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

3). Therapeutic strategies

Radiation treatment of Tumors

Ischemic Heart Diseases

2). Pathogenesis

1). Metabolic control in cells

Oxidant / Antioxidant dynamics

Methods for Oxygen Measurements In Tissues

Fluorescent Probes

Ru(bpy)33+

hRu(bpy)3

3+* O2

O2

*

A silicone matrix at the tip of a 230 m fiber contains the oxygen-sensitive fluorescent dye.

Clarks Electrodes

O2 H2O4e

Microelectrode (10-100mic) is inserted into the tissue at the desired location and the oxygen reduction current is measured.

Magnetic Resonance Methods

Nuclear Magnetic Resonance (NMR)

Electron Paramagnetic Resonance (EPR)

1.

2.

Paramagnetic nucleus relaxation is induced by the molecular oxygen

Paramagnetic single electron relaxation is increased by the the molecular oxygen

• BOLD MRI -- T2* effect

• 19F MRS(I) --- T1 effect

Molecular oxygen is paramagnetic Ö2

2p (O) 2p(O)

y*=z*

y=z

x*

x

Molecular oxygen has two unpaired electrons

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

±½

En

erg

y

Magnetic Field

Electron Paramagnetic Resonance (EPR)Oxymetry Electron

Spin, S

ms = +½

ms = -½

E=h=gB

OxygenOxygen

Magnetic Field

EPR Oxymetry - ProbesEPR Oxymetry - Probes

Solid state probesSoluble probesLithium Phthalocyanine Sugar charsFusiniteCoalIndia ink

NitroxidesTrityl radicals

Requirement to be a good oxymetry Probe:

• Higher T2 (Sharp EPR spectrum)• Preferably single EPR line (No hyperfine splitting)

LW = e( 1T2

+1

2T1

)

The Principle of EPR oxymetry

T2 – spin-spin relaxation

LW

S

S

S

S

S

S

S

SS

SS

S

EPR spectrum of S

S

S

SS

SS

EPR spectrum of S

S

S

SS

SS

OO

O

O

OO

O

O

EPR spectrum of S

S O

LW =

e

2

3

= 4Rp[O2] DS + DO2

R = interaction radius between A and B

DA and DB = Diffusion coefficients of A and B

P = the probability of relaxation

LW

[O2]

R

(Smoluchowski eqn.)

.

HO

C

SS

OO

OHO

H

CO

O-

S

SO

O

HO

Tryaryl methyl radical

(TAM)

Example 1: Soluble Spin Probes for EPR oximetry

SS

OO

OH

OH

CO

O-

S

S

OO

HO

S

SO

O

OHOH

COO-

S

S

O

OHO

HO

HO

EPR spectrum in the presence of room air (21%O2)

EPR spectrum at anoxic condition (N2)

p O 2 / mmHg

0 20 40 60 80

LW

/ m

G

0

200

400

600

a

Calibration Curve

NN

NN NN

NN

NN

NN

NN

NN

LLii

••NN NN

NNNN

NN

NN NN

NN

LLii

Lithium PhthalocyanineLithium Phthalocyanine Lithium NaphthalocyanineLithium Naphthalocyanine(LiPc)(LiPc) (LiNc)(LiNc)

EPR OXIMETRY: PROBESEPR OXIMETRY: PROBESMicrocrystalline particulatesMicrocrystalline particulates

Ilangovan et al, Ilangovan et al, J. Phys. Chem.BJ. Phys. Chem.B,, 2000 2000, 104, 4047, 104, 4047 20002000, 104, 9404, 104, 9404 20012001, 105, 5323, 105, 5323 20022002, 106, 11929, 106, 11929 J. Magn. ResonJ. Magn. Reson. . 20042004, 170, 42-48, 170, 42-48

Ilangovan et al, Ilangovan et al, Free Rad. Biol. MedFree Rad. Biol. Med, , 20022002, 32, 139, 32, 139 20032003, 35, 1138, 35, 1138Ilangovan et al, Ilangovan et al, J. Magn. Magnt. MaterJ. Magn. Magnt. Mater, , 20012001, 233, L131, 233, L131

Transport of Molecular O2 into the Particulate Spin probes

The Knudson flux Jk is defined as*

Jk = Dk (pO2)

l R Twhere

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

l(pO2)start (pO2)end

Di f

fus i

on

Adsorption

S S

Gas Phase

Schematics of Gas transport

* Frank-Kameneskii, D.A., Diffusion and heat Transfer in Chemical Kinetics, II nd Edition, Plenum, NY, 1969

