Biochemical methods for measuring metabolic fitness of tissues or whole organisms

• US 20070248540A1
• Filed: 04/26/2007
• Published: 10/25/2007
• Est. Priority Date: 09/16/2002
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Hellerstein, Marc
• Application Number:
  US11/796,438
• Publication Number:
  US 20070248540A1
• Time in Patent Office: Days
• US Class Current: 424/1.610
• CPC Class Codes: A61K 49/0004 Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
• CPC Class Codes: A61K 51/02 characterised by the carrier , i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The present invention relates to biochemical methods for assessing metabolic fitness and/or aerobic demands of a living system. Specifically, the rate of synthesis and turnover of the molecular components of mitochondrial mass are used to determine the aerobic capacity and/or aerobic demand of tissues or living organisms. The direct measurement of metabolic fitness and/or aerobic demand by this means can be used as an index of the efficacy of an exercise training program or other therapeutic intervention; as a medical risk factor for predicting the risk of cardiovascular disease, diabetes, death or other health outcome; or as an aid to pharmaceutical companies for drug discovery in the area of metabolic fitness, deconditioning, and oxidative biology.

Claims

1. A method for assessing metabolic fitness or aerobic demand of a living system, comprising:

a) administering ²H₂O to the living system in a manner sufficient to achieve a ²H₂O enrichment level of 0.5% to 15% of the total water in the living system;

b) allowing sufficient time for the ²H label to be incorporated into a mitochondrial molecule in the living system;

c) measuring the isotopic content, isotopic pattern, rate of change of isotopic content, or rate of change of the isotopic pattern, of the mitochondrial molecule; and

c) calculating the rate of synthesis or degradation of the mitochondrial molecule to assess the metabolic fitness or the aerobic demand of the living system.

2. The method of claim 1, wherein the ²H₂O enrichment level achieved is 1% to 10% of the total water in the living system.

3. The method of claim 1, wherein the ²H₂O is administered orally as two or more doses over time.

4. The method of claim 1, wherein the mitochondrial molecule is a deoxyribonucleic acid (DNA) molecule.

5. The method of claim 1, wherein the mitochondrial molecule is a ribonucleic acid (RNA) molecule selected from the group consisting of ribosomal RNA, transfer RNA, and messenger RNA.

6. The method of claim 1, wherein the mitochondrial molecule is a protein selected from the group consisting of a subunit of cytochrome c oxidase, a subunit of F₀ ATPase, a subunit of F₁ ATPase, a subunit of cytochrome c reductase, and a subunit of NADH-CoQ reductase.

7. The method of claim 1, wherein the mitochondrial molecules is a phospholipid selected from the group consisting of cardiolipin, phosphatidylcholine, phosphatidylethanolamine, and a mixture thereof.

8. The method of claim 1, wherein the living system is selected from the group consisting of skeletal muscle tissue cardiac muscle tissue, and adipose tissue.

9. The method of claim 1, wherein the living system is a mammal selected from the group consisting of a rodent and a human.

10. The method of claim 1, wherein the living system is a cell selected from the group consisting of a platelet and a cultured cell in a high-throughput screening assay system.

11. The method of claim 1, wherein the step of measuring isotopic content, pattern or rate of change of isotopic content, or pattern is performed by mass spectroscopy, NMR spectroscopy, or liquid scintillation counting.

12. A method of identifying a drug agent capable of altering metabolic fitness or aerobic demand of a living system, comprising:

a) assessing the metabolic fitness or aerobic demand of the living system according to claim 1;

b) administering the drug agent to said living system; and

c) re-assessing the metabolic fitness or aerobic demand of the living system according to claim 1 after the administration of the drug; and

d) comparing the metabolic fitness or aerobic demand of the living system before and after administration of the drug, wherein a change in the metabolic fitness or aerobic demand identifies the drug agent as capable of altering the metabolic fitness or aerobic demand of the living system.

13. A method of identifying a drug agent capable of altering metabolic fitness or aerobic demand of a first living system, comprising:

a) assessing the metabolic fitness or aerobic demand, according to the method of claim 1, of a first living system to which the drug agent has been administered;

b) assessing the metabolic fitness or aerobic demand, according to the method of claim 1, of a second living system to which the drug agent has not been administered; and

c) comparing the metabolic fitness or aerobic demand of the first living system and of the second living system, wherein a change in the metabolic fitness or aerobic demand of the first and second living systems identifies the drug agent as capable of altering the metabolic fitness or aerobic demand of the first living system.

14. A method for assessing deconditioning of a living system, comprising:

a) administering an isotopically labeled precursor molecule to the living system;

b) allowing for a period of time sufficient for the label of the isotopically labeled precursor molecule to be incorporated into a mitochondrial molecule in the living system;

c) measuring the isotopic content, isotopic pattern, rate of change of isotopic content, or rate of change of isotopic pattern of the isotopically labeled precursor molecule in the living system; and

d) calculating the rate of synthesis or degradation of the isotopically labeled precursor molecule to assess the initial metabolic fitness or aerobic demand of the living system;

e) subjecting the living system to a deconditioning event, resulting in a deconditioned living system;

f) administering the isotopically labeled precursor molecule to the deconditioned living system;

g) allowing for a period of time sufficient for the label of the isotopically labeled precursor molecule to be incorporated into a mitochondrial molecule in the deconditioned living system;

h) measuring the isotopic content, isotopic pattern, rate of change of isotopic content, or rate of change of isotopic pattern of the isotopically labeled precursor molecule in the deconditioned living system; and

i) calculating the rate of synthesis or degradation of the isotopically labeled precursor molecule to assess the deconditioning of the deconditioned living system.

15. The method of claim 14, wherein the isotopically labeled precursor molecule is labeled with a stable isotope selected from the group consisting of ²H-labeled glucose, ¹³C-labeled glucose, a ²H-labeled amino acid, a ¹⁵N-labeled amino acid, a ¹³C-labeled amino acid, ²H-labeled acetate, ¹³C-labeled acetate, a ²H-labeled ribonucleoside, a ¹³C-labeled ribonucleoside, a ¹⁵N-labeled ribonucleoside, a ²H-labeled deoxyribonucleoside, a ¹³C-labeled deoxyribonucleoside, a ¹⁵N-labeled deoxyribonucleoside, a ²H-labeled fatty acid, and a ¹³C-labeled fatty acid.

16. The method of claim 14, wherein the isotopically labeled precursor molecule is ²H₂O that is administered orally as two or more doses over time.

17. The method of claim 14, wherein the label of the isotopically labeled precursor is a radioactive isotope selected from the group consisting of ³H-labeled glucose, ¹⁴C-labeled glucose, a ³H-labeled amino acids, a ¹⁴C-labeled amino acid, ³H-labeled acetate, ¹⁴C-labeled acetate, a ³H-labeled ribonucleoside, a ¹⁴C-labeled ribonucleoside, a ³H-labeled deoxyribonucleoside, a ¹⁴C-labeled deoxyribonucleoside, a ³H-labeled fatty acid, and a ¹⁴C-labeled fatty acid.

18. The method of claim 14, wherein the mitochondrial molecule is a deoxyribonucleic acid (DNA) molecule.

19. The method of claim 14, wherein the mitochondrial molecule is a ribonucleic acid (RNA) molecule selected from the group consisting of ribosomal RNA, transfer RNA, and messenger RNA.

20. The method of claim 14, wherein the mitochondrial molecule is a protein is selected from the group consisting of a subunit of cytochrome c oxidase, a subunit of F₀ ATPase, a subunit of F₁ ATPase, a subunit of cytochrome c reductase, and a subunit of NADH-CoQ reductase.

21. The method of claim 14, wherein the mitochondrial molecule is a phospholipid selected from the group consisting of cardiolipin, phosphatidylcholine, phosphatidylethanolamine, and a mixture thereof.

22. The method of claim 14, wherein the living system is selected from the group consisting of skeletal muscle tissue, cardiac muscle tissue, and adipose tissue.

23. The method of claim 14, wherein the living system is a mammal selected the groups consisting of a rodent and a human.

24. The method of claim 14, wherein the living system is a cell selected from the group consisting of a platelet and a cultured cell in a high-throughput screening assay system.

25. The method of claim 14, wherein the step of measuring isotopic content, pattern or rate of change of isotopic content, or pattern is performed by mass spectroscopy, NMR spectroscopy, or liquid scintillation counting.

26. An isolated, ²H labeled mitochondrial molecule with its ²H label derived from ²H labeled water (²H₂O), wherein said isolated, ²H labeled mitochondrial molecule is produced by:

a) administering the ²H₂O to a living system wherein said ²H₂O is labeled with a detectable amount of ²H, wherein the ²H is incorporated into the mitochondrial molecule of the living system; and

b) isolating said ²H labeled mitochondrial molecule, wherein the isolated, ²H labeled mitochondrial molecule is a molecule selected from the group consisting of DNA, RNA, a subunit of cytochrome c oxidase, a subunit of F₀ ATPase, a subunit of F₁ ATPase, a subunit of cytochrome c reductase, a subunit of NADH-CoQ reductase, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine.

27. A kit for assessing the metabolic fitness or aerobic demand of a living system, the kit comprising:

a) ²H₂O, and

b) instructions for use of the kit,

wherein the kit is used to assess the metabolic fitness or aerobic demand of a living system.