Periannan Kuppusamy, PhD Center for Biomedical EPR Spectroscopy & Imaging

EPR Spectroscopy Spying on unpaired electrons - What information can we get? Periannan Kuppusamy, PhD Center for Biomedical EPR Spectroscopy & Imaging Davis Heart & Lung Research Institute Ohio State University, Columbus, OH E-mail: Kuppusamy.1@osu.edu Sunrise Free Radical School Oxygen-2002 ::Nov 21, 2002

Electron Paramagnetic Resonance (EPR) Electron Spin Resonance (ESR) Electron Magnetic Resonance (EMR) EPR ~ ESR ~ EMR What is EPR? Ms = +½ DBpp ±½ Energy DE=hn=gbB Ms = -½ B = 0 B > 0 Magnetic Field (B) h Planck’s constant 6.626196 x 10-27 erg.sec n frequency (GHz or MHz) g g-factor (approximately 2.0) b Bohr magneton (9.2741 x 10-21 erg.Gauss-1) B magnetic field (Gauss or mT) EPR is the resonant absorption of microwave radiation by paramagnetic systems in the presence of an applied magnetic field • hn = gbB • = (gb/h)B = 2.8024 x B MHz • for B = 3480 G n = 9.75 GHz (X-band) • for B = 420 G n = 1.2 GHz (L-band) • for B = 110 G n = 300 MHz (Radiofrequency)

Selection Rule DMS = ±1; DMI = 0 Hyperfine Coupling Electron S(½) Nucleus I (½) S=½; I=½ Doublet MI=+½ MS=+½ MI=-½ hfc MS=±½ MI=-½ MS=-½ MI=+½ Magnetic Field

Hyperfine Coupling Nucleus I (1) MI=0,±1 Electron S (½) MS = ±½ S=½; I=1 Triplet MI=+1 MS=+½ MI= 0 MI=-1 MS=±½ hfc hfc MI=-1 MS=-½ MI= 0 MI=+1 Magnetic Field Selection Rule DMS = ±1; DMI = 0

What do we do with EPR? We can detect & measure free radicals and paramagnetic species • High sensitivity (nanomolar concentrations) • No background • Definitive & quantitative Direct detection e.g.: semiquinones, nitroxides, trityls Indirect detection Spin-trapping Species: superoxide, hydroxyl, alkyl, NO Spin-traps: DMPO, PBN, DEPMPO, Fe-DTCs Chemical modifications Spin-formation : hydroxylamines (Dikhalov et al) Spin-change : nitronylnitroxides ( Kalyanaraman et al) Spin-loss : trityl radicals

Can we use EPR to measure free radicals from biological systems (in vivo or ex vivo)? What do we do with it? Yes! Intact tissues, organs or whole-body can be measured. But there is a catch! Biological samples contain large proportion of water. They are aqueous and highly dielectric. Conventional EPR spectrometers operate at X-band ((9-10 GHz) frequencies, which result in (i) ‘non-resonant’ absorption (heating) of energy and (ii) poor penetration of samples. Hence the frequency of the instrumentation needs to be reduced. What is the optimum frequency? - depends on sample size

What else can we do with EPR? We can use free radicals as “spying probes” to obtain information from biological systems • A known free radical probes is infused or injected into the animal • The change in the EPR line-shape profile, which is correlated to some physiological function, is then monitored as a function of time or any other parameter. • The measurements can be performed in real-time and in vivo to obtain ‘functional parameters’.

Oxygen, pO2 Acidosis, pH Viscosity Molecular motion width Splitting Redox status Cell viability Tissue perfusion amplitude Functional parameters from an EPR spectrum In vivo EPR spectroscopy is capable of providing useful physiologic and metabolic (functional) information from tissues Oxygen, pO2 Redox status Acidosis, pH Thiols (GSH) Cell viability Viscosity Tissue perfusion Molecular motion

Can we image free radicals in biological systems? Spatially-resolved information (mapping) can be obtained using EPR imaging (EPRI) techniques

Can we image free radicals in biological systems? NMREPR Spin Probes Tissue protons Free radicals (endogenous) (>50 M) (<< nM) Probe Stability Ideal < nanoseconds Relaxation time (LW) m sec < m sec (LW: 1 G) No suitable endogenous spin probes for EPRI Nothing to image Fast electronics needed for EPRI. No way to image Eaton & Eaton (1989)

EPR Spectroscopy Spatial Imaging Spectral-spatial Imaging Spatially-unresolved Spatially-resolved Spin density Spatially-resolved Spectral shape 0 + 1 dimensional 3 + 0 dimensional 3 + 1 dimensional EPRI is capable of measuring the distribution of paramagnetic and free radical species in tissues

