1. 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 EPR Spectroscopy Spying on unpaired electrons - What information can we get? Sunrise Free Radical School Oxygen-2002 ::Nov 21, 2002

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

3. M S =±½ M S =-½ M S =+½ Electron S(½) M I =+½ M I =-½ M I =+½ Nucleus I (½) Selection Rule  M S = ±1;  M I = 0 Magnetic Field S=½; I=½ Doublet hfc Hyperfine Coupling

4. M S =±½ M S =-½ M S =+½ Electron S (½) M S = ±½ M I =+1 M I =-1 M I =+1 Nucleus I (1) M I =0,±1 Selection Rule  M S = ±1;  M I = 0 Magnetic Field S=½; I=1 Triplet hfc Hyperfine Coupling M I = 0 hfc

5. We can detect & measure free radicals and paramagnetic species 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 High sensitivity (nanomolar concentrations) No background Definitive & quantitative What do we do with EPR?

6. What do we do with it? Yes! Intact tissues, organs or whole-body can be measured. But there is a catch! What is the optimum frequency? - depends on sample size 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. Can we use EPR to measure free radicals from biological systems (in vivo or ex vivo)? Frequency~300 MHz~750 MHz1-2 GHz~3 GHz9-10 GHz Penetration Depth > 10 cm6-8 cm1-1.5 cm1-3 mm1 mm ObjectsMouse, ratMouseMouse, rat heart Mouse tail Topical (skin) In vitro samples (~100 uL vol.) PioneersHalpern et al Krishna et al Zweier et alHyde et al Swartz et al Zweier et al Hyde et al Zweier et al

7. What else can we do with EPR? 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’. We can use free radicals as “spying probes” to obtain information from biological systems

8. amplitude Redox status Cell viability Tissue perfusion width Oxygen, pO 2 Acidosis, pH Viscosity Molecular motion Splittin g Oxygen, pO 2 Redox status Acidosis, pH Thiols (GSH) Cell viability Viscosity Tissue perfusion Molecular motion Functional parameters from an EPR spectrum In vivo EPR spectroscopy is capable of providing useful physiologic and metabolic (functional) information from tissues

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

10. Can we image free radicals in biological systems? NMREPR Spin ProbesTissue protonsFree radicals (endogenous)(>50 M)(<< nM) Probe StabilityIdeal< nanoseconds Relaxation time (LW)m sec<  sec (LW: 1 G) No suitable endogenous spin probes for EPRINothing to image Fast electronics needed for EPRI.No way to image Eaton & Eaton (1989)

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

12. 1D SPATIAL 2D SPATIAL 3D SPATIAL

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

14. Gradient Magnetic Field ISOFIELD LINE B1B1 B2B2 B 1 > B 2 Gradient Vector In the conventional CW EPR sweep mode, spins at B 1 will come into resonance first.

15. s2s2 s3s3 s1s1  s = cos  + y sin  p(  ) s Projectio n x y

16.  Field Sweep 020020030 Field Sweep  0 2 0 0 3 0 0 2 0  Field Sweep 0 0 2 0 3 0 1 0 1 Field Sweep  1 0 1 0 2 0 2 0 1 Field Gradient Ray Spin density Projection  = 0 o )  = 45 o )  = 90 o )  =135 o ) Projection Acquisition

17. 020020030 020030020 0 0 20 301 0 1 10102 02 0 1 222222133 262282292 222222341 122223231 3833103393 221322131 321121132 282262282 123131231 P (  =45 o ) P (  =0 o ) P (  =90 o ) P (  =135 o ) 123456789 1 2 3 4 5 6 7 8 9 x y Image Reconstruction by Backprojection

18. 12 mm 02560 12 mm 3D composite viewA 2D projection of the image Proj., 1024; gradient, 10 G/cm; acq. time, 51.6 min; resolution, 100 mm. 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. 3D IMAGING OF A SPIRAL PHANTOM

19. A B PA C LAD LV apex Ao LV LM Ao 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. 3D Image of a rat heart perfused with glucose char

20. Gated Imaging of Rat Heart Transverse slices systolicdiastolic Longitudinal slices

21. A1A2A3 A6A5A4 a c d b B1B2 B3 d a c Representative slices (24x24 mm 2, 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 IMAGING OF RAT KIDNEY PERFUSED WITH TAM Proj, 1024; grad, 25.0 G/cm; acq. time, 76.8 min; resolution, 200 um Intensity

22. NORMAL RIF-1 SCCVII SLICES (0.3 MM) FROM 3D IMAGES OF MURINE TUMORS (3-CP; 100 mg/kg) C3H mice; gradient 20 G/cm;144 projections;10x10 mm 2 Intensity --> 3D IMAGING OF NITROXIDE DISTRIBUTION IN TUMORS Kuppusamy, P., et al. Cancer Research, 58, 1562-1568 (1998).

