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
Oxygenation from a clinical perspective
•
•
•
•
•
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Lung function
Oxygen saturation
Hemoglobin levels
Vasomotor tone
Regional distribution
Shunting
Oxygen
content
Cardiac
output
Regional
blood
flow
Blood
pressure
•
•
•
•
Venous return
Intravascular volume
Pump function / valves
Heart rate / rythm
•
•
Vasomotor tone
Vascular resistance
Meet Sara! Happy and Healthy
Sara = Septic
Treatment of sepsis
Antibiotics
Fluid resuscitation
Mechanical ventilation
After Resuscitation
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.
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
Classical respirometry: isolated mitochondria / cells
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
Towards in vivo respirometry
‘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).
Mitochondrial oxygen tension measurements
by
Protoporphyrin IX (PpIX) Triplet state lifetime measurement
E.G. Mik, 2006. Nat Methods
Mitochondrial localization of PpIX
E.G. Mik, 2006. Nat Methods
emission intensity
PpIX shows delayed fluorescence
A
High PO2
30 s delay
100 s delay
600
650
700
750
800
850
emission wavelenght (nm)
E.G. Mik, 2006. Nat Methods
Low PO2
emission intensity
Prompt fluorescence PpIX
Excitation 405 nm
B
100 s delay
500 s delay
1 ms delay
600
650
700
750
800
emission wavelenght (nm)
850
Oxygen-dependent delayed fluorescence
Exitatie: 510 nm
Emissie: 600-700 nm
1
PO2 
1 M PpIX in 4% BSA
Temp 37˚C, pH = 7.4
E.G. Mik, 2006. Nat Methods


1
0
kq
Oxygen-dependent delayed fluorescence
O2
QUENCHING
no light
green light
DEL. FLUORESCENCE
red light
“The old” setup
“New” in vivo setup
reset
Monochromator
Lens
Tissue
PMT
Integrator
Pulsed tunable laser
Fiber based method
In vivo delayed fluorescence
Skin paw
4 hrs after intravenous ALA injection
Rectus abdominis muscle
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).
Rat liver in vivo
EG Mik et al., Biophys J 95: 3977-90 (2008).
Aim of my PhD project
Towards clinical monitoring of mitochondrial function in sepsis
Towards clinical monitoring of mitochondrial function
1. Non invasive measurements on the skin.
2. Validation of PpIX TSLM for mitoPO2 measurements in
the skin.
3. In vivo respirometry in the skin.
4. In vivo respirometry in sepsis.
5. Mitochondrial respirometry on the skin of a human
volunteer.
Non invasive measurements on the skin.
Applied on
Harms FA, et al. (2011) J. Biophotonics.
Towards clinical monitoring of mitochondrial function
1. Non invasive measurements on the skin.
2. Validation of PpIX TSLM for mitoPO2 measurements in
the skin
3. In vivo respirometry in the skin.
4. In vivo respirometry in sepsis.
5. Mitochondrial respirometry on the skin of a human
volunteer.
Validation of PpIX TSLM for mitoPO2 measurements in the skin
Normal tissue
Nonrespiring tissue
Harms FA, et al. (2012) Optics Letters
Tissue hypoxia during
100% nitrogen ventilation
Towards clinical monitoring of mitochondrial function
1. Non invasive measurements on the skin.
2. Validation of PpIX TSLM for mitoPO2 measurements in
the skin
3. In vivo respirometry in the skin.
4. In vivo respirometry in sepsis.
5. Mitochondrial respirometry on the skin of a human
volunteer.
In vivo respirometry in the skin
In vivo respirometry in the skin
Example measurement
In vivo respirometry in the skin
Tested in 8 anesthetized and mechanical ventilated wistar rats
MitoPO2 (mmHg)
6
60
4
40
2
20
0
0
1
2
3
4
5
Rats
6
7
8
Mitochondrial V0 (mmHg/s)
80
Mitochondrial oxygen tension
Mitochondrial V0 (dPO2/dt)
The reliable monitor is developed!!!
Co-workers
Bert Mik, MD PhD
Head of laboratory
ErasmusMC
Harold Raat, PhD
Blood substitutes
Baxter, ErasmusMC
Sander Bodmer, MD
PNVs technology
NVA
Tanja Johannes, MD PhD
Experimental sepsis
ErasmusMC
Sander v.d. Heuvel, MD
Beademing en IAH
ErasmusMC
Sjoerd Niehof, PhD
Thermography
ErasmusMC
Jaqueline Voorbeijtel
Biotechnician
ErasmusMC
Patricia Specht
Biotechnician
ErasmusMC
Marit van Velzen
Pletysmography
ErasmusMC
Questions