G.R.Eaton, S.S.Eaton, K.Ohno, EPR imaging and In vivo EPR Introduction to EPR/ESR Spectroscopy and Imaging Suggested reading: C.P.Poole, Electron Spin Resonance, A comprehensive Treatise on Experimental Techniques J.A.Weil, J.R.Bolton, J.E.Wertz, Electron Paramagnetic Resonance: Elementary Theory and Practical Applications G.R.Eaton, S.S.Eaton, K.Ohno, EPR imaging and In vivo EPR

Magnetic momentum of an add electron s = gS N L = gL  N = 1838 This is the ratio of rest mass of proton to the rest mass m of electron Thus EPR energies are generally about 2000 times as big as NMR energies

NMR – EPR comparison of energies Radio wave in the range : 90 – 700 MHz Field value : 2 - 14 T Relaxation time : 10-3 to 10 sec EPR Microwave in the range : 1.2 GHz – 100 GHz Field : 0.03 – 0.3 T Relaxation time : 10-9 – 10-6 sec “Additional problems with biological EPR spectroscopy is the microwave absorption H2O in biological objects.”

A serious limitation for FT-EPR spectroscopy Dead Time

Principle of EPR spectroscopy  Relaxation T1 – Spin lattice relaxation E = g(B0+B1) T2 – Spin-spin relaxation T2* – Spin-spin relaxation  B0 Expt. Obtained spectrum Absorption spectrum

Oscillating Magnetic field Field (B1) modulation in EPR Why: Absorption signal is weak, compared NMR, and buried under equally amplified noise. Modulation frequency Modulation amplitude B1 Oscillating Magnetic field Unmodulated Modulated

Phase Sensitive Detection in EPR 3 2 Max 1 3 4 2 5 -Max 1 4 5 Field Field

Nuclear magnetic coupling – “Hyperfine splitting” -1 +1 - 1 2 + N O. S = 1 for 14N 2S+1 = 3

Secondary Hyperfine Splittings - 1 2 + - 1 2 +1 -1 + N O. H Expected Experimentally measured

EPR spin trapping Many free radicals, generated by enzymatic reactions are not stable enough to detect by EPR spectroscopy. Superoxide radical (O2.-) Hydroxyl radical (OH.) Nitric oxide (NO:) They need to be stabilized to detect by EPR: “Spin trapping” Spin trap + Unstable radical Stable radical (?) (No EPR signal) (No EPR signal) (EPR signal)

Superoxide trapping: Example 1 Xanthine / Xanthine oxidase DEPMPO DEPMPO-OOH Xanthine Hypoxanthine + xo EPR spect. of DMPO-OH

Trapping Nitric Oxide Although NO is paramagnetic, it is impossible to detect by EPR directly, because being small, it relaxes very fast as in the case of O2. Thus special approaches are required to restrict its motion to get reasonable spectrum. Fe complexes of dithiocarbamate and its derivatives

Fe(MGD) Fe(MGD)-NO

Superoxide trapping: Example 1 Nitric oxide synthase (NOS) Fe-MGD DMPO-OO-

EPR Imaging

EPR Imaging – Concept of gradient Field 1 2 MAGNET MAGNET 3 4 Bo Field is being uniform (g(B0+B1)) all the four spin pockets come to resonance frequency at a time

Principle of cw EPR Imaging Gradient Direction Projection 2D image Re-construction Projections Gradient generation Bo 1 2 3 4 1- 4 N S Bo 1 2 3 4 (x+Bo) (x-Bo) N S Bo 1, 3 2, 4 Bo 1 2 3 4 x+Bo x-Bo N S 3 1,4 2

Pros and Cons of EPR imaging Not adequate concentration of radicals available in biological systems Needs exogenous infusion of stable radicals species in organs or whole body imaging Needs significant reduction of microwave frequency to avoid microwave absorption. This significantly compromises the sensitivity But…. It is an unique technique to study redox status of tissues, organs or in whole body, which cannot be achieved by other techniques

NORMAL TISSUE RIF-1 TUMOR 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0 16.5 RESONATOR NORMAL TISSUE RIF-1 TUMOR 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0 16.5 Time (min) 256 Kuppusamy et al, Canc. Res, 1998, 58, 1562

Breathing Mouse (pO2=2.5 mmHg) Carbogen Breathing Mouse (pO2= 95 mmHg) Pharmacokinetics of Nitroxides at different Oxygenation of RIF-1 Tumor Room air Breathing Mouse (pO2=2.5 mmHg) Carbogen Breathing Mouse (pO2= 95 mmHg) 15N-TPL and LiPc 0.5 min Nitroxide intensity -> 10 min Nitroxide intensity -> 100 Frequency 10 20 30 0.05 0.10 Rate constant (min-1) 0.15 0.0 40 3-CP room air 60 3-CP Carbogen 40 10 Frequency I/I0 x 100 15N-TPL room air 20 15N-TPL Carbogen 0.0 0.05 0.10 0.15 1 Rate constant (min-1) 10 20 30 40 Time (minutes) Ilangovan, G. et al Mol. Cell. Biochem., 2002, 234, 393

Example 1 In vivo Imaging of NO generation Fe-MGD + NO Fe-MGD-NO No EPR signal No EPR signal Strong EPR signal NO generated in the thoracic region of a mouse, subjected to cardiopulmonary arrest