Monitoring superoxide levels in skeletal muscle and cell models by capillary electrophoresis with laser-induced fluorescence detection

As the primary reactive oxygen species (ROS) generated in skeletal muscle, the production of superoxide has been associated with increased oxidative stress during aging. Such association is still obscure partly due to the lack of sensitive and specific methods for analyzing superoxide in biological systems. This thesis describes the development of technologies for monitoring superoxide levels in skeletal muscle systems including isolated mitochondria, single muscle fibers and myoblasts.The method for detecting superoxide is based on the reaction of triphenylphosphonium hydroethidine (TPP-HE) with superoxide and the subsequent separation and detection of the specific product hydroxytriphenylphosphonium ethidium (OH-TPP-E +) by micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF). The method has proved to be effective at detecting superoxide release at both sides of the inner membrane of isolated mitochondria in the presence and the absence of inhibitors. The method was applied to investigate the mitochondrial superoxide production of rat soleus (type I) and semimembranosus (type II) muscles in response to aging. The results indicated that superoxide release is complex as it occurs asymmetrically and such release has muscle type and temporal specificity.Due to the heterogeneous and complex nature of skeletal muscle, it is difficult to investigating ROS production and their effects on specific muscle fiber types. A novel method was developed for the analysis of superoxide production in single fibers isolated from the soleus muscle. The suitability of the method to monitor changes in superoxide production at the single-fiber level was confirmed by inhibiting and enhancing superoxide generation with various treatments.Previous studies based on the use of TPP-HE are not quantitative because the dependence of TPP-HE accumulation on the mitochondrial membrane potential was not taken into account. A new approach was developed for the quantitation of mitochondrial superoxide levels in single myoblasts. The myoblasts are simultaneously labeled with TPP-HE and rhodamine 123 (R123). R123 is used as an internal mitochondrial membrane potential calibrant. The OH-TPP-E + and R123 contents released from each myoblast were analyzed by chemical cytometry based on MEKC-LIF. The results from single cells in the presence and absence of respiratory inhibitors demonstrate the feasibility of this method.