In vivo EPR oximetry using an isotopically-substituted nitroxide: Potential for quantitative measurement of tissue oxygen

Variations in brain oxygen (O₂) concentration can have profound effects on brain physiology. Thus, the ability to quantitate local O₂ concentrations noninvasively in vivo could significantly enhance understanding of several brain pathologies. However, quantitative O₂ mapping in the brain has proven difficult. The electron paramagnetic resonance (EPR) spectra of nitroxides are sensitive to molecular O₂ and can be used to estimate O₂ concentrations in aqueous media. We recently synthesized labile-ester-containing nitroxides, such as 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 4), which accumulate in cerebral tissue after in situ hydrolysis, and thus enable spatial mapping of O₂ concentrations in the mouse brain by EPR imaging. In an effort to improve O₂ quantitation, we prepared 3-acetoxymethoxycarbonyl-2,2,5,5-tetra(²H₃)methyl-1-(3,4,4-²H₃,1-¹⁵N)pyrrolidinyloxyl (nitroxide 2), which proved to be a more sensitive probe than its normo-isotopic version for quantifying O₂ in aqueous solutions of various O₂ concentrations. We now demonstrate that this isotopically substituted nitroxide is ∼2-fold more sensitive in vivo than the normo-isotopic nitroxide 4. Moreover, in vitro and in vivo EPR spectral-spatial imaging results with nitroxide 2 demonstrate significant improvement in resolution, reconstruction and spectral response to local O₂ concentrations in cerebral tissue. Thus, isotopic-substituted nitroxides, such as 2, are excellent sensors for in vivo O₂ quantitation in tissues, such as the brain.