Knudson Diffusion is dominated since the mean free path of O2 is higher than pores in LiPc

Ilangovan, G. et al Ilangovan, G. et al J. Phys. Chem.,J. Phys. Chem., 20002000, 104, 4047, 104, 4047

Effect of Oxygen on the Effect of Oxygen on the EPR Spectrum of LiPc EPR Spectrum of LiPc

WithWithNitrogenNitrogen

WithWithroom airroom air

Re-saturatedRe-saturatedwith nitrogenwith nitrogen

0.5 G0.5 G

Oxygen Sensing ProbeOxygen Sensing Probe

Lithium Phthalocyanine (LiPc) Lithium Phthalocyanine (LiPc)

Line width Vs. pO2Line width Vs. pO2

pOpO22 (mmHg) (mmHg)00 5050 100100 150150 200200

Lin

e w

idt h

(G

)L

ine

wid

th (

G)

00

0.50.5

1.01.0

1.51.5

2.02.0

2.52.5

5µm

8mm8mm

5050µµL quartz L quartz microtube as microtube as reaction vesselreaction vessel

Magnified view of the micro Magnified view of the micro tube containing the LiPc tube containing the LiPc microcrystals (oxymetry microcrystals (oxymetry probe) probe)

Schematics of EPR oxymetry experimental Schematics of EPR oxymetry experimental set up. The sample tube could be either set up. The sample tube could be either quarts microtube or gas permeable teflon quarts microtube or gas permeable teflon tubetube

LiPcLiPc

OutletOutlet

CottonCottonsupportsupport

Gas mixture Gas mixture inletinlet

MA

GN

ET

MA

GN

ET

MA

GN

ET

MA

GN

ET

TMTM110110CavityCavity

SampleSample

Experimental Set-up Experimental Set-up

Ilangovan et al, Ilangovan et al, Methods In Enzymology, Methods In Enzymology, 2004,381, 7472004,381, 747

An EPR Based method for Simultaneous An EPR Based method for Simultaneous measurements of Omeasurements of O22 and Free radicals and Free radicals

generationgeneration

Effect of NO Addition n on BAEC RespirationEffect of NO Addition n on BAEC Respiration

Complex I Complex I

Co

nC

on

ΔΔ O

.D./

min

O.D

./m

in

0.0000.000

0.0010.001

0.0020.002

0.0030.003

21

%2

1%

21

%2

1%

Co

nC

on

Complex II/III Complex II/III

ΔΔ O

.D./

min

O.D

./m

in

0.0000.000

0.0010.001

0.0020.002

0.0030.003

21

%2

1%

21

%2

1%

NO

NO

Complex IVComplex IV

0.000.00

0.010.01

0.020.02

0.030.03

ΔΔ O

.D./

min

O.D

./m

in

Co

nC

on

21

%2

1%

21

%2

1%

.5%

.5%

.5%

.5%

* P < 0.001* P < 0.001

NO

NO

NO

NO

ETC Complex ActivityETC Complex Activity

NADHNADH

I IIICyt c

II IVUQ Inner membrane

SuccinateSuccinateOO22

Complex INADH:Ubiquinone

Complex II/III Succi-Cyto Reductase

Complex IVCyt c Oxidase

00 2020 4040 6060 808000

2020

4040

6060

8080

100100

120120

140140

160160

Control Control

NO addedNO added

Time (min)Time (min)

pO

pO

22 (m

mH

g)

(m

mH

g)

4x104x1066cellscells21%0.5%

pOpO22 (mmHg) (mmHg)d

pO

dp

O22/

dt(

mm

Hg

/min

)/d

t(m

mH

g/m

in)

00 2020 4040 6060 8080 100100 120120 140140 16016000

11

22

33

55 1010 1515 2020 2525

1.01.0

2.02.0

3.03.0

NO addedNO added

VVO

2max

O2m

ax (

mm

Hg

/min

) (

mm

Hg

/min

)

00

11

22

33

44

55

p50

(m

mH

g)

p50

(m

mH

g)

00

22

44

66

88

Co

nC

on

NO

NO

Co

nC

on

NO

NO

**

**

NO

Mechanism

H2O

I IIIII

IV

NOO2

ONOO-

Reversible inhibitionby NO

Irreversible inhibitionby ONOO-

This inhibition is

pO2 dependent

O2.-

NOS

(Not present at low pO2)(Present irrespective of pO2)

NOS generated NO causes attenuation of respiration via irreversible CuB binding; yet, no competitive binding at the a3 site; p50 remains unchanged

OO22

OO22-.-.

