Study of metabolic pathway by using radio isotope
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Metabolic Pathways:
Radioisotopes are frequently used for tracing metabolic pathways. This usually involves adding a radioactive substrate, taking samples of the experimental material at various times, extracting and chromatographically or otherwise separating the products.
Radioactivity detectors can be attached to gas liquid chromatography or high performance liquid chromatography columns to monitor radioactivity coming off the column during separation.
Alternatively, radioactivity can be located on paper or thin-layer chromatography with either a Geiger-Muller chromatograph scanner or with autoradiography. If it is suspected that a particular compound is metabolized by a pathway, then radioisotope can also be used to confirm this. For instance, it is possible to predict the fate of individual carbon atoms of [14C] acetate through the tricarboxylic cycle, or Krebs cycle. Methods have been developed whereby intermediates can be ascertained. This is the so-called specific label ling pattern. Should the actual pattern coincide with the theoretical pattern, then this is a very good evidence for the mode of operation of the Krebs cycle.
Another example of the use of radioisotopes to confirm the mode of operation, or other-wise, of a metabolic pathway is in studies carried out on glucose catabolism. There are numerous ways whereby glucose can be oxidized, the two most important ones in aerobic organisms being glycolysis followed by Krebs cycle together with the pentose phosphate pathway.
Frequently, organisms or tissues posses the necessary enzymes for both pathways to occur and it is of interest to establish the relative contribution of each to glucose oxidation. Both pathways involve the complete oxidation of glucose to carbon dioxide, but the origin of the carbon dioxide in terms of the six carbon atoms of glucose is different (at least in the initial stages of respiration of exogenously added substrate), thus it is possible to trap the carbon dioxide evolved during the respiration of specifically labelled glucose (e.g., [6-14C] glucose or [ 6-14C] glucose in which only the C-6 atom is radioactive) and obtain an evaluation of the contribution of each pathway to glucose oxidation. The use of radioisotopes in studying the operation of Krebs cycle or in evaluating the pathway of glucose catabolism are just two examples of how such isotopes can be used to confirm metabolic pathways.
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Radioisotopes are frequently used for tracing metabolic pathways. This usually involves adding a radioactive substrate, taking samples of the experimental material at various times, extracting and chromatographically or otherwise separating the products.
Radioactivity detectors can be attached to gas liquid chromatography or high performance liquid chromatography columns to monitor radioactivity coming off the column during separation.
Alternatively, radioactivity can be located on paper or thin-layer chromatography with either a Geiger-Muller chromatograph scanner or with autoradiography. If it is suspected that a particular compound is metabolized by a pathway, then radioisotope can also be used to confirm this. For instance, it is possible to predict the fate of individual carbon atoms of [14C] acetate through the tricarboxylic cycle, or Krebs cycle. Methods have been developed whereby intermediates can be ascertained. This is the so-called specific label ling pattern. Should the actual pattern coincide with the theoretical pattern, then this is a very good evidence for the mode of operation of the Krebs cycle.
Another example of the use of radioisotopes to confirm the mode of operation, or other-wise, of a metabolic pathway is in studies carried out on glucose catabolism. There are numerous ways whereby glucose can be oxidized, the two most important ones in aerobic organisms being glycolysis followed by Krebs cycle together with the pentose phosphate pathway.
Frequently, organisms or tissues posses the necessary enzymes for both pathways to occur and it is of interest to establish the relative contribution of each to glucose oxidation. Both pathways involve the complete oxidation of glucose to carbon dioxide, but the origin of the carbon dioxide in terms of the six carbon atoms of glucose is different (at least in the initial stages of respiration of exogenously added substrate), thus it is possible to trap the carbon dioxide evolved during the respiration of specifically labelled glucose (e.g., [6-14C] glucose or [ 6-14C] glucose in which only the C-6 atom is radioactive) and obtain an evaluation of the contribution of each pathway to glucose oxidation. The use of radioisotopes in studying the operation of Krebs cycle or in evaluating the pathway of glucose catabolism are just two examples of how such isotopes can be used to confirm metabolic pathways.
I hope dis was helpful 2 u...
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