The following are several approaches to studying metabolic reactions in cells:
Whole organism - Tools, such as nuclear magnetic resonance (NMR) spectroscopy, allow scientists to monitor processes occurring inside of living tissue. For example, Figure 12.14 shows the effect of anaerobic exercise on 31P NMR spectra of human forearm muscle.
Isolated or perfused organ - Organs can be isolated and bathed in an appropriate solution to study the effects of desired materials. Perfusion involves pumping a buffered isotonic solution containing nutrients, drugs, or hormones through the organ.
Whole cells - Methods are now available for disaggregating an organ or a tissue into its component cells. Liver, kidney, and heart cells can be prepared by treatment of the organ with trypsin or collagenase to break down the extracellular matrix that holds the organ together. The complex mixture of cell types in an organ can be fractionated by size (centrifugation) or by a fluorescence-activated cell sorter. Here, a cell suspension is treated with a fluorescent-tagged antibody to a cell surface antigen, which is present in different amounts among different cell types. Cells pass in single file through a laser beam and are physically separated according to the amount of fluorscence recorded from each cell.
Uniformity of cell populations is often achieved by growth of cells in tissue culture. Disaggregated cells of an organ or a tissue can, with special care, be induced to grow in a medium containing cell nutrients and protein growth factors. The cells grow and divide independently of one another, much like the cells in a bacterial culture. Although animal cells usually cease growth after a certain number of divisions in culture, variant lines arise that are capable of indefinite growth, as long as they are adequately nourished. In such cultures, clonal cell lines can be generated in which all of the cells in a line are derived from a single cell, so that they are genetically and metabolically uniform. This uniformity has been a boon for many biochemical investigations because it reduces the number of variables in an experiment and gives results that may be unique to one specific genetic phenotype, but not others.
Cell-free Systems - Cells can be easily broken open and the contents, such as the organelles, can be crudely fractionated by centrifugation.
Purified components - Multiple factors for a metabolic process can be isolated and then mixed back together to test their ability to perform the reaction. This process is called reconstitution.
See also: Reconstitution