(419b) Monitoring Hydroxyl Radical Production In Mitochondria with Micellar Electrokinetic Chromatography

Reactive oxygen species (ROS) are thought to be a significant contributing factor to the aging process at a cellular level. Of particular interest are the ROS generated by the mitochondria, as research has pegged these to be a significant source of oxidative damage. Current methodology to investigate ROS production is heavily biased towards fluorescence microscopy. This method, however, lacks low limits of detection and the ability to distinguish between components fluorescing at the same wavelength. To overcome these drawbacks, we are developing sensitive capillary electrophoresis techniques with laser-induced fluorescence detection (CE-LIF) for ROS analysis. The treatment of cells and mitochondria with probes selective for a ROS of interest and subsequent separation will enable us to monitor low levels of specific ROS present in biological samples. The current focus of our work is the use of 2-[6-(4 -hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) to monitor the production of hydroxyl radicals. In vitro controls have verified the selectivity of the probe, and the analysis of isolated mitochondria from L6 myoblasts shows our ability to measure basal and stimulated levels of hydroxyl radicals. The initial CE-LIF method development will be presented here, as well as results from control mitochondria. The application of HPF combined with CE-LIF to look at hydroxyl radical levels in mitochondria isolated from adipocytes will also be discussed.