1D SPATIAL 2D SPATIAL 3D SPATIAL

Gradient Magnetic Field Homogeneous (Spectroscopy) Inhomogeneous (Not useful) Gradient (Imaging) Distance ---> Distance ---> Distance ---> <----- Magnetic Field

Gradient Magnetic Field B1 > B2 ISOFIELD LINE In the conventional CW EPR sweep mode, spins at B1 will come into resonance first. B1 B2 Gradient Vector

Projection s Projection p(q) y s1 q x s2 s3 s = cos q + y sin q

(q = 0o) Projection Acquisition (q = 45o) Spin density Field Gradient 0 0 Ray 2 0 3 0 Projection 0 2 0 0 2 0 0 3 0 1  Field Sweep 0  Field Sweep 1 (q =135o) 0 2 0 (q = 90o) 0 1 Field Sweep  0 3 2 0 0 0 2 0 2 Field Sweep  1 0 0 1

2 3 1 3 1 2 2 2 2 2 2 8 2 2 2 6 2 8 2 1 3 3 2 2 2 1 2 2 2 1 3 2 2 1 2 3 10 2 6 2 3 8 2 2 2 2 2 3 2 2 1 2 3 1 3 2 2 2 1 1 3 2 1 9 3 3 8 3 4 9 3 3 3 2 1 1 1 2 1 2 3 1 2 3 4 5 6 7 8 9 0 2 1 1 0 1 0 2 0 0 2 0 0 3 0 0 2 0 0 2 0 0 2 0 0 3 0 0 0 3 0 1 0 2 0 1 Image Reconstruction by Backprojection x y 1 2 3 P (q =90o) 4 5 6 7 8 9 P (q =135o) P (q =45o) P (q =0o)

3D IMAGING OF A SPIRAL PHANTOM A pack of three identical tubes (i.d.: 3 mm) and a polyethylene tubing (id: 1.1 mm) wound around the pack. The tubes were filled with 0.5 mM solution of TAM. 12 mm 12 mm 12 mm 0 256 0 256 3D composite view A 2D projection of the image Proj., 1024; gradient, 10 G/cm; acq. time, 51.6 min; resolution, 100 mm.

3D Image of a rat heart perfused with glucose char B C A PA Ao Ao LM LAD LV LV apex LV apex 3D EPR image of an ischemic rat heart infused with glucose char suspension oximetry label. A: Full view B: A longitudinal cutout showing the internal structure of the heart. Ao, aortic root; C, cannula; PA, pulmonary artery; LM, left main coronary artery; LAD, left anterior descending artery; LV, left ventricular cavity.

Gated Imaging of Rat Heart Transverse slices Longitudinal slices systolic diastolic

IMAGING OF RAT KIDNEY PERFUSED WITH TAM A1 A2 A3 c b d a Representative slices (24x24 mm2, thickness, 0.19 mm) obtained from a 3D spatial image. A1-A6: Vertical slices B1-B3: Transverse slices a - cannula b - renal artery c - cortex d - calyses A4 A5 A6 c B1 B2 a B3 Proj, 1024; grad, 25.0 G/cm; acq. time, 76.8 min; resolution, 200 um d Intensity

3D IMAGING OF NITROXIDE DISTRIBUTION IN TUMORS NORMAL RIF-1 SCCVII Intensity --> SLICES (0.3 MM) FROM 3D IMAGES OF MURINE TUMORS (3-CP; 100 mg/kg) C3H mice; gradient 20 G/cm;144 projections;10x10 mm2 Kuppusamy, P., et al. Cancer Research, 58, 1562-1568 (1998).

Molecular oxygen is paramagnetic sx* py*=pz* 2p (O) 2p(O) py=pz sx Molecular oxygen has two unpaired electrons • Oxygen gives strong EPR signals in the gas phase • However, no EPR spectrum has been reported 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, molecular oxygen can be measured and quantified indirectly using spin-label EPR oximetry

Particulate (Solid) probes What is reported? Lithium phthalocyanine (LiPc) Sugar chars Fusinite Coal India ink • pO2 (mmHg/Torr) • Localized Measurement • Resolution < 0.2 mmHg • Repeated Measurements • Stable for days to weeks • Independent of medium Soluble probes Nitroxides Trityl radicals EPR oximetry probes • Concentration (mM) of dissolved oxygen in the bulk volume • Resolution 2-10 mmHg

Principle of EPR oximetry The collision frequency w, according to the hard sphere theory of Smoluchowski is R Ö2 Ö2 w = 4pRp(DSL + DO2) [O2] which translates to EPR line-broadening as . N Ö2 Ö2 Dw = k DO2 [O2] O Ö2 SL Bimolecular collision between SL and oxygenleads to Heisenberg spin exchange

Object Data Information xy Spectral-Spatial EPR image Spin density e.g. Oxygen Mapping of Oxygen Mapping of oxygen in biological tissues is possible by spectral-spatial or ‘spectroscopic’ EPR imaging.

