of endogenous levels of SP and metabolites, the limits of detection ranged from 2.5 to 28.5 nM and were low enough for the studies conducted in this dissertation.; This method was then applied to investigate SP metabolism. Metabolite formation was monitored in rat striatum using microdialysis sampling and upon exposure to the bovine brain microvessel endothelial cell (BBMEC) culture system, a model of the BBB. Correlation of results obtained by in vivo and in vitro systems is of great interest to the pharmaceutical industry for drug screening, because standard in vivo methods are frequently too difficult, costly and time-consuming for use as simple drug screening tools. In our studies, similar metabolites were formed in both systems.; Finally, this method was applied to investigate the transport properties of SP at the BBB using the BBMEC model. Although SP has been shown to cross into the brain after injection into the periphery, the transport mechanism of the peptide has not been elucidated. An in vitro model, such as the BBMEC system, allows the examination of the BBB at the cellular level without the complexities and high costs involved in animal studies. In general, a good qualitative correlation is seen between the in situ/in vivo and in vitro permeabilities. The results of our studies indicate that SP crosses the BBB in both directions via an energy-dependent pathway. This permeation was further shown to involve an endocytic pathway from the apical side.