Development and validation of a LC/MS-based method for the measurement of intracellular superoxide anion.

Superoxide anion (O2.-), as the first generated reactive oxygen species (ROS), has been considered to be highly deleterious to cell functions. The measurement of intracellular O2.- level is of great importance to uncover its roles in a variety of oxidative damage diseases. Hydroethidium (HE) fluorescence-based method is dominating intracellular O2.- assay by monitoring the unique product 2-OH-E+ of HE/O2.- reaction. However, the avoid-less cross-interference of red fluorescence limited its ability to provide trustworthy information on intracellular O2.- formation. By the detection of 2-OH-E+, we herein developed and validated an improved LC/MS-based method for the measurement of intracellular O2.-. Firstly, we demonstrated the proportionality of HE/O2.- reaction. Secondly, ungerimine was used as internal standard to eliminate daily basis and matrix effect in the LC/MS-based detection of 2-OH-E+. Afterward, the total protein concentration was utilized for cell number normalization. Accordingly, an equation was further proposed to calculate the relative abundance (RA) of intracellular O2.-. Finally, the developed method has been successfully utilized to evaluate the inhibitory effects of natural compounds on O2.- generation, the result of which was validated by the HE-based fluorescent measurement. Compared with the fluorescent measurement, the LC/MS-based intracellular O2.- assay method is more sensitive, selective and accurate.

Primary fibroblasts of NDUFS4(-/-) mice display increased ROS levels and aberrant mitochondrial morphology.

The human NDUFS4 gene encodes an accessory subunit of the first mitochondrial oxidative phosphorylation complex (CI) and, when mutated, is associated with progressive neurological disorders. Here we analyzed primary muscle and skin fibroblasts from NDUFS4(-/-) mice with respect to reactive oxygen species (ROS) levels and mitochondrial morphology. NDUFS4(-/-) fibroblasts displayed an inactive CI subcomplex on native gels but proliferated normally and showed no obvious signs of apoptosis. Oxidation of the ROS sensor hydroethidium was increased and mitochondria were less branched and/or shorter in NDUFS4(-/-) fibroblasts. We discuss the relevance of these findings with respect to previous results and therapy development.