EPR Oxegen Mapping (EPROM) [PDT, mM] [Oxygen] Phantom of tubes with 15N-PDT 0.25 38% 38% 0.50 0.50 21% 0.25 21% 40% 30% Amplitude Map Oxygen Map Spin Density Map 20% Oxygen 10% 0% S. Sendhil Velan, R. G. S. Spencer,J. L. Zweier & P. Kuppusamy, Magn. Reson. Med. 43, 804-809 (2000)

Lateral arteries Tendon Lateral veins Bone Cutaneous layer Ventral vein Ventral artery Amplitude Map Spin Density Map Oxygen Map 300 225 3-D spectral-spatial L-band, 3-CP probe Oxygen Concentration (mM) 150 75 0 Mapping of arterio-venous oxygenation in a rat tail, in vivo Sendhil Velan, S., Spencer, R.G.S., Zweier, J. L. & Kuppusamy P. Magn. Reson. Med. 43, 804-809 (2000)

Response to Oxygen Oxygen Time Response 0.6 Air N2 Air N2 Air slope = 8.9 mG / mmHg 21% 0.4 EPR Line-width (G) Oxygen 0.2 0 % 0.0 0 10 20 30 40 50 60 0 25 50 75 100 125 150 Time (sec) Oxygen (pO2) LiPc (Lithium Phthalocyanine) Oxygen sensitive (T2) EPR probe N N N N N Li N N N Ilangovan, G., Li, H., Zweier, J.L., Kuppusamy, P. J. Phys. Chem. B 104, 4047 (2000); 104, 9404 (2000); 105, 5323 (2001)

Carbogen breathing Room Air breathing Carbogen breathing Room Air breathing Carbogen breathing 120 100 80 60 40 20 0 0 20 40 60 80 100 120 Time (min) Oxygenation of RIF-1 Tumor (Carbogen-breathing) Air-breathing Carbogen-breathing pO2 (mmHg) 444 446 448 Magnetic Field (Gauss)

Co-imaging of the oximetry probe in the tumor tissue RESONATOR Perfusion image of a nitroxide Image of LiPc powder deposit 20 mm x 20 mm Normal Leg Tissue RIF-1 Tumor (14x 16 mm) pO2 = 17.6 mmHg pO2 = 4.6 mmHg Oxygen Measurements using LiPc, a Particulate EPR Probe

Control : Muscle Tissue of Non-tumor bearing mice Normal Muscle Tissue of RIF-1 Mice Tumor Tissue of RIF-1 Mice In vivo measurements of pO2 from tumor and normal gastrocnemius muscle tissues of RIF-1 tumor-bearing mice. 30 25 20 LiPc particles were implanted in the tumor on the right leg and normal muscle on the left leg and tissue pO2 values were repeatedly measured on the same animals for up to 8 days using EPR oximetry. (N = 5) 15 Tissue pO2 (mmHg) 10 5 0 0 2 4 6 8 Days Post-Implantation of LiPc

Redox Status applies to a set of redox couples. It is the summation of the products of the reduction potential and reducing capacity of all the redox couples present GSSG/2GSH NADP+/NADPH TrxSS/Trx(SH)2 Redox Status =  [Reduction potential x concentration]i Redox Status Redox State describe the ratio of the interconvertible oxidized and reduced form of a specific redox couple GSSG/2GSH GSSG + 2H+ + 2e- 2GSH Ehc = E0– (RT/nF) log([GSH]2/[GSSG]) Redox State = Reduction potential x concentration Schaefer, F. Q. & Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191-1212 (2001)

EPR Intensity t½ ~ min Time (min) Nitroxides as probes of tissue redox status O O + e- NH2 NH2 - e- Redox conversion in tissues N N O OH Nitroxide Hydroxylamine EPR 'active' EPR 'inactive' Swartz et al, Free Radic. Res. Commun., 9, 399-405 (1990) Kuppusamy et al, Cancer Research, 58, 1562-1568 (1998) Krishna et al, Breast Disease, 10, 209-220 (1998)

NORMAL TISSUE (LEFT LEG) RIF-1 TUMOR (RIGHT LEG) 3.0 4.5 6.0 7.5 9.0 10.5 12.0 13.5 15.0 16.5 Time (min) Intensity --> Reduction of 3-CP in the Normal & Tumor Tissue C3H mice with RIF-1 tumor; ~30 g bw; dose: 100 mg/kg, iv; Measured in vivo using surface resonator at L-band (1.25 GHz); Images: 10x10 mm2 Kuppusamy, P., et al. Cancer Research, 58, 1562-1568 (1998).

