Isotope Tracers

substances whose isotopic composition differs from the natural composition, owing to which they are used as tags in the study of the most varied processes. Stable or radioactive isotopes of chemical elements, which can be readily detected and determined quantitatively, serve as isotope tags. The high sensitivity and specificity of isotope tracers make it possible to trace them in the complex processes of transfer, distribution, and transformation of substances even in extremely complex systems, including living organisms.

The isotope tracer technique (also called the method of tagged atoms) was first proposed by G.C. de Hevesy and F.A. Paneth in 1913. The wide use of isotope tracers became possible with the development of nuclear technology, which enabled isotopes to be obtained on a mass scale.

The technique is based on the fact that the chemical properties of the various isotopes of an element are almost identical (owing to which the behavior of the tagged atoms in the processes studied essentially does not differ from that of other atoms of the same element) and on the ease of detecting isotopes, particularly radioisotopes. When using the technique, it is necessary to take into account the possible reactions of isotope exchange, which lead to the redistribution of the tagged atoms (and consequently to the loss of the tag by a compound), and sometimes also the radiation effects related to the influence of radiation on the course of the process. The isotope used as a tag is introduced into the composition of the compounds being studied. Stable, as well as radioactive, isotopes can be used.

The advantages of stable isotopes are their stability and the absence of nuclear radiation. However, only a small number of elements have suitable stable isotopes. The unavailability of the latter and the comparatively complex technique of detection are the disadvantages of using stable isotopes. The advantages of using radioisotopes are the possibility of obtaining them for practically all elements of the periodic system; the high degree of sensitivity, specificity, and accuracy of their detection; and the simplicity and availability of the measuring devices. Hence, most studies using the isotope tracer technique have been done with radioisotopes.

Such elements as hydrogen, carbon, sulfur, chlorine, and lead have convenient stable isotopes (²H, ¹³C, ³⁴S, ³⁵C1, ³⁷C1, ²⁰⁴Pb) as well as radioisotopes (³H, ^UC, ¹⁴C, ³⁵S, ³⁶C1, ²¹²Pb). The stable isotopes ¹⁵N and ¹⁸0 are most often used as isotopes of nitrogen and oxygen. Stable isotope tracers are obtained by enriching natural isotopic mixtures by means of the multiple repetition of the operation of separation (distillation, diffusion, thermal diffusion, isotope exchange, electrolysis; seeISOTOPE SEPARATION); they are also obtained in mass spectrometer installations and during nuclear reactions.

For those elements that exist in nature in the form of only one isotope (Be, F, Na, Al, P, I) only artifically produced radioisotopes are used as tagged atoms. Examples of commonly used radioisotopes are ³H, ¹⁴C, ³²P, ³⁵S, ⁴⁵Ca, ⁵¹Cr, ⁵⁹Fe, ⁶⁰Co, ⁸⁹Sr, ⁹⁵Zr, ⁹⁵Nb, ¹¹⁰Ag, and ¹³¹I. The selection of a radioisotope is determined by its nuclear characteristics—its half-life and the nature and intensity of its radiation. Suitable as tracers are radioisotopes whose half-life is not very short, which makes it possible to work in the time necessary for the experiment, and not very long, which makes it possible to work with extremely small quantities of the tracer.

The principal method of analysis of stable isotopes is mass spectrometry (sensitivity of 10 ^-4 percent of the isotope with an accuracy of 0.1–1 percent for experiments with samples of fractions of a mg). Spectral methods and paramagnetic resonance are finding ever greater use. The content of deuterium, ¹⁸O, and several other isotopes is determined by the change in the index of refraction, thermal conductivity, and density of the element itself, as well as of its compounds. Radioisotopes are analyzed by measuring their radiation using Geiger counters or scintillation counters. Thus, using a Geiger counter one can detect a radiation of 10^-11 g of carbon ^l4C, 10^-16 g of phosphorus ³²P and iodine ¹³¹I, and 10~¹⁹ g of carbon ¹¹C. Modern liquid scintillation counters have high efficiency and accuracy in the isotope determination of weak beta emitters (³H, ¹⁴C, ³⁵S). The introduction of this method of isotope analysis into practice increases its productivity and permits working with insignificant activities approaching those of the cosmic background. The method of autoradiography has received wide use in biology. In working with radioisotopes it is necessary to observe standard precautions.

Various methods of synthesizing labeled compounds are known. The reactions of isotope exchange and biological synthesis are used along with ordinary chemical synthesis. In most cases, the isotope tag occupies a definite position in the molecule; for example, the carbon of propionic acid may be tagged in three ways: ¹⁴CH₃CH₂COOH, CH₃¹⁴CH₂COOH, or CH₃CH₂^l4COOH.

There are three principal uses of isotope tracers. The isotope tracer technique is used to study the nature of the distribution of substances and the means of their transfer. An isotope tracer is introduced into a given system, and its presence in various parts of the system is established at the end of definite time intervals. The most graphic pictures of distribution are obtained without destruction of the sample by means of radioautographs.

Another use of isotope tracers is in quantitative analysis. One of the simplest and most common variants of the isotope tracer technique is the isotope dilution technique, in which a measured quantity of the isotope tracer is added to the substance being analyzed, and the initial quantity of the substance is judged from the degree of dilution of the isotope tracer. This method makes it possible to determine infinitesimally small quantities of substances that are difficult to determine and of large masses of material; it also makes it possible to analyze complex mixtures, whose analysis and separation would be impossible by other methods. Activation analysis, a method related to the isotope tracer technique, is distinguished by its extensive possibilities. In this method, the tag is the isotope of another element, which formed from the original one as a result of a nuclear reaction. This method has especially great value in determining trace elements in metals, alloys, minerals, and textiles during rapid inspection of technological processