PORPHYRIN-BASED IN VIVO MITOCHONDRIAL RESPIROMETRY

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PORPHYRIN-BASED IN VIVO MITOCHONDRIAL
RESPIROMETRY

• Floor Harms, Sander Bodmer,
• Robert Jan Stolker, Bert Mik

Department of Anesthesiology, Laboratory of
Experimental Anesthesiology, ErasmusMC
University Medical Center Rotterdam, the
Netherlands
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Oxygenation from a clinical perspective
Oxygen content
Cardiac output
Regional blood flow
Blood pressure
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Meet Sara! Happy and Healthy
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Sara Septic
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Treatment of sepsis
Antibiotics
Fluid resuscitation
Mechanical ventilation
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After Resuscitation
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Mitochondrial Dysfunction

• Mitochondrial dysfunction in sepsis is a well
  known phenomenon called
• Cytopathic Hypoxia
• ? function of the mitochondrial respiratory chain
  
• Resulting in
• ? production of adenosine triphosphate (ATP)
• ? cell metabolism.

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Mitochondrial Dysfunction

• The current knowledge of mitochondrial
  dysfunction is based on
• Swelling and disruption of normal mitochondrial
  architecture
• ? Tissue oxygen tension
• ? Respiratory rates of isolated mitochondria

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Classical respirometry isolated mitochondria /
cells
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Mitochondrial dysfunction in vivo

• In vivo data about the mitochondrial oxygen
  metabolism is NOT is available.
• Due to the lack of an adequate in vivo
  measurement method

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Towards in vivo respirometry
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In vivo monitoring of mitochondrial function

• In vivo measurement of the mitochondrial oxygen
  tension (mitoPO2)
• by the
• Protoporphyrin IX Triplet state lifetime
  measurement (PpIX-TSLM).

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Mitochondrial oxygen tension measurements
by Protoporphyrin IX (PpIX) Triplet state
lifetime measurement
E.G. Mik, 2006. Nat Methods
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Mitochondrial localization of PpIX
E.G. Mik, 2006. Nat Methods
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PpIX shows delayed fluorescence
High PO2
Low PO2
Prompt fluorescence PpIX Excitation 405 nm

• E.G. Mik, 2006. Nat Methods

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Oxygen-dependent delayed fluorescence
Exitatie 510 nm Emissie 600-700 nm
1 mM PpIX in 4 BSA Temp 37C, pH 7.4
E.G. Mik, 2006. Nat Methods
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Oxygen-dependent delayed fluorescence
O2
QUENCHING
collision
no light
green light
relaxation
DEL. FLUORESCENCE
red light
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The old setup
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New in vivo setup
Fiber
Pulsed tunable laser
Mono- chromator
Tissue
Lens
PMT
Integrator
reset
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Fiber based method
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In vivo delayed fluorescence
Rectus abdominis muscle
Skin paw
4 hrs after intravenous ALA injection
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Isolated organs
Rat liver
Rat heart
EG Mik et al., Biophys J 95 3977-90 (2008). EG
Mik et al., J Mol Cell Cardiol 46 943-51 (2009).
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Rat liver in vivo
EG Mik et al., Biophys J 95 3977-90 (2008).
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Aim of my PhD project

• Towards clinical monitoring of mitochondrial
  function in sepsis

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Towards clinical monitoring of mitochondrial
function

• Non invasive measurements on the skin.
• Validation of PpIX TSLM for mitoPO2 measurements
  in the skin.
• In vivo respirometry in the skin.
• In vivo respirometry in sepsis.
• Mitochondrial respirometry on the skin of a human
  volunteer.

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Non invasive measurements on the skin.
Applied on
Harms FA, et al. (2011) J. Biophotonics.
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Towards clinical monitoring of mitochondrial
function

• Non invasive measurements on the skin.
• Validation of PpIX TSLM for mitoPO2 measurements
  in the skin
• In vivo respirometry in the skin.
• In vivo respirometry in sepsis.
• Mitochondrial respirometry on the skin of a human
  volunteer.

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Validation of PpIX TSLM for mitoPO2 measurements
in the skin
Tissue hypoxia during 100 nitrogen ventilation
Normal tissue
Nonrespiring tissue

• Harms FA, et al. (2012) Optics Letters

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Towards clinical monitoring of mitochondrial
function

• Non invasive measurements on the skin.
• Validation of PpIX TSLM for mitoPO2 measurements
  in the skin
• In vivo respirometry in the skin.
• In vivo respirometry in sepsis.
• Mitochondrial respirometry on the skin of a human
  volunteer.

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In vivo respirometry in the skin
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In vivo respirometry in the skin

• Example measurement

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In vivo respirometry in the skin
Tested in 8 anesthetized and mechanical
ventilated wistar rats
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The reliable monitor is developed!!!
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Co-workers
Bert Mik, MD PhD Head of laboratory ErasmusMC
Harold Raat, PhD Blood substitutes Baxter,
ErasmusMC
Jaqueline Voorbeijtel Biotechnician ErasmusMC
Tanja Johannes, MD PhD Experimental
sepsis ErasmusMC
Sander Bodmer, MD PNVs technology NVA
Patricia Specht Biotechnician ErasmusMC
Sjoerd Niehof, PhD Thermography ErasmusMC
Sander v.d. Heuvel, MD Beademing en IAH ErasmusMC
Marit van Velzen Pletysmography ErasmusMC
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Questions
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