Intensity Time Reconstruction of Tissue ‘Redox Status’ Image ‘kx,y’ x,y x,y Reduction constant, kx,y Redox Map Intensity Map [64x64] 8-12 points, first order [64x64] Kuppusamy, P., & Krishna, MC., Curr. Topics in Biophys. (2002)

REDOX MAPPING BY EPR IMAGING (VALIDATION EXPERIMENT) A B 3.6 min 5.8 min 8.2 min 10.5 min X + XO TPL TPH D C GSH TPL + XO 13.5 min 15.8 min 18.2 min 20.5 min + 4 XO + 2 XO A 120 + XO 100 B 25 0.43 min-1 Frequency 20 TPL 15 C D 10 + 4 XO 5 0 0.00 min-1 0.0 0.1 0.2 0.3 0.4 Reduction Rate (min-1) + XO (x2)

Redox Mapping of Tumor: Effect of BSO (GSH Depletion) 40 30 Median: 0.054 min-1 RIF-1 Frequency 20 10 0 88 75 63 RIF-1 +BSO 50 Frequency Median: 0.030 min-1 38 25 13 0 0.0 0.05 0.10 0.15 0.0 0.05 0.10 0.15 Rate constant (min-1) Rate constant (min-1) Kuppusamy et al Cancer Research (2002)

Redox Status 0.08 0.06 Rate Constant (min-1) 0.04 0.02 0.00 Normal Muscle RIF-1 RIF-1 +BSO Rate constants of nitroxide clearance in leg muscle (Normal) and RIF-1 tumors of untreated and BSO-treated (6-hrs post-treatment of 2.25 mmol/kg of BSO, ip) tumor-bearing mice. (N=5) REDOX STATUS & GSH LEVELS IN RIF-1 TUMOR GSH Level 5 4 3 GSH (mmol/g Tissue) 2 1 0 Normal Muscle RIF-1 RIF-1 +BSO GSH levels in leg muscle (Normal) and RIF-1 tumors of untreated and BSO-treated (6-hrs post-treatment of 2.25 mmol/kg of BSO, ip) tumor-bearing mice. (N=7)

EFFECT OF DIETHYLMALEATE (DEM) ON TUMOR REDOX STATUS Tumor 4 3 GSH in Tumor Tissue Median 0.053 min-1 Frequency 2 2.5 1 2.0 0 0 0.04 0.08 0.12 0.16 Reduction rate (min-1) 1.5 GSH (µg/mg tissue) 1.0 3 Tumor+DEM 0.5 2 Frequency 0.0 Median 0.034 min-1 Tumor (N=5) Tumor+DEM (N=8) 1 0 0 0.04 0.08 0.12 0.16 Reduction rate (min-1) Yamada et al Acta Radiol (2002)

40 30 Frequency 20 Room air 10 0 60 40 Frequency Carbogen 20 0 0.0 0.05 0.10 0.15 0.0 0.05 0.10 0.15 Rate constant (min-1) Rate constant (min-1) Nitroxide intensity -> Redox mapping of tumor: Effect of Carbogen-Breathing (Oxygenation) Ilangovan G., Li, H., Zweier, J. L., Krishna M. C., Mitchell J. B. Kuppusamy, P. Magn. Reson. Med. (2002))

EPR detection of SH-groups (ESR analogs of Ellman’s reagent) Reaction with GSH Berliner, L.J., et al, Unique In VivoApplications of Spin Traps, Free Rad.Biol.Med.30(5): 489-499. Khramtsov,V.V. et al. 1997, J.Biochem. Biophys. Methods 35: 115

Summary • EPR spectroscopy is a direct & definitive technique for detection and quantitation of free radicals and paramagnetic species. • Low-frequency EPR spectroscopy enables measurement of free radicals (endogenous/exogenous) in biological systems including intact tissues, isolated organs and small animals. • In vivo EPR spectroscopy and imaging methods enable noninvasive measurement and mapping of tissue pO2, redox status and pH.

