nmr spectrocopy

8/9/2019 nmr spectrocopy

1/38

Magnetic Resonance Imaging to Assess Tissue

Oxygenation and Redox Status

.

Hyperpolarized (by EPR) MRI

Electron Paramagnetic Resonance (EPR) Imaging

Redox Sensitive Paramagnetic Contrast Agents in MRI

Murali C Krishna

Radiation Biology Branch

Center for Cancer Research

National Cancer Institute

NIH, Bethesda, MD


2/38

EPR spectroscopy is similar to NMR

spectroscopy.

NMR spectroscopy detects nuclei with magnetic

moments.

ex: 1H, 13 C, 19 F, 31 P etc.

EPR spectroscopy detects species with unpaired

electrons.

ex: free radicals, transition metal complexes

At a given magnetic field, EPR is more sensitive

than NMR

Electron Paramagnetic Resonance


3/38

NMR spectroscopy detects nuclei with magnetic moments.

ex: 1H, 13 C, 19 F, 31 P etc.

EPR spectroscopy is similar to NMR spectroscopy.

EPR spectroscopy detects species with unpaired electrons.

ex: free radicals, transition metal complexes

At a given magnetic field, EPR is more sensitive than NMR

In MRI, proton NMR spectra are used for anatomic imaging.Reason: simple NMR spectrum.

For EPR Imaging we need species with simple EPR spectrum.

candidates: free radicals


4/38

Can we image free radicals in biological systems?

Spatially-resolved (anatomical) information can be

obtained using EPR imaging, similar to MRI

MRI EPR imaging

Spin Probes Tissue protons Free radicals

(endogenous) (>50 M) (


5/38

Molecular Oxygen Provides Contrast to Paramagnetic Probes inEPRI

Molecular oxygen is paramagnetic and provides contrast to paramagnetic probes.

This causes spectral broadening (increase in line width)

Line width changes from oxygen contrast > 200 % in EPRIn NMR such changes may be ~10%

It is possible to image spatial distribution of paramagnetic spin

probe by EPR and obtain pO2 information

Oxygen (pO2)

0 25 50 75 100

EPR

Line-w

idth(mG)

0

200

400

600

EPR Line Width vs pO2


6/38

Desirable Features

Chemical and spectroscopic:

Water soluble

Kinetically and metabolically stable

Single and narrow line resonance

Linewidth pO2

Toxicology:

Non-toxic at concentrations required for imaging

In vivo life time imaging time

Pharmacologic:

EPR Imaging with infusible probes may provide a more

global assessment of pO2


7/38

Overhauser, A; Phys. Rev. (1953)Dynamic nuclear polarizationapplicable to conduction electrons in alkali metals

Hyperpolarized MRI using EPR and Paramagnetic Contrast AgentsLurie DJ, Bussell DM, Bell LH, Mallard JR,

Proton- Electron Double Magnetic-Resonance Imaging of Free-Radical Solutions

J. Magnetic Resonance 76, 366-370 (1988)

C

.

Physical Basis for Hyperpolarization of Nuclei

Dynamic Nuclear Polarization with Paramagnetic Agents

Overhauser Effect
http://store.aip.org/OA_HTML/ecl.jsp?mode=detail&item=24804


8/38

Trityl Radicals

Gomberg, M; JACS. (1900)

Triphenyl methyl: A case of trivalent carbon. I reserve the field for myself.

Golman, K et. al.

Overhauser-enhanced MR imaging (OMRI)Acta Radiologica 39, 10-17(1998)

Contrast Agents for Hyperpolarization

of1H, 13 C


9/38

Contrast Agent - Trityl Radical

Mouse MTD = 2.5 - 5 mmol/kg

T1/2 in mouse blood and

kidney: 9-10 min

Linewidth: 100 - 300 mG

Oxygen tension: 0 - 21 %

.C

OX063

=CH2OH

CH2OH

HOCH2

COO-

Na+

S

S

S

S

HOCH2 Nycomed Imaging

Nycomed Amersham

AmershamGE


10/38

Overhauser MRI

Combination of EPRI + MRI

MRI for anatomy and EPR for Spectral information

MR Imaging of Hyperpolarized Water Protons by EPR

Low magnetic field (~10 mT)

Uses Free Radical Paramagnetic contrast agentsCoil tuned to both NMR and EPR frequencies


11/38

Scanner

Field strengths and Pulse Frequenci

EPR NMR

Magnet (mT): 8 15

Resonators: 226 MHz 640 kHz

Magnet and resonator dimensions

Resonator Magnet

Diameter (cm): 2.5 80

Length (cm): 8 125

Frequency Encoding

Gradient: 1.5 G/cm

Phase Encoding

Gradient 64 steps


12/38

Int

0.6

1.2

1.8

2.4

3.0

MRI Contrast Agent MRI + Contrast Agent

Kidney

Bladder

Tumor

Image Intensity enhanced by:

1) Contrast Agent concentration

2) Hypoxic/Ischemic regions


13/38

% Oxygen inbreathing air

Time

(min.)

