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

2. 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

3. 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.

4. Magnetic Resonance Methods1.
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

5. 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

6. 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

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

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

10. 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

11. . 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

12. 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

13. 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 , 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. 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
pO = 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

15. 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

16. 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

17. Effect of NO Addition n on BAEC Respiration20 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

18. 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

19. 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

20. 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

21. 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

22. 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

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

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

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

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

29. 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

30. 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

31. 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

32. 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

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 ?