BOOKS Rosen, G. M., Britigan, B. E., Halpern, H. J., and Pou, S. Free Radicals: Biology and Detection by Spin Trapping. New York: Oxford University Press, 1999 Eaton, G. R., Eaton, S. S., and Ohno, K. EPR imaging and in vivo EPR: CRC Press, Inc, 1991. REFERENCES Buettner, G. R. Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med, 3: 259-303, 1987. Berliner, L. J., Khramtsov, V., Fujii, H., and Clanton, T. L. Unique in vivo applications of spin traps. Free Radic Biol Med, 30: 489-499, 2001. McCay, P. B. Application of ESR spectroscopy in toxicology. Arch Toxicol, 60: 133-137, 1987. Kuppusamy, P., Chzhan, M., Vij, K., Shteynbuk, M., Lefer, D. J., Giannella, E., and Zweier, J. L. Three-dimensional spectral-spatial EPR imaging of free radicals in the heart: a technique for imaging tissue metabolism and oxygenation. Proc Natl Acad Sci U S A, 91: 3388-3392, 1994. Kuppusamy, P., Ohnishi, S. T., Numagami, Y., Ohnishi, T., and Zweier, J. L. Three-dimensional imaging of nitric oxide production in the rat brain subjected to ischemia-hypoxia. J Cereb Blood Flow Metab, 15: 899-903, 1995. Kuppusamy, P., Wang, P., and Zweier, J. L. Three-dimensional spatial EPR imaging of the rat heart. Magn Reson Med, 34: 99-105, 1995. Kuppusamy, P., Chzhan, M., Wang, P., and Zweier, J. L. Three-dimensional gated EPR imaging of the beating heart: time-resolved measurements of free radical distribution during the cardiac contractile cycle. Magn Reson Med, 35: 323-328, 1996. Kuppusamy, P., Wang, P., Samouilov, A., and Zweier, J. L. Spatial mapping of nitric oxide generation in the ischemic heart using electron paramagnetic resonance imaging. Magn Reson Med, 36: 212-218, 1996. Kuppusamy, P., Wang, P., Zweier, J. L., Krishna, M. C., Mitchell, J. B., Ma, L., Trimble, C. E., and Hsia, C. J. Electron paramagnetic resonance imaging of rat heart with nitroxide and polynitroxyl-albumin. Biochemistry, 35: 7051-7057, 1996. Khramtsov, V. V., Yelinova, V. I., Glazachev Yu, I., Reznikov, V. A., and Zimmer, G. Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label. J Biochem Biophys Methods, 35: 115-128, 1997. Kuppusamy, P., Afeworki, M., Shankar, R. A., Coffin, D., Krishna, M. C., Hahn, S. M., Mitchell, J. B., and Zweier, J. L. In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model. Cancer Res, 58: 1562-1568, 1998. Kuppusamy, P., Shankar, R. A., and Zweier, J. L. In vivo measurement of arterial and venous oxygenation in the rat using 3D spectral-spatial electron paramagnetic resonance imaging. Phys Med Biol, 43: 1837-1844, 1998. Velan, S. S., Spencer, R. G., Zweier, J. L., and Kuppusamy, P. Electron paramagnetic resonance oxygen mapping (EPROM): direct visualization of oxygen concentration in tissue. Magn Reson Med, 43: 804-809, 2000. Kuppusamy, P., Shankar, R. A., Roubaud, V. M., and Zweier, J. L. Whole body detection and imaging of nitric oxide generation in mice following cardiopulmonary arrest: detection of intrinsic nitrosoheme complexes. Magn Reson Med, 45: 700-707, 2001. Ilangovan, G., Li, H., Zweier, J. L., Krishna, M. C., Mitchell, J. B., and Kuppusamy, P. In vivo measurement of regional oxygenation and imaging of redox status in RIF-1 murine tumor: Effect of carbogen-breathing. Magn Reson Med, 48: 723-730, 2002. Kuppusamy, P., Li, H., Ilangovan, G., Cardounel, A. J., Zweier, J. L., Yamada, K., Krishna, M. C., and Mitchell, J. B. Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels. Cancer Res, 62: 307-312, 2002. Yamada, K. I., Kuppusamy, P., English, S., Yoo, J., Irie, A., Subramanian, S., Mitchell, J. B., and Krishna, M. C. Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging. Acta Radiol, 43: 433-440, 2002. WEB SITES The Illinois EPR Research Center: http://ierc.scs.uiuc.edu/ The National Biomedical EPR Center at the Medical College of Wisconsin : http://www.biophysics.mcw.edu/bri-epr/bri-epr.html NIEHS - Spin Trap Database: http://epr.niehs.nih.gov/stdb.html

Periannan Kuppusamy, PhD Center for Biomedical EPR Spectroscopy & Imaging