Electron paramagnetic resonance spectroscopy for analysis of free radicals in zebrafish

Abstract

Electron paramagnetic resonance (EPR) is an excellent choice for detecting free radicals in biological samples. Biologically relevant radicals are extremely short-lived and cannot be detected directly, emphasizing the need for an appropriate compound to generate stable adducts that can be measured by EPR. Spin trapping with nitrone compounds like 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is a method commonly employed for detecting free radicals. However, due to the instability of nitrone radical adducts, using the cell-permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl pyrrolidine (CMH) appears to be a more effective approach within biological tissues. Here, we compare the use of DMPO and CMH to detect the most abundant reactive oxygen species radical, superoxide (O2⋅-), in zebrafish and present an optimized protocol for performing EPR with a CMH spin probe in both zebrafish hearts and larvae. Together, our data suggest that EPR using the CMH probe is a reliable method to detect O2⋅- in zebrafish pathologies linked to oxidative stress, such as cardiovascular diseases.