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

• Venous return• Intravascular volume• Pump function / valves• Heart rate / rythm

• Vasomotor tone• Vascular resistance

• Lung function• Oxygen saturation• Hemoglobin levels

• Vasomotor tone• Regional distribution• Shunting

Oxygenation from a clinical perspective

Oxygencontent

Cardiacoutput

Regionalbloodflow

Bloodpressure

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

• ‘In vivo’ data about the mitochondrial oxygen metabolism is NOT is available.

• Due to the lack of an adequate in vivo measurement method

Mitochondrial dysfunction “in vivo”

Voeding

Zuurstof

WaterCO

2

Energie

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

PpIX shows delayed fluorescence

E.G. Mik, 2006. Nat Methods

Prompt fluorescence PpIXExcitation 405 nm

High PO2

Low PO2

600 650 700 750 800 850

emission wavelenght (nm)

emis

sion

inte

nsity

30 s delay

100 s delay

600 650 700 750 800 850

emission wavelenght (nm)

emis

sion

inte

nsity 100 s delay

500 s delay

1 ms delay

A

B

Oxygen-dependent delayed fluorescence

qkPO 0

2

11

1 M PpIX in 4% BSATemp 37˚C, pH = 7.4

Exitatie: 510 nmEmissie: 600-700 nm

E.G. Mik, 2006. Nat Methods

Oxygen-dependent delayed fluorescence

O2

collis

ion

relaxation

no light

red light

green light

QUENCHING

DEL. FLUORESCENCE

“The old” setup

“New” in vivo setup

LensMono-chromatorP

MT

Inte

grat

or

reset

Pulsed tunable laser Fiber

Tiss

ue

Fiber based method

In vivo delayed fluorescence

Rectus abdominis muscleSkin paw

4 hrs after intravenous ALA injection

Isolated organs

EG Mik et al., Biophys J 95: 3977-90 (2008).EG Mik et al., J Mol Cell Cardiol 46: 943-51 (2009).

Rat liver Rat heart

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

Harms FA, et al. (2012) Optics Letters

Normal tissue Nonrespiring tissue 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

1 2 3 4 5 6 7 80

20

40

60

80

0

2

4

6

Mitochondrial V0 (dPO2/dt)

Mitochondrial oxygen tension

Rats

Mito

PO2

(mm

Hg)

Mitochondrial V

0 (mm

Hg/s)

The reliable monitor is developed!!!

Co-workers

Bert Mik, MD PhDHead of laboratoryErasmusMC

Sander Bodmer, MDPNVs technologyNVA

Harold Raat, PhDBlood substitutesBaxter, ErasmusMC

Marit van VelzenPletysmographyErasmusMC

Jaqueline VoorbeijtelBiotechnicianErasmusMC

Patricia SpechtBiotechnicianErasmusMC

Tanja Johannes, MD PhDExperimental sepsisErasmusMC

Sjoerd Niehof, PhDThermographyErasmusMC

Sander v.d. Heuvel, MDBeademing en IAHErasmusMC

Questions