Determination of In Vivo Nitric Oxide Levels in Animal Tissues Using a Novel Spin Trapping Technology

Abstract

It has been established that microdialysis ensured by the passage of aqueous solutions of Fe3+ complexes with N-methyl-d-glucamine dithiocarbamate (MGDMGD ) through fine dialysis fibers permeable for compounds with molecular weights below 5 kDa. These fibers can be implanted into heart, liver, and kidney tissues, enabling effective binding of Fe3+–MGD complexes to nitric oxide generated in interstitial fluids of narcotized rats in vivo. Subsequent treatment of dialyzate samples (60 μL) with sodium dithionite favors conversion of newly formed diamagnetic NO–Fe3+–MGD complexes into electron paramagnetic resonance-detectable NO–Fe2+–MGD complexes. The basal levels of NO determined from the concentrations of the complexes in the respective tissues are similar (1 μМ). The microdialysis data suggest that treatment of rats with a water-soluble analogue of nitroglycerine or a dinitrosyl iron complex with thiosulfate induces a long-lasting (>1 h) increase in the steady-state level of NO in animal tissues. This novel technology can be used for comparative analyses of production rates of NO and reactive oxygen species when using iron–dithiocarbamate complexes and spin traps for reactive oxygen species, respectively.