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
Levels of organization by which metabolism can be studied
Whole organism Isolated or perfused organs Isolated cells Cell-free systems Purified components Metabolic probes
whole animal studies Measures food and
water intake (may be radiolabled precursors
Measures metabolites from urine output/blood samples (biochemical assays)
Metabolic cage
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
Isotope Tracers as Probes
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
Cardiovascular pharmacology
Isolated organ studies Isolated perfused
rabbit heart Isolated auricles Isolated aortic rings
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)
isolated cells as test system Suspension of cells or culture of cells
derived from animal tissues or from continuously replicating cells lines.
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
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
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
Metabolic probes Metabolic inhibitors- help to identify role of
a reaction Mutation – identify the role of genes/
proteins in metabolism
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”)cThe 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.
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.