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

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

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

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 2. Electron Paramagnetic Resonance (EPR) Paramagnetic single electron relaxation is increased by the the molecular oxygen

Ö2 Molecular oxygen is paramagnetic 2p (O) 2p(O) 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

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

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)

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

S EPR spectrum of S EPR spectrum of S O EPR spectrum of

O S R 2  LW = LW [O2] 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

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

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

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

Transport of Molecular O2 into the Particulate Spin probes The Knudson flux Jk is defined as* Diffusion Adsorption S Gas Phase Schematics of Gas transport Dk (pO2) Jk = l R 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 * Frank-Kameneskii, D.A., Diffusion and heat Transfer in Chemical Kinetics, II nd Edition, Plenum, NY, 1969

Effect of Oxygen on the EPR Spectrum of LiPc 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 50 100 150 200 pO2 (mmHg) 0.5 G Ilangovan, G. et al J. Phys. Chem., 2000, 104, 4047

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

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

Irreversible inhibition Reversible inhibition Mechanism Irreversible inhibition by ONOO- NOS 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

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

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

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

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

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

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

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

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

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

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 2 4 6 2 4 6 Number of days Number of days Animal 1 Animal 2 Animal 3 Animal 4 Animal 5 On the day of LiPc was implanted the tumor size was of the size 8 x 8 x 6 mm

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

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 Tumor volume (mm3) 100 200 300 pO2 (mmHg) 4 8 12 16 20 40 60 80 30 LiPc with RIF-1 Cells 300 25 250 20 200 pO2 (mmHg) 15 Tumor Volume (mm)3 150 10 100 5 50 Z X 2 4 6 8 10 12 14 Y Days post injection

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

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

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.

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 ?