DEPMPODEPMPO DEPMPO-OOHDEPMPO-OOH

SubstrateSubstrateProductProduct

+

Enz

yme

N

O -

CH3

P(O) (OEt )2

H

N

O

H

O2

HOO P(O)(OEt)2

CH3

Free Radicals MeasurementFree Radicals Measurement

Simultaneous measurement approachSimultaneous measurement approach

Oxygen MeasurementOxygen Measurement

Simultaneous measurement of O2-. trapping

using DMPO

30 min30 min 45 min45 min

60 min60 min 90 min90 min

Experimental spectraExperimental spectra Simulated DMPO - OOH SpectraSimulated DMPO - OOH Spectra

Co

nce

ntr

atio

n /

Co

nce

ntr

atio

n /

MM

00

4040

8080

120120

160160

200200

240240

TIME /STIME /S

00 200200 400400 600600 800800 10001000 12001200 14001400 16001600 18001800

A

O2 Concentration

DEPMPO-OOH Concentration

Decomposed product of spin adduct

Kinetic analysis and concentration profiles of Kinetic analysis and concentration profiles of OO22 and DEPMPO-OOH and its decomposed and DEPMPO-OOH and its decomposed product product

5mm

Inserted LiPc in the heart

Micrograph of LiPc microcrystals

10 m14 mm

Cable to Power supply

To Microwave source

PerfusedHeart

Pre-ischemicequilibrium

5 min after ischemia

10 min after ischemia

15 min after ischemia

10m

l/m

in

FL

OW

RA

TE

100m

mH

g

DE

VE

LO

PE

D P

RE

SS

UR

E

60 90 120

150

Time (min)

RPP (bpm. mmHg)

0 10000 20000 30000 40000

Q(n

mol

/g/m

in)

0

2000

4000

6000

8000

10000

Correlation of Heart Injury to Oxygen consumption

Oxygen Measurements in RIF-1 Murine Tumor Model

Biomedical Applications

Implanting the LiPc microcrystals into the Gastrocnemius muscle and Tumor Tissues

Shaft LiPc

EPR imaging instrumentation, consisting of an L-band EPR spectrometer, three sets of water-cooled gradient coils and a bridged-loop surface resonator.

Male C3H anesthetized mouse with Implanted LiPc into the gastrocnemius muscle.

RESONATOR

25 mm

100 mm

MAGNET MAGNET

GRADIENTCOILS

[Z Y X]

GRADIENTCOILS

[X Y Z]

MODULATIONCOILS

HALL PROBES

Number of days0 2 4 6

pO

2 /

mm

Hg

0

5

10

15

20

25

30

Number of days0 2 4 6

pO

2/ m

mH

g

0

5

10

15

20

25

30

Animal 1 Animal 2 Animal 3 Animal 4 Animal 5

TUMOR NORMAL TISSUE

pO2 measurement in RIF-1 Tumor bearing mice

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)

Time / min0 20 40 60 80 100 120 140

pO

2/ m

mH

g

0

20

40

60

80

100

120

Car

bo

gen

Ro

om

air

1st cycle 2nd cycle 3rd cycle

Initial pO2

<5mmHg

While Carbogen breathing100 mmHg

When switched to room air 20 mmHg

0.5G

CarbogenRoom air

Tumor is oxygenated to the level of normal tissue at least for 2hrs, after termination of carbogen breathing treatment

Ilangovan, G. et al Magn. Reson. Med., 2002, 48, 723

Tumor volume (mm3)0 100 200 300

pO

2 (m

mH

g)

0

4

8

12

16

0 20 40 60 800

4

8

12

16

20

Tumor Growth Vs pO2 relationship

Tu

mo

r V

olu

me

(mm

)3

100

200

0

50

150

250

300

Days post injection0 2 4 6 8 10 12 14

pO

2 (m

mH

g)

0

5

10

15

20

25

30LiPc with RIF-1 Cells

Suspensions of LiPc in Saline or LiPc with RIF-1 cells were injected into the gastrocnemius muscle of right hind leg.

XY

Z

LiPc & RIF cell suspension

Oxymetry in Wound Healing

SKC1 mouse wound with LiPc in the periphery of the wound

Mouse in the EPR machine

EPR image of the LiPc in the wound

2mm

0 7500

2D EPR image

0

5

10

15

20

25

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day7 Day 8

Time Post Wounding

Control

Stress

STRESS AND OXYGEN

* P< 0.05

* **

**

pO

2 (

mm

Hg

)

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.