PORPHYRIN-BASED IN VIVO MITOCHONDRIAL RESPIROMETRY

1. 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

2. Oxygenation from a clinical perspective • Lung function • Oxygen saturation • Hemoglobin levels • Venous return • Intravascular volume • Pump function / valves • Heart rate / rythm Oxygen content Cardiac output Regional blood flow Blood pressure • Vasomotor tone • Regional distribution • Shunting • Vasomotor tone • Vascular resistance

3. Meet Sara! Happy and Healthy

4. Sara = Septic

5. Treatment of sepsis Antibiotics Fluid resuscitation Mechanical ventilation

6. After Resuscitation

7. 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.

8. 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

9. Classical respirometry: isolated mitochondria / cells

10. Mitochondrial dysfunction “in vivo” • ‘In vivo’ data about the mitochondrial oxygen metabolism is NOTis available. • Due to the lack of an adequate in vivo measurement method Zuurstof Voeding Water Energie CO2

11. Towards in vivo respirometry

12. ‘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).

13. Mitochondrial oxygen tension measurements by Protoporphyrin IX (PpIX) Triplet state lifetime measurement E.G. Mik, 2006. Nat Methods

14. Mitochondrial localization of PpIX E.G. Mik, 2006. Nat Methods

15. PpIX shows delayed fluorescence High PO2 Low PO2 Prompt fluorescence PpIX Excitation 405 nm E.G. Mik, 2006. Nat Methods

16. Oxygen-dependent delayed fluorescence Exitatie: 510 nm Emissie: 600-700 nm 1 mM PpIX in 4% BSA Temp 37˚C, pH = 7.4 E.G. Mik, 2006. Nat Methods

17. Oxygen-dependent delayed fluorescence O2 QUENCHING collision no light green light relaxation DEL. FLUORESCENCE red light

18. “The old” setup

19. “New” in vivo setup Fiber Pulsed tunable laser Mono- chromator Tissue Lens PMT Integrator reset

20. Fiber based method

21. In vivo delayed fluorescence Skin paw Rectus abdominis muscle 4 hrs after intravenous ALA injection

22. 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).

23. Rat liver in vivo EG Mik et al., Biophys J 95: 3977-90 (2008).

24. Aim of my PhD project Towards clinical monitoring of mitochondrial function in sepsis

25. 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.

26. Non invasive measurements on the skin. Applied on Harms FA, et al. (2011) J. Biophotonics.

27. 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.

28. Validation of PpIX TSLM for mitoPO2 measurements in the skin Harms FA, et al. (2012) Optics Letters Tissue hypoxia during 100% nitrogen ventilation Normal tissue Nonrespiring tissue

29. 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.

30. In vivo respirometry in the skin

32. In vivo respirometry in the skin Example measurement

33. In vivo respirometry in the skin Tested in 8 anesthetized and mechanical ventilated wistar rats

34. The reliable monitor is developed!!!

35. 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 Sander v.d. Heuvel, MD Beademing en IAH ErasmusMC Sjoerd Niehof, PhD Thermography ErasmusMC Marit van Velzen Pletysmography ErasmusMC

36. Questions