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

Abstract

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-acetoxymethox ycarbonyl-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.

Analysis of Nitroxide‐Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy

“…The extended lifetimeo fanitroxide radicalm ight also be important for in vivob iomedical assays that require long nitroxide incubation times. [19,21,23] With the addition of K 3 Fe(CN) 6 ,w eh ave extended the feasible incubation time for DEER data by 50 %. Notably,i fn itroxider eduction is the only contribution to the loss in DEER signal, then DEER should be feasible after 2h even without K 3 Fe(CN) 6 .T hus far,n itroxidebased distance measurements have been made either by flashfreezing immediately after microinjection or flash-freezinga fter am ore limited time of incubation.…”

In vivo electron paramagnetic resonance oximetry and applications in the brain

“…Complete isotopic substitution of 1 H, 14 N (spin I = 1) with 2 H, 15 N (spin I = 1/2) significantly reduces EPR signal linewidth; thereby, increasing the spectral peak intensity and sensitivity for measuring and mapping O 2 . Proof-of-concept in vitro phantom spectral spatial EPR images demonstrate that 2 H, 15 N-substituted nitroxides offers significant improvement in spectral resolution and reconstruction made possible by the higher SNR and ~2-fold narrower linewidth 899499. Additionally, similar to non-isotopic nitroxides, labile-ester-containing 2 H, 15 N-substituted nitroxides can diffuse across the BBB to enter brain tissue, where it is entrapped for EPRI,5894 and we have demonstrated that in vivo O 2 -dependent line broadening of the corresponding carboxy- 2 H, 15 N-substituted nitroxide after injection of the labile-ester-containing 2 H, 15 N-substituted nitroxide is greater than that of its 1 H, 14 N-nitroxides counterpart.…”

“…Additionally, similar to non-isotopic nitroxides, labile-ester-containing 2 H, 15 N-substituted nitroxides can diffuse across the BBB to enter brain tissue, where it is entrapped for EPRI,5894 and we have demonstrated that in vivo O 2 -dependent line broadening of the corresponding carboxy- 2 H, 15 N-substituted nitroxide after injection of the labile-ester-containing 2 H, 15 N-substituted nitroxide is greater than that of its 1 H, 14 N-nitroxides counterpart. At 1 GHz, the in vivo peak amplitude of the carboxy- 2 H, 15 N-substituted nitroxide is ~2-fold higher than non-isotopic version in the brain and this ~2-fold increase of in vivo peak amplitude significantly improves the sensitivity, resolution, reconstruction and spectral response to cerebral pO 2 ; thus, improving cerebral pO 2 mapping by EPRI 94. These results establish that in vivo EPRI with 2 H, 15 N-substituted nitroxide provides images with sufficiently short acquisition time and sufficiently high spatial resolution for in vivo O 2 quantification in the brain and motivates their use for superior and higher-resolution O 2 mapping in pathological states in the brain where O 2 levels are compromised brain compared to 1 H, 14 N-nitroxides.…”

Effect of body temperature on the pharmacokinetics of a triarylmethyl‐type paramagnetic contrast agent used in EPR oximetry

“…The trityl free radical, tri[8-carboxy-2,2,6,6-tetrakis(2hydroxymethyl)benzo [1,2-d:4,5-d']bis (1,3)dithio-4-yl]methyl radical (Oxo63), is used commonly as a paramagnetic probe for EPR oxymetric imaging. The concentration of the Oxo63 should be sufficient to provide adequate signal-to-noise ratio.…”