23. 2p (O) 2p(O)  y *=  z * y=zy=z x*x* xx 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 Molecular oxygen is paramagnetic

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

25. .N.N O SL Ö2Ö2 Ö2Ö2 Ö2Ö2 Ö2Ö2 Ö2Ö2 Bimolecular collision between SL and oxygen leads to Heisenberg spin exchange R Principle of EPR oximetry which translates to EPR line-broadening as  = 4  Rp(D SL + D O 2 ) [O 2 ] The collision frequency , according to the hard sphere theory of Smoluchowski is  w = k D O 2 [O 2 ]

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

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

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

29. NN NN N NN N Li LiPc (Lithium Phthalocyanine) Oxygen sensitive (T 2 ) EPR probe Time (sec) 0102030405060 21% 0 % Oxygen Oxygen Time Response Air N2N2 N2N2 Oxygen (pO 2 ) 0255075100125150 EPR Line-width (G) 0.0 0.2 0.4 0.6 Response to Oxygen slope = 8.9 mG / mmHg Ilangovan, G., Li, H., Zweier, J.L., Kuppusamy, P. J. Phys. Chem. B 104, 4047 (2000); 104, 9404 (2000); 105, 5323 (2001)

30. Oxygenation of RIF-1 Tumor (Carbogen-breathing) pO 2 (mmHg) Time (min) 020406080100120 0 20 40 60 80 100 120 Room Air breathing Carbogen breathing Room Air breathing Carbogen breathing Carbogen breathing Magnetic Field (Gauss) 444446448 Carboge n- breathin g Air-breathing

31. RESONATOR 20 mm x 20 mm RIF-1 Tumor (14x 16 mm) Normal Leg Tissue pO 2 = 4.6 mmHgpO 2 = 17.6 mmHg Oxygen Measurements using LiPc, a Particulate EPR Probe Co-imaging of the oximetry probe in the tumor tissue Image of LiPc powder deposit Perfusion image of a nitroxide

32. Days Post-Implantation of LiPc 02468 Tissue pO 2 (mmHg) 0 5 10 15 20 25 30 Normal Muscle Tissue of RIF-1 Mice Tumor Tissue of RIF-1 Mice Control : Muscle Tissue of Non-tumor bearing mice In vivo measurements of pO 2 from tumor and normal gastrocnemius muscle tissues of RIF-1 tumor-bearing mice. LiPc particles were implanted in the tumor on the right leg and normal muscle on the left leg and tissue pO 2 values were repeatedly measured on the same animals for up to 8 days using EPR oximetry. (N = 5)

33. 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 E hc = E 0 – (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) 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

34. Nitroxides as probes of tissue redox status NitroxideHydroxylamin e Redox conversion in tissues EPR 'active'EPR 'inactive' + e - - e - 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) N O NH 2 O N OH NH 2 O

35. NORMAL TISSUE (LEFT LEG) RIF-1 TUMOR (RIGHT LEG) 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 mm 2 3.04.56.07.59.010.512.013.515.016.5 Time (min) Intensity --> Kuppusamy, P., et al. Cancer Research, 58, 1562-1568 (1998).

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

37. 3.6 min5.8 min8.2 min10.5 min 13.5 min15.8 min18.2 min 20.5 min Frequency 0.00.10.20.3 0 0.4 5 Reduction Rate (min -1 ) 25 120 20 15 10 B C D 100 A + 4 XO TPL + XO (x2) + XO 0.00 min -1 0.43 min -1 REDOX MAPPING BY EPR IMAGING (VALIDATION EXPERIMENT) A B CD + XO + 2 XO + 4 XO TPL TPL TPH X + XO GSH

38. Frequency 0 10 20 30 40 RIF-1 0 0.00.050.100.15 Rate constant (min -1 ) RIF-1 +BSO 0.050.10 Rate constant (min -1 ) 0.150.0 13 25 38 50 63 75 88 Frequency Redox Mapping of Tumor: Effect of BSO (GSH Depletion) Median: 0.054 min -1 Median: 0.030 min -1 Kuppusamy et al Cancer Research (2002)

39. 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) Normal Muscle RIF-1 +BSO GSH (  mol/g Tissue) 0 1 2 3 4 5 GSH Level Normal Muscle RIF-1 +BSO Rate Constant (min -1 ) 0.00 0.02 0.04 0.06 0.08 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 REDOX STATUS & GSH LEVELS IN RIF-1 TUMOR

40. EFFECT OF DIETHYLMALEATE (DEM) ON TUMOR REDOX STATUS 0 1 2 3 4 0.080.120.1600.04 Frequency Reduction rate (min -1 ) Tumor Median 0.053 min -1 1 2 3 0 0.080.120.1600.04 Frequency Reduction rate (min -1 ) Tumor+DEM Median 0.034 min -1 0.0 0.5 1.0 1.5 2.0 2.5 GSH (µg/mg tissue) GSH in Tumor Tissue Tumor+DEM (N=8) Tumor (N=5) Yamada et al Acta Radiol (2002)

41. Nitroxide intensity -> Carbogen 0.00.050.100.15 Rate constant (min -1 ) Frequency 0.050.10 0 20 40 60 0.150.0 Room air Frequency 0 10 20 30 40 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))

42. 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

43. Summary 1.EPR spectroscopy is a direct & definitive technique for detection and quantitation of free radicals and paramagnetic species. 2.Low-frequency EPR spectroscopy enables measurement of free radicals (endogenous/exogenous) in biological systems including intact tissues, isolated organs and small animals. 3.In vivo EPR spectroscopy and imaging methods enable noninvasive measurement and mapping of tissue pO 2, redox status and pH.

44. 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/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 http://www.biophysics.mcw.edu/bri-epr/bri-epr.html NIEHS - Spin Trap Database: http://epr.niehs.nih.gov/stdb.html

45. 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/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 http://www.biophysics.mcw.edu/bri-epr/bri-epr.html NIEHS - Spin Trap Database: http://epr.niehs.nih.gov/stdb.html