This article throws light upon the various applications of radioisotopes in the biological science.
(A) Investigating Aspects of Metabolism:
1. 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.
2. Metabolic Turnover Times:
Radioisotopes provide a convenient method of ascertaining turnover times for particular compounds. As an example, the turnover of proteins in rats will be considered. A group of rats is injected with a radioactive amino acid and left for 24 h, during which time most of the amino acid is assimilated into proteins.
The rats are then killed at suitable time intervals and radioactivity in organs or tissues of interest is determined. In this way it is possible to ascertain the rate of metabolic turnover of protein. Using this sort of method, it has been shown that liver protein is turned over in 7-14 days, while skin and muscle proteins are turned over every 8-12 weeks, and collagen is turned over at a rate of less than 10% per annum.
3. Studies of Absorption, Accumulation and Translocation:
Radioisotopes have been very widely used in this study of the mechanisms and rates of absorption, accumulation and translocation of inorganic and organic compounds by both plants and animal. Such experiments are generally simple to perform and can also yield evidence on the route of translocation and sites of accumulation of molecules of biological interest.
4. Pharmacological Studies:
Another field where radioisotopes are widely used is in the development of new drugs. This is a particularly complicated process, because, besides showing whether a drug has a desirable effect, much more must be ascertained before it can be used in the treatment of clinic al conditions. For instance, the site of drug accumulation, the rate of accumulation, the rate of metabolism and the metabolic products must all be determined.
In each of these areas of study, radiotracers are extreme v useful, if not indispensable. For instance, autoradiography on whole sections of experimental anima yields information on the site and rate of accumulation, while typical techniques used in metabolic studies can be used to follow the rate and products of metabolism.
(B) Analytical Applications:
1. Enzyme and Ligand Binding Studies:
Virtually any enzyme reaction can be assayed using radiotracer methods provided that a radioactive form of the substrate is available. Radiotracer-based enzyme assays are more expensive than other methods, but frequently have the advantage of a higher degree of sensitivity. Radioisotopes have also been used in the study of the mechanism of enzyme action and in the studies of ligand binding to membrane receptors.
2. Isotope Dilution Analysis:
There are many compounds present in living organisms that cannot be accurately assayed by conventional means because they are present in such low amounts and in mixtures of similar compounds. Isotope dilution analysis offers a convenient and accurate way of overcoming this problem and avoids the necessity of quantitative isolation.
For instance, if the amount of iron in a protein preparation is to be determined, this may be difficult using normal methods, but it can also be done if a source of 59Fe is available. This is mixed with the protein and a sample of iron is subsequently isolated, assayed for total iron and the radioactivity is determined.
If the original specific activity was 10000 d.p.m. (10 mg)-1 and the specific activity of the isolated iron was 9000 d.p.m (10 mg)-1, then the difference is due to the iron in the protein (x), i.e.,
9000/1 = 10000/10+x
x = 1.1 mg.
This technique is widely used, for instance, in studies on trace elements.
3. Radioimmunoassay:
One of the most significant advances in biochemical technique in recent years has been the development of the radioimmunoassay.
4. Radio Dating:
A quite different analytical use for radioisotopes is in the dating (i.e., determining the age) of rocks, fossils and sediments. In this technique it is assumed that the proportion of an element that is naturally radioactive has been the same throughout time. From the time of fossilization or deposition the radioactive isotope will decay.
By determining the amount of radioisotope remaining (or by examining the amount of the decay product) and from the knowledge of the half-life, it is possible to date the sample. For instance, if the radioisotope normally comprises 1% of the element and it is found that the sample actually contains 0.25%, then two half-lives can be assumed to have elapsed since deposition. If the half-life is one million years then the sample can be dated as being two million years old.
(C) Other Applications:
1. Molecular Biology Techniques:
Recent advances in molecular biology that have led to advances in genetic manipulation have dependent heavily upon use of radioisotope in DNA and RNA sequencing, DNA replication, transcription, synthesis of complementary DNA, recombinant DNA technology and many similar studies. Many of these techniques are more fully discussed in other chapters of the book.
2. Clinical Diagnosis:
Radioisotopes are very widely used in medicine, in particular for diagnostic tests. Lung function tests routinely made using xenon-133 (133Xe) are particularly useful in diagnosis of malfunctions of lung ventilation. Kidney function tests using [133] iodohippuric acid are used in diagnoses of kidney infection, kidney blockages or imbalance of function between the two kidneys. Various aspects of hematology are also studied by using radioisotopes. These include such aspects as blood cell lifetimes, blood volumes and blood circulation times, all of which may vary in particular clinical conditions.
3. Ecological Studies:
The bulk of radiotracer work is carried out in biochemical, clinical or pharmacological laboratories; nevertheless, radiotracers are also of use to ecologists. In particular, migratory patterns and behaviour patterns of many animals can be monitored using radiotracers. Another ecological application is in the examination of food chains where the primary producers can be made radioactive and the path of radioactivity followed throughout the resulting food chain.
4. Sterilization of Food and Equipment:
Very strong у-emitters are now widely used in the food industry for sterilization of pre-packed foods such as milk and meats. Normally either 60Co or 137Ce is used, but care has to be taken in some cases to ensure that the food product itself is not affected in any way.
Thus doses often have to be reduced to an extent where sterilization is not complete but nevertheless food spoilage can be greatly reduced. 60Co and 137Ce are also used in sterilization of plastic disposable equipment such as Petridishes and syringes, and in sterilization of drugs that are administered by injection.
5. Mutagens:
Radioisotopes may cause mutations, particularly in micro-organisms. In various microbiological studies mutants are desirable, especially in industrial microbiology. For instance, developments of new strains of a micro-organism that produce higher yields of a desired microbial product frequently involve mutagenesis by radioisotopes.