Methods and devices for assessment of mitochondrial function

Patent metadata

Field	Value
Publication number	US-10920260-B2
Application number	US-200913059225-A
Country	US
Kind code	B2
Filing date	Aug 14, 2009
Priority date	Aug 15, 2008
Publication date	Feb 16, 2021
Grant date	Feb 16, 2021

Abstract

Mitochondrial function is measured. Repetitive or continuous measurements are performed of prompt red fluorescence, emerging from the skin due to PpIX build up, and/or delayed fluorescence of PpIX. An estimate of the rate of PpIX generation is used as an indicator of mitochondrial integrity and ATP availability. Mitochondrial oxygen tension is determined from the delayed fluorescence lifetime of PpIX. When blood supply to the measurement volume is interrupted or reduced, the resulting changes to the mitochondrial oxygen tension allow an estimation of information about the kinetics of oxygen consumption in the mitochondria, such as the maximum rate of oxygen consumption as well as the Michaelis-Menten constant, providing information about the oxygen affinity of the mitochondrial respiratory chain.

First claim

The invention claimed is:

1. A non-invasive method of measuring the lifetime of the first excited triplet-state (TAUT1) of endogenous protoporphyrin IX (PpIX) ex vivo or in vivo in mitochondria in cells in a sample volume of skin tissue of a subject, the method comprising:
administering 5-aminolevulinic acid to the skin cells in said sample volume;
repetitively or continuously measuring fluorescence of protoporphyrin IX (PpIX) in mitochondria in the skin cells in said sample volume of skin tissue, using a device comprising an excitation light source directed to illuminate said sample volume, a light detector arranged to detect fluorescence from said sample volume, and a pressure pad for applying local pressure on said sample volume;
restricting or ceasing the supply of oxygen to the cells in said sample volume of skin tissue by applying local, positive pressure on the tissue to thereby occlude microvessels comprised in said said sample volume of skin;
repetitively or continuously measuring the lifetime of the first excited triplet state (TAUT1) of endogenous protoporphyrin IX (PpIX) in mitochondria in cells in said sample volume of skin tissue using said device, whereby at least two measurements are performed during said restricting or ceasing the supply of oxygen to the cells in said sample volume of skin tissue;
discontinuing applying local, positive pressure to said sample volume of skin tissue through said pressure pad to thereby discontinue said restricting or ceasing the supply of oxygen to the cells in said sample volume of skin tissue; and
repetitively or continuously measuring the lifetime of the first excited triplet state (TAUT1) of endogenous protoporphyrin IX (PpIX) in mitochondria in cells in said sample volume of skin tissue.

2. The method according to claim 1 ,
wherein multiple measurements of the lifetime of the first excited triplet state (TAUT1) of endogenous protoporphyrin IX (PpIX) in mitochondria in cells in said sample volume of skin tissue are performed to provide information on the maximal oxygen consumption VO 2,max and/or the Michaelis-Menten constant P 50 of the mitochondrial respiratory chain in said cells in said sample volume of skin tissue.

3. The method according to claim 1 , wherein the tissue comprises in vivo skin or organ tissue and wherein oxygen supply to a measurement volume is restricted or reduced by outside pressure on the tissue higher than the venous capillary closing pressure but lower than arterial capillary closing pressure to impede return flow.

4. The method according to claim 1 , wherein oxygen supply to a sample volume is restricted or reduced by outside pressure applied to a region including or surrounding the sample volume higher than arterial capillary closing pressure.

5. The method according to claim 2 , wherein the same instrument is used for the exertion of local pressure on the microvessels of the tissue and the measurement of the lifetime of the first excited triplet state (TAUT1) of PpIX.

6. The method according to claim 2 , wherein the measurement of the lifetime of the first excited triplet state (TAUT1) of PpIX is performed with an instrument that comprises at least one measuring unit selected from the group consisting of a detector; an optical fiber connected to a detector; and a system composed of at least one of lenses, filters and mirrors that projects an image of a part of the measurement volume onto a detector; and
wherein the same instrument is used for the exertion of local pressure on the microvessels of said tissue.

7. The method according to claim 1 , wherein said pressure pad is a movable fiberoptic member, and wherein the measurement is carried out with said fiberoptic member.

8. The method according to claim 1 , wherein repetitively or continuously measuring comprises repetitively or continuously providing an excitation signal to protoporphyrin IX (PpIX) present in the tissue comprising the occluded microvessels.

9. The method according to claim 2 , wherein the combination of mitochondrial oxygen consumption, VO 2,max and P 50 is used to differentiate between disorders of oxygen supply and oxygen utilization.

10. The method according to claim 9 , wherein a reduction of VO 2,max compared to values at other times or in healthy individuals is associated with partial blockage or dysfunction of the mitochondrial respiratory chain.

11. The method according to claim 9 , wherein an increase in VO 2,max compared to values at other times or in healthy individuals is associated with uncoupling of the mitochondrial respiratory chain.

12. The method according to claim 10 , wherein said assessment provides an indication of the mechanism by which the oxygen consumption is reduced, wherein an increase in P 50 is associated with competitive inhibition by nitric oxide, and wherein a reduced VO 2,max without a significant alteration in P 50 is associated with a non-competitive inhibition by toxins.

13. The method according to claim 8 , wherein the assessment of mitochondrial function is in vivo and further comprises an assessment of mitochondrial outer membrane integrity and intracellular ATP availability, wherein said assessment of mitochondrial outer membrane integrity and intracellular ATP availability comprises steps wherein ALA is administered and kinetics of prompt PpIX fluorescence are measured.

14. The method according to claim 1 , wherein an elevated mitochondrial oxygen consumption well above normal values indicates mitochondrial uncoupling and wherein a low mitochondrial oxygen consumption indicates mitochondrial dysfunction.

15. The method of claim 1 , wherein the method comprises estimating the rate of extension of said lifetime as an indicator of oxygen consumption, said assessment provides information on the maximal oxygen consumption VO 2,max .

16. The method of claim 1 , wherein the step of repetitively or continuously measuring the lifetime of the first excited triplet state (TAUT1) of endogenous protoporphyrin IX (PpIX) in mitochondria in cells of said tissue is performed using a device for measurement of mitochondrial function, the device comprising:
an excitation light source directed to illuminate a sample volume of said tissue;
a light detector arranged to detect fluorescence or other luminescence from said sample volume or absorption by said sample volume; and
a control unit configured to obtain repetitive or continuous measurements of mitochondrial oxygen tension, wherein the control unit is configured to use the measurements after the step of restricting or ceasing the supply of oxygen to said tissue to deduce the Michaelis-Menten constant and/or the maximal oxygen consumption of the mitochondrial respiratory chain in said tissue.

17. The method of claim 16 , whereby the temperature of the tissue is regulated.

18. The method of claim 16 , whereby the temperature of the tissue is regulated to a value in the range of 35-44° C.

19. The method of claim 16 , whereby the at least two measurements are performed by using an excitation light source illuminating said sample volume with blue (˜410 nm), green (˜510 nm) or red (˜632 nm) light and by using a light detector for detecting delayed fluorescence at 630-710 nm.

20. The method of claim 1 , whereby the temperature of the tissue is regulated to a value in the range of 35-44° C.

21. The method of claim 1 , whereby the lifetime of the first excited triplet state (TAUT1) of endogenous protoporphyrin IX is determined by photo excitation with blue (˜410 nm), green

Assignees

• Mik Egbert G
• Univ Erasmus Med Ct Rotterdam

Inventors

• Mik Egbert G

Classifications

• G01N33/5302

  Apparatus specially adapted for immunological test procedures · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N2021/6413

  Distinction short and delayed fluorescence or phosphorescence · CPC title

• G01N2021/6484

  Optical fibres · CPC title

• G01N21/6408

  with measurement of decay time, time resolved fluorescence · CPC title