0:45 6:20 12:00 17:30 23:20

21% 9.5% 21% 9.5% 21%

0

100

mm Hg

Oxygen Mapping with OMRI

Rat, respiratory model1.5 mmol/kg

Oxygen Maps from OMRI Correspond with Changes in Tissue Oxygenation

Dynamic changes in pO2 can be monitored


14/38

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0

(O

xyge

n,mm

Hg

)

(Oxyge

n

,mm

Hg)

Air

Carbogen


15/38

Overhauser MRI/Summary

Currently implemented in mice, rats.

For human applications:

Contrast agents/Safety

SAR/Localized coils


16/38

Challenges in Imaging of13 C labeled molecules with MRI.

1) Lower concentration compared to protons

2) Lower magnetic moment than protons (25%)

3) Lower Polarization than protons (25%)

Hyperpolarized 13 C MRI

Implications for Molecular Imaging


17/38

1H 13 C 13 C Hyperpolarized

C, M 80 0.1 0.1

(MHz/T) 42.5 10.7 10.7

Polariz., P 1.10-5 2.10-6 0.5

c P 0.034 2.14.10-6 0.535

Sensitivity Considerations in MRI of1H and 13 C at 3 Tesla

Magnetic Field

Molecular Imaging With Endogenous Substances.Golman et al: PNAS Vol 100, 10158-10163, 2003.

Golman et al: PNAS Vol 100, 10435-10439, 2003.


18/38

Strategy for Hyperpolarization of13 C labeled Molecules by EPRfor in vivo imaging

Mix the 13 C labeled compound with trityl radical

Freeze to 1. 4 K and put it in 2.7 T magnet Irradiate with 95 GHz radiation (EPR) Thaw it to room temperature, inject and image in< 2min!

Golman et al PNAS 2003

Ardenkjaer et al PNAS 2003


19/38


20/38

A)

B)

NMR Spectrum of13 C Urea (natural Abundance)

9.4 Tesla (400 MHz)

Single Shot NMR Spectrum

Acquisition time: 1s

Polarization of13 C Urea 20%

13 C Urea at normal polarization

Acquisition time: 65 HoursPolarization: 7.5 ppm


21/38

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Time (s)

Signal Loss of hyperpolarized 13 C Urea in a mouse after iv bolus

There is ~ minute for image data

acquisition before polarization is lost.


22/38

Metabolic fates of

pyruvate

Alan

ine

Oxaloacetate

Lactate

Acetyl-CoA

PyruvateTransamination

Carboxylation Oxidative

decarboxylation

Reduction

Pyruvate is converted into lactate, alanine,

oxaloacetate or Acetyl-CoA depending on the

needs of the tissues.

With suitable hyperpolarized molecules,

it is possible to distinguish breakdown

products based on their chemical shiftsby 13 C MRI.


23/38


24/38

3D surface rendering

In vivo metabolic mapping

using 13 C-pyruvate


25/38

Imaging pO2 using EPR Imaging and

paramagnetic moleculesDirect detection of contrast agent by EPRI

Image collection using static magnetic field gradients

Images of spin probe distribution

pO2 maps obtained by T2* weighted imaging


26/38

frequency

FT

pulse width10 - 100 ns

dead time0.3 - 1.0 s

1.0 - 4.0 s

FID

TIME DOMAIN EPR EXPERIMENTS

The EPR Signals last ~ microseconds after RF pulse


27/38

Gradient ramping and pseudo-echo

r

FT

Frequency ( 1D spatial profile)

RFp u

ls e

Dea

d

tim

e time

Gx

Gy

Gz

EPR/Single Point Imaging

After the dead time, the phase of one point after a fixed time delay is monitored at

different gradient magnitudes per direction.

The resultant envelope is equivalent of a Gradient Recalled Echo.

FT of this envelop gives the spatial projection

Resolution independent of line width.

Fourier reconstruction possible with static gradients

We implemented Fourier Imaging Capabilities in EPRI.


28/38

Time (ns) Frequency (MHz)

FT

EPR Experiment in Time-Domain.

Line width, = 1/( T2)Dead-time

We have Developed EPR Instrumentation withnanosecond time resolution

EPR can be used for pO imaging by post processing for T * effects


29/38

EPR can be used for pO2 imaging by post-processing for T2* effects.

RFp u

ls e

time

cm

Int.

Using several times points in the echo for image reconstruction

it is possible to estimate oxygen dependent line width of the

contrast agent

Gx

O P id E ll t T2 C t t t


30/38

0 % 1 %

2.5 %5 %

Oxygen Provides Excellent T2 Contrast to

Paramagnetic Contrast AgentsPhantom Schematic

mm

mm

Intensity Image

-20 -15 -10 -5 0 5 10 15 20

-20

-15

-10

-5

0

5

10

15

20

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

mm

mm

LineWidthMapping

-20 -15 -10 -5 0 5 10 15 20

-20

-15

-10

-5

0

5

10

15

20

150

160

170

180

190

200

210

220

230

240

250

pO2 dependent T2 Map

150

160

170

180

190

200

210

220230

240

250

0 1 2 3 4 5Oxygen concentration %

Lw

inmG


31/38

Intensity ImageIntensity Image

Sagital View ( 1mm Slices)Sagital View ( 1mm Slices)


32/38

Line Width ImageLine Width Image

Sagital View ( 1mm Slices)Sagital View ( 1mm Slices)


33/38

N

O

R

N

O H

R

.

Reduction

Oxidation

n = 1, piperidine nitroxides eg. Tempol, Tempo, Temponen = 0, pyrrolidine nitroxides. Eg. Carbamoyl proxyl, carboxy proxyl

(n) (n)

Nitroxide radical Hydroxylamine

Paramagnetic Diamagnetic

Provides enhancement in T1 based MRI Does not provide T1 contrast in MRI

Nitroxides can provide redox status dependent contrast in MRI

Time Course of Tempol-Induced


34/38

%Difference in Intensity Green: +; Red: -

Evo = 1/60

Tempol-Induced T1 contrast in

Tumor Bearing Mouse

Image Intensity in Tumor Bearing

Mouse

Tempol-Induced T1 contrast in Tumor Bearing Mouse changes with time after administration.

Time-intensity profiles in muscle and tumor are different

Evo = 1/60

GEFI: TR75, TE3, FA45

Evo = 1/60 Evo = 8/60 Evo = 15/60

Evo = 22/60 Evo = 29/60 Evo = 60/60

Tumor Normal

B C D

E F G

A


35/38

y = -0.8592x + 5.2778

R2 = 0.9954

0

1

2

3

4

0 1 2 3 4 5 6 7 8

Time (min)

ln(%c

hange)

y = -0.2735x + 3.126

R2 = 0.7954

0

1

2

3

4

0 1 2 3 4 5 6 7 8

Time (min)

ln(%c

hange)

Tumor

Normal

Change in Tempol-induced MR Intensity Enhancement as a Function of Time

Intensity change with time is faster in tumor than normal tissue.

Nitroxides are reduced faster in tumors than in normal tissue

Time Course of 3-CP-Induced


36/38

MSME: TR4000, TE450

Evo = 1/60


Evo = 1/60 Evo = 8/60 Evo = 15/60

Evo = 22/60 Evo = 29/60 Evo = 60/60

%Difference

Image Intensity in Tumor Bearing

Mouse3CP-Induced T1 contrast in

Tumor Bearing Mouse

3CP-Induced T1 contrast in Tumor Bearing Mouse changes with time after administration.

Time-intensity profiles in muscle and tumor are different

Evo = 1/60

Tumor Normal



37/38

y = -0.1074x + 4.074

R2 = 0.9978

0

1

2

3

4

0 5 10 15 20

Time (min)

ln(%c

hange)

y = -0.0698x + 3.9395

R2 = 0.9933

0

1

2

3

4

0 5 10 15 20

Time (min)

ln(%c

hange)

Tumor

Normal


38/38

John Cook, Ph. D

Deva Devasahayam, MS (EE)

Fuminori Hyodo, Ph. DJanusz Koscielniak, Ph. D

Atsuko Matsumoto M.S

Ken-Ichiro Matsumoto, Ph. D

Sankaran Subramanian, Ph. D

James B. Mitchell, Ph. D

David Wink, Ph. D

Angelo Russo, Ph. D, M. DAmram Samuni, Ph. D

Klaes Golman, Amersham, Sweden

Jan-Henrik Ardenkjaer, Amersham,

Sweden

ACKNOWLEDGEMENTS

nmr spectrocopy

Documents