Using Fluorescence Derivatization and Analytical Separations to Investigate Biomarkers of Oxidative Stress

Abstract

Oxidative stress occurs when there is an overproduction of reactive nitrogen and oxygen species in the body. This condition has been linked to many prominent diseases. This thesis details analytical methods that use fluorescence derivatization in order to detect small molecule biomarkers of oxidative stress as they relate to epilepsy, inflammation, and traumatic brain injury. A liquid chromatography-fluorescence method was developed for the detection of reactive aldehyde biomarkers, which are by-products of oxidative stress following epileptic seizures. The method was applied to an animal model of epilepsy and used to monitor concentrations of the analytes over several days in urine samples. This study revealed a circadian cycling of these aldehydes, as well as elucidating which ones were present at increased levels after a seizure. A microchip electrophoresis-fluorescence method was then developed for the detection of carnosine, an endogenous dipeptide that is involved in oxidative stress-induced inflammation following prolonged activation of macrophage cells. The method was used to measure the native presence of carnosine in these cells, as well as investigate its uptake under normal and pro-oxidative conditions. This study indicated that carnosine’s antioxidant properties might lead to its increased uptake by macrophage cells under inflammatory conditions. Another microchip electrophoresis-fluorescence method was developed to examine excitatory amino acid neurotransmitters involved in neuronal damage following traumatic brain injury. The microchip electrophoresis chip was coupled to microdialysis sampling with on-line derivatization for continuous monitoring of these analytes with a total lag time of twelve minutes and 90 second temporal resolution. Further work with this system will focus on improving the limits of detection so that it could be used by medical professionals to monitor the neurochemical health of a TBI patient.