PhD Thesis in Medical Technology of Wibeke Nordhøy

Manganese and the Heart Manganese and the Heart Intracellular MR relaxation and Intracellular MR relaxation and

water exchange across the cardiac water exchange across the cardiac cell membranecell membrane

PhD Thesis PhD Thesis in Medical Technologyin Medical Technology

ofofWibeke NordhøyWibeke Nordhøy

17.12.200417.12.2004 PhD Thesis of Wibeke NordhøyPhD Thesis of Wibeke Nordhøy 22

ContentContent

• General theoryGeneral theory

• Paper I-IIIPaper I-III

• Main conclusionsMain conclusions


Hydrogen/Proton: Hydrogen/Proton: A spinning topA spinning top


Randomly oriented protonsRandomly oriented protons


MM00

BB00zz

Aligned with the external magnetic Aligned with the external magnetic fieldfield

MM00 = the netto magnetization vector= the netto magnetization vector Resonance Frequency: 0 = B0


B

X

Y

Zo

time

S

time

Mz

MMzz(t) = (t) = MM00 ( 1 - 2e ( 1 - 2e-t/-t/TT11 ) ) TT11 and and TT22 relaxationrelaxation

Mo

B1 T2 (T2*)MMxyxy(t) = (t) = MM00 e e-t/-t/TT22**


MR and relaxationMR and relaxation

• Magnetic properties of protons in Magnetic properties of protons in 11H-MRIH-MRI

(H), (H), TT11 and and TT22 are essential factors in MRI are essential factors in MRI

• Paramagnetic contrast agents (CA) as MnParamagnetic contrast agents (CA) as Mn2+2+ act act indirectly by decreasing indirectly by decreasing TT11 and and TT22 of water of water protons in near proximity protons in near proximity

• TT11 –weighted imaging, where –weighted imaging, where TT22 is decreased is decreased


Contrast enhancementContrast enhancementShorter and shorter Shorter and shorter TT11 or stronger and stronger signal in a or stronger and stronger signal in a TT11 weighted image where the CA is present weighted image where the CA is present


H2OecH2Oic

ec-1

ec: Extracellular

ic: Intracellular

p ic T1ic

p ec T1ec

ic-1

TT11 and water transport and water transport

SymbolsSymbols-1-1 = water exchange = water exchange

pp = population = population fraction of waterfraction of water

TT11 = longitudinal = longitudinal relaxation timerelaxation time

Intracellular valuesIntracellular valuesicic

-1-1 ppicic T T1ic1ic

Extracellular valuesExtracellular valuesecec

-1-1 ppecec T T1ec1ec


Water exchange rateWater exchange rate


Fast exchangeFast exchange

MMzz(t) = (t) = MM00 ( 1 - 2e ( 1 - 2e-t/-t/TT11 ) )

Monoexponential Monoexponential signalsignal


MMzz (t) = (t) = ppicic ( 1 - 2e ( 1 - 2e-t/-t/TT1ic1ic ) + ) + ppecec ( 1 - 2e ( 1 - 2e-t/-t/TT1ec1ec )) ))

Biexponential Biexponential signalsignal

Slow exchangeSlow exchange


Intermediate exchangeIntermediate exchange

A more complicated model: A more complicated model: Two-site water exchange (2SX)Two-site water exchange (2SX)

Ref: Springer Ref: Springer et al.et al.


Main goals of Paper I-IIIMain goals of Paper I-III• To establish a model for To establish a model for TT1 1 measurements in measurements in

myocardiummyocardium

• The examine the influence of water exchange across The examine the influence of water exchange across the cardiac cell membranethe cardiac cell membrane

• To calculate the To calculate the TT11 efficacy in each tissue compartment efficacy in each tissue compartment

• To study the interaction beetween MnTo study the interaction beetween Mn2+2+ and Ca and Ca2+2+: : uptake and retention of Mnuptake and retention of Mn2+2+-ions-ions


MnMn2+2+

Mn content from freeze-dried hearts

LVDP, HRLVDP, HR

Experimental setupExperimental setup

http://rds.yahoo.com/S=96062883/K=beating+heart/v=2/SID=e/l=IVI/SIG=11omlcmvb/*-http%3A//www.stmonicasea.org/images/beating.heart.gif

http://rds.yahoo.com/S=96062883/K=beating+heart/v=2/SID=e/l=IVI/SIG=11omlcmvb/*-http%3A//www.stmonicasea.org/images/beating.heart.gif


Mn administrationMn administration

• Langendorff-perfused heartsLangendorff-perfused hearts– control perfusioncontrol perfusion– MnMn2+2+ ‘wash-in’ ‘wash-in’– MnMn2+2+ ‘wash-out’ ‘wash-out’

• Single wash-in and wash-out (Paper I and II)Single wash-in and wash-out (Paper I and II)

• Repeated wash-in and wash-out with Mn-Repeated wash-in and wash-out with Mn-accumulation (Paper III)accumulation (Paper III)


Manganese ions as intracellular Manganese ions as intracellular contrast agents: proton relaxation contrast agents: proton relaxation

and calcium interactions in rat and calcium interactions in rat myocardiummyocardium

NMR in biomedicineNMR in biomedicine16(2): 82-95 (2003)16(2): 82-95 (2003)

Paper IPaper I


TT11 relaxation in the hearts without Mn relaxation in the hearts without Mn (control): which model is most suited?(control): which model is most suited?


TT11 relaxation in hearts with relaxation in hearts with 100 µM MnCl100 µM MnCl22 hearts: hearts:


0 250 500 750 10000

5

10

15

R1-2r=0.953

r=0.730

R1-1

Mn content (nmol/g dry wt.)

R1

(s-1

)Strong correlation (Strong correlation (rr) between ic ) between ic RR1-11-1 ((RR1ic1ic) )

and the Mn contentand the Mn content

RR1-21-2 = 1/= 1/TT1-21-2

RR1-11-1 = 1/ = 1/TT1-11-1


TT11 model of rat myocardium model of rat myocardium• Two-components of Two-components of TT11::

– TT1-11-1 rapid, share ~ 60 % (ic component)rapid, share ~ 60 % (ic component)

– TT1-21-2 slow, share ~ 40 % (ec component) slow, share ~ 40 % (ec component)

• An assumed slow water exchange situationAn assumed slow water exchange situation

Fast exchangeFast exchangeSlow exchangeSlow exchange

Mito-chondrial

Inter-stitial

Intra-vasc-ular

Cytosolic

Intracellular Extracellular

Mito-chondrial

Inter-stitial

Intra-vasc-ular

Cytosolic


Mito-chondrial

Inter-stitial

Intra-vasc-ular

Cytosolic



•Multiexponential analyses of Multiexponential analyses of TT11 revealed two water revealed two water compartments (ic and ec) with different chemical compartments (ic and ec) with different chemical environments in the rat myocardiumenvironments in the rat myocardium

•The intracellularThe intracellular R R11 correlated highly with tissue Mn correlated highly with tissue Mn content, which increased content, which increased RR11 effectively effectively

•These These TT11 components were detectable with a 0.47 T MR components were detectable with a 0.47 T MR spectrometer due to a slow-to-intermediate water exchange spectrometer due to a slow-to-intermediate water exchange across the cardiac cell membraneacross the cardiac cell membrane

Conclusions of Paper IConclusions of Paper I


Intracellular manganese ions Intracellular manganese ions provide strong provide strong TT11 relaxation in rat relaxation in rat

myocardiummyocardium

Magnetic Resonance in Medicine Magnetic Resonance in Medicine 52: 506 - 514 (2004)52: 506 - 514 (2004)

Paper IIPaper II


Mn dipyridoxyl-diphosphate (MnDPDP)

O- MnN N

O

OO O

O

+NH

O P O-

O-

O

+NH

OP

O

OH

O


Relaxation rate constants vs. Mn contentRelaxation rate constants vs. Mn content

25 75 125 175 225 2750

2

4

6

r =0.921

r =0.691

R1-1R1-2

Mn content (nmol/g dry wt.)

R 1 (s

-1)


2SX water exchange analysis2SX water exchange analysis

0 100 200 3000.0

2.5

5.0

7.5R1ic

Mn content (nmol /g dry wt.)

R1-

1(s

-1)

0 100 200 3000.0

0.5

1.0

1.5

2.0R1ec


R1-

2(s

-1)

0 100 200 3000.00

0.25

0.50

0.75

1.00pec


p 02


0 25 50 75 100 125 150 175 2000

5

10

15MnCl2MnDPDP

Mn2+ concentrations ex vivo (µM)

R1-

1 (s

-1)

High intracellular relaxivity withHigh intracellular relaxivity with both MnClboth MnCl22 and MnDPDP and MnDPDP

In vitro r1 ofMnCl2

In vitro r1 ofMnDPDP

r1-1 ~ 60 (s mM)-1


Conclusions of Paper IIConclusions of Paper II• TT11 relaxographyrelaxography and 2SX analyses revealed two and 2SX analyses revealed two

compartments representing ic- and ec- watercompartments representing ic- and ec- water

• The ic relaxivity of MnDPDP was as high as for MnClThe ic relaxivity of MnDPDP was as high as for MnCl22

• Protein binding may explain the remarkably high Protein binding may explain the remarkably high intracellular relaxivity of Mnintracellular relaxivity of Mn2+2+ ions ions – Increased correlation time (Increased correlation time (cc) between proton- and electron ) between proton- and electron

spins of Mnspins of Mn2+2+-ions due to increased rotational correlation time -ions due to increased rotational correlation time ((RR) of bound protons) of bound protons

– Rapid water exchange (Rapid water exchange (MM-1-1) within Mn) within Mn2+2+ sites sites


Manganese-Calcium interactions with Manganese-Calcium interactions with contrast media for cardiac MRI: A contrast media for cardiac MRI: A

study of manganese chloride study of manganese chloride supplemented with calcium gluconate supplemented with calcium gluconate

in isolated guinea pig heartsin isolated guinea pig hearts

In Press March 2005: In Press March 2005: Investigative RadiologyInvestigative Radiology

Paper IIIPaper III


Background and GoalsBackground and Goals• What will be the optimal formulation of MnWhat will be the optimal formulation of Mn2+2+--

releasing contrast media? releasing contrast media? • ‘‘efficacy’ versus ‘safety’?efficacy’ versus ‘safety’?• Authors have suggested different combinations of Authors have suggested different combinations of

CaCa2+2+- and Mn- and Mn2+2+-salts (10:1 or 8:1)-salts (10:1 or 8:1)

Three possible MnThree possible Mn2+2+-releasing agents:-releasing agents:1.1. A slow-release MnA slow-release Mn2+2+ chelate like MnDPDP chelate like MnDPDP2.2. Add a ‘cardioprotective’ CaAdd a ‘cardioprotective’ Ca2+2+ salt to a salt to a ‘ ‘MR effective’ MnMR effective’ Mn2+ 2+ saltsalt3.3. Avoid cardiodepression by controlled infusion of a Avoid cardiodepression by controlled infusion of a

rapidly dissolving Mnrapidly dissolving Mn2+ 2+ salt like MnClsalt like MnCl22


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

020406080100120140160180

Time (min)

LVD

P (%

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

020406080100120140160180

Time (min)

HR (%

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

020406080

100120140160180

Time (min)

LVD

P x

HR

(%)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

020406080

100120140160180

Time (min)

LVD

P (%

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

020406080

100120140160180

Time (min)

HR (%

)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

020406080

100120140160180

Time (min)

LVD

P x

HR

(%)

a) b) c)

d) e) f)

LVDP, HR and LVDPxHR

Manganese

Manganese-Calcium


• Normal cell metabolism in all groups (CrP, ATP)Normal cell metabolism in all groups (CrP, ATP)

• Manganese Manganese (660 µM):(660 µM):– Reduced myocardial contractility (-53 %)Reduced myocardial contractility (-53 %)– Reduced heart rate (-18 %)Reduced heart rate (-18 %)– Large Mn metal content (93 times control)Large Mn metal content (93 times control)

• Manganese-Calcium Manganese-Calcium (660 µM)(660 µM): : – Increased myocardial contractility (+56 %)Increased myocardial contractility (+56 %)– Large Mn metal content (41 times control)Large Mn metal content (41 times control)

• Slow water exchange and biexponential TSlow water exchange and biexponential T11

ResultsResults


Conclusions of Paper IIIConclusions of Paper III• Alternative 1: High addition of CaAlternative 1: High addition of Ca2+2+ to Mn to Mn2+2+

(10:1) increases contractility, but reduces Mn (10:1) increases contractility, but reduces Mn uptake uptake

• MnDPDP is more suited than alternative 1 for MnDPDP is more suited than alternative 1 for clinical MRI studies on the heartclinical MRI studies on the heart

• Depression of the contractile force can also be Depression of the contractile force can also be avoided by using a slow infusion of MnClavoided by using a slow infusion of MnCl22


• A biexponential model is best suited for A biexponential model is best suited for TT11 analyses analyses

• A slow-intermediate water exchange across the A slow-intermediate water exchange across the cardiac cell membrane was confirmed for both rat cardiac cell membrane was confirmed for both rat and guinea pig heartsand guinea pig hearts

• MnMn2+2+ entry dependent on Ca entry dependent on Ca2+2+ channel activity channel activity

• The contractile force was not significantly reduced The contractile force was not significantly reduced for clinically relevant Mnfor clinically relevant Mn2+2+ concentrations concentrations

• Close correlation between tissue Mn content and Close correlation between tissue Mn content and relaxation parameters, especially for relaxation parameters, especially for TT1ic1ic and high and high intracellular efficacyintracellular efficacy

Main conclusionsMain conclusions

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PhD Thesis in Medical Technology of Wibeke Nordhøy