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EPR Oxymetry - Biomedical Applications

EPR Oxymetry - Biomedical Applications. Suggested Reading:. G.R. Eaton, S.S.Eaton and K.Ohno, EPR Imaging and In vivo EPR. 1). Metabolic control in cells. Oxidant / Antioxidant dynamics. 2). Pathogenesis. Hypoxia / Ischemic Heart Diseases. 3). Therapeutic strategies.

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EPR Oxymetry - Biomedical Applications

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  1. EPR Oxymetry - Biomedical Applications Suggested Reading: G.R. Eaton, S.S.Eaton and K.Ohno, EPR Imaging and In vivo EPR

  2. 1). Metabolic control in cells Oxidant / Antioxidant dynamics 2). Pathogenesis Hypoxia /Ischemic Heart Diseases 3). Therapeutic strategies Radiation treatment of Tumors Oxygen Measurements in Tissues

  3. Clarks Electrodes Fluorescent Probes 4e O2 H2O A silicone matrix at the tip of a 230 m fiber contains the oxygen-sensitive fluorescent dye. Microelectrode (10-100mic) is inserted into the tissue at the desired location and the oxygen reduction current is measured. * * O2 Ru(bpy)3 3+ h Ru(bpy)3 O2 3+ Methods for Oxygen Measurements In Tissues

  4. Magnetic Resonance Methods 1. Nuclear Magnetic Resonance (NMR) Paramagnetic nucleus relaxation is induced by the molecular oxygen • BOLD MRI -- T2* effect • 19F MRS(I) --- T1 effect Electron Paramagnetic Resonance (EPR) 2. Paramagnetic single electron relaxation is increased by the the molecular oxygen

  5. Ö2 Molecular oxygen isparamagnetic sx* • No EPR spectra have been observed for oxygen dissolved in fluids. (too broad) • Thus, there seems to be no possibility for direct detection of oxygen in biological systems using EPR • However, oxygen can be measured and quantified indirectly using spin-label (EPR) oximetry pz* py* 2p (O) 2p(O) pz py sx Molecular oxygen has two unpaired electrons

  6. Electron Spin, S ms = +½ ±½ Energy DE=hn=gbB ms = -½ Magnetic Field Magnetic Field Electron Paramagnetic Resonance (EPR) Oxymetry Oxygen

  7. EPR Oxymetry - Probes Particulate probes Soluble probes Lithium Phthalocyanine Sugar chars Fusinite Coal India ink Nitroxides Trityl radicals Requirement to be a good oxymetry Probe: • Higher T2 (Sharp EPR spectrum) • Preferably single EPR line (No hyperfine splitting)

  8. 1 1 LW = e( ) + T2 2T1 The Principle of EPR oxymetry LW T2 – spin-spin relaxation

  9. O O O O O O O O S S S S S S S S S S S S S S S S S S S S S S S S S S S EPR spectrum of EPR spectrum of EPR spectrum of

  10. LW [O2] R O S 2  LW = e 3  = 4Rp[O2] DS + DO2 (Smoluchowski eqn.) R = interaction radius between A and B P = the probability of relaxation DA and DB = Diffusion coefficients of A and B

  11. Example 1: Soluble Spin Probes for EPR oximetry Tryaryl methyl radical (TAM) COO- O OH S S OH O O S HO S O HO OH HO OH HO O O . O O C S S S S COO- S S S COO- S O O O O HO OH HO OH

  12. 600 400 LW / mG a 200 0 40 60 80 0 20 p O / mmHg 2 EPR spectrum at anoxic condition (N2) EPR spectrum in the presence of room air (21%O2) Calibration Curve

  13. N N N N N N L i N • N N N L i N N N N N N EPR OXIMETRY: PROBES Microcrystalline particulates Lithium Phthalocyanine Lithium Naphthalocyanine (LiPc) (LiNc) Ilangovan et al, J. Phys. Chem.B, 2000, 104, 4047 2000, 104, 9404 2001, 105, 5323 2002, 106, 11929 J. Magn. Reson. 2004, 170, 42-48 Ilangovan et al, Free Rad. Biol. Med, 2002, 32, 139 2003, 35, 1138 Ilangovan et al, J. Magn. Magnt. Mater, 2001, 233, L131

  14. Schematics of Gas transport Gas Phase Diffusion Adsorption S S * Frank-Kameneskii, D.A., Diffusion and heat Transfer in Chemical Kinetics, II nd Edition, Plenum, NY, 1969 Transport of Molecular O2 into the Particulate Spin probes The Knudson flux Jk is defined as* Dk (pO2) Jk = lR T where pO2 = O2 pressure difference between the start and end of pore; [(pO2)start - (pO2)end ] l = the length of the pore Dk = Knudson diffusion coefficient Knudson Diffusion is dominated since the mean free path of O2 is higher than pores in LiPc l (pO2)start (pO2)end

  15. Oxygen Sensing Probe Effect of Oxygen on the EPR Spectrum of LiPc Lithium Phthalocyanine (LiPc) With Nitrogen With room air 5µm 2.5 2.0 1.5 Re-saturated with nitrogen Line width (G) 1.0 0.5 Line width Vs. pO2 0 0 50 100 150 200 pO2 (mmHg) 0.5 G Ilangovan, G. et al J. Phys. Chem.,2000, 104, 4047

  16. Gas mixture inlet Cotton Sample support MAGNET MAGNET TM Cavity 110 Outlet An EPR Based method for Simultaneous measurements of O2 and Free radicals generation Experimental Set-up LiPc 8mm Schematics of EPR oxymetry experimental set up. The sample tube could be either quarts microtube or gas permeable teflon tube Magnified view of the micro tube containing the LiPc microcrystals (oxymetry probe) 50µL quartz microtube as reaction vessel Ilangovan et al, Methods In Enzymology, 2004,381, 747

  17. Effect of NO Addition n on BAEC Respiration 21% 0.5% 8 160 ** 4x106cells 5 3 140 6 4 120 100 2 VO2max (mmHg/min) 3.0 NO added ** 3 pO2 (mmHg) 4 p50 (mmHg) 80 dpO2/dt(mmHg/min) 2.0 2 60 NO added 1 2 1.0 40 1 Control 20 25 5 10 15 20 0 0 0 0 0 20 40 60 80 100 120 140 160 Con Con NO 0 20 40 60 80 NO pO2 (mmHg) Time (min) Cyt c III UQ I Inner membrane IV II O2 NADH Succinate Complex I NADH:Ubiquinone Complex II/III Succi-Cyto Reductase Complex IV Cyt c Oxidase ETC Complex Activity NO 0.003 0.003 0.03 * P < 0.001 0.002 0.02 0.002 Δ O.D./min ΔO.D./min ΔO.D./min 0.01 0.001 0.001 21% .5% 21% 21% 21% 21% 21% .5% 0.00 0.000 0.000 Con Con NO Con NO NO Complex I Complex II/III Complex IV

  18. Mechanism NOS Irreversible inhibition by ONOO- Reversible inhibition by NO (Present irrespective of pO2) (Not present at low pO2) ONOO- NO O2.- O2 This inhibition is pO2 dependent H2O III I II IV NOS generated NO causes attenuation of respiration via irreversible CuB binding; yet, no competitive binding at the a3 site; p50 remains unchanged

  19. P ( O ) ( O E t ) 2 H N C H 3 O - Simultaneous measurement approach Substrate Product HOO P(O)(OEt)2 O2-. + O 2 CH3 H N Enzyme DEPMPO-OOH O DEPMPO O2 Free Radicals Measurement Oxygen Measurement

  20. Simultaneous measurement of O2-. trapping using DMPO [DEPMPO-OOH] Change [Oxygen ] Change 30 min 45 min 50 G 90 min 60 min Experimental spectra Simulated DMPO - OOH Spectra

  21. O Concentration 2 Decomposed product of spin adduct DEPMPO-OOH Concentration Kinetic analysis and concentration profiles of O2 and DEPMPO-OOH and its decomposed product 240 200 160 Concentration /M 120 80 40 A 0 0 200 400 600 800 1000 1200 1400 1600 1800 TIME /S

  22. Perfused Heart 5mm Inserted LiPc in the heart To Microwave source 14 mm 10 m Cable to Power supply Micrograph of LiPc microcrystals

  23. Pre-ischemic equilibrium 10 min after ischemia 15 min after ischemia 5 min after ischemia

  24. FLOW RATE 10ml/min 100mmHg DEVELOPED PRESSURE 150 60 90 120 Time (min)

  25. Correlation of Heart Injury to Oxygen consumption 10000 8000 6000 Q(nmol/g/min) 4000 2000 0 0 10000 20000 30000 40000 RPP (bpm. mmHg)

  26. Other Applications Oxygen Measurements in RIF-1 Murine Tumor Model

  27. Implanting the LiPc microcrystals into the Gastrocnemius muscle and Tumor Tissues Shaft LiPc

  28. Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 pO2 measurement in RIF-1 Tumor bearing mice NORMAL TISSUE TUMOR 30 30 25 25 20 20 pO2/ mmHg pO2 / mmHg 15 15 10 10 5 5 0 0 0 2 4 6 0 2 4 6 Number of days Number of days On the day of LiPc was implanted the tumor size was of the size 8 x 8 x 6 mm

  29. Room air Carbogen 0.5G Oxygenation of RIF-1 Tumor by breathing Carbogen (95%O2 + 5%CO2) Initial pO2 <5mmHg While Carbogen breathing 100 mmHg When switched to room air  20 mmHg 120 1st cycle 2nd cycle 3rd cycle 100 80 Carbogen pO2/ mmHg 60 Tumor is oxygenated to the level of normal tissue at least for 2hrs, after termination of carbogen breathing treatment Room air 40 20 0 0 20 40 60 80 100 120 140 Time / min Ilangovan, G. et al Magn. Reson. Med.,2002,48, 723

  30. 20 16 16 12 12 8 pO2 (mmHg) 4 8 0 0 20 40 60 80 4 0 0 100 200 300 Tumor volume (mm3) Tumor Growth Vs pO2 relationship Suspensions of LiPc in Saline or LiPc with RIF-1 cells were injected into the gastrocnemius muscle of right hind leg. LiPc & RIF cell suspension 30 LiPc with RIF-1 Cells 300 25 250 20 200 pO2 (mmHg) 15 Tumor Volume (mm)3 150 10 100 5 Z 50 0 X 0 Y 0 2 4 6 8 10 12 14 Days post injection

  31. 0 7500 Oxymetry in Wound Healing 2D EPR image 2mm EPR image of the LiPc in the wound SKC1 mouse wound with LiPc in the periphery of the wound Mouse in the EPR machine

  32. STRESS AND OXYGEN 25 * * Control * * Stress 20 * 15 pO2 (mmHg) 10 5 * P< 0.05 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day7 Day 8 Time Post Wounding

  33. CLINICAL APPLICATIONS Hal Swartz works on one of his tattooed volunteers, in an effort to use the carbon particles in tattoo ink to measure the oxygen content of tissues.

  34. Discussion points… What are the advantages with EPR oximetry, over other conventional methods, in measuring tissue oxygen changes? What is your opinion about the potential use of particulate probes for EPR oximetry, compared soluble probes ?

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