1. Experimental strategies in the study of Metabolism

2. goals Identify components in a reaction
Understand regulation of reactions at the tissue of origin
Identify physiological function of each reaction pathway
Understand the control mechanisms in a pathway

3. Levels of organization by which metabolism can be studied
Whole organism
Isolated or perfused organs
Isolated cells
Cell-free systems
Purified components
Metabolic probes

4. whole animal studies
Measures food and water intake (may be radiolabled precursors
Measures metabolites from urine output/blood samples (biochemical assays)
Metabolic cage

5. Isotope Tracers as Probes
Substrates labeled with an isotopic form of some element can be fed to cells or whole organism and used to elucidate metabolic sequences
Radioactive isotopes: 14C, 3H, 31P
Stable ‘heavy’ isotopes: 18O, 15N

6. Disadvantages Impractical for screening purposes
Requires a large number of animals

7. Isolated Tissues
                      
                      
                      
Transducer
Organ bath
Printer

8. Isolated tissues
Tissues are isolated from a newly sacrificed animal (guinea pig, rabbit, rats or mice)
Proteins expressed by the cells in the tissues is similar to the proteins of the same tissues in the intact animal
Effects observed in tissue or isolated organ assays may be predictive of effects in vivo

9. Cardiovascular pharmacology
Isolated organ studies
Isolated perfused rabbit heart
Isolated auricles
Isolated aortic rings

10. Limitations of the assay
The value of the observed responses can be limited by the inability of an isolated tissue to react with other reciprocating organ-body systems
(isolated tissues/organs excludes the effects of gastrointestinal system which influences the absorption of the test substance in vivo)

11. isolated cells as test system
Suspension of cells or culture of cells derived from animal tissues or from continuously replicating cells lines.

12. Advantages of isolated cells
Test substance can be examined in direct contact with the cell type of interest
The concentration of of test substance can be rigorously controlled
The secondary effects of extracellular influences such as metabolic factors , matrix and cell to cell contact can be excluded or controlled

13. Disadvantages
Isolated cells may undergo mutation when kept for a long term growth
All cell types do not respond similarly to a single test substance even if the cells originate from the cell organ

14. Cell free systems

15. Purified components Isolate enzyme from a specific pathway
Allow reaction to proceed in the presence of substrate
Duplicate a particular reaction in a pathway
Reaction limited to those that do not require cellular structure

16. Metabolic probes
Metabolic inhibitors- help to identify role of a reaction
Mutation – identify the role of genes/ proteins in metabolism

18. Transgenic animal technology
Animal whose genome has been modified
-insertion of a new gene (“knock-in gene”)
- target mutation of functional gene (‘knock-out genes”)c
The objective is to produce animals which possess the transferred gene in their germ cells (sperm or ova). Such animals are able to act as "founder" stock to produce many offspring that carry a desirable gene or genes.

19. Applications
Nude mice production for in vivo tumorigenicity

21. Pronuclear microinjection
Pronuclear microinjection. Genetic material is injected directly into the fertilized egg which is then implanted back into a mouse and allowed to come to term.

22. Pronuclear microinjection
Pronuclear microinjection. Genetic material is injected directly into the fertilized egg which is then implanted back into a mouse and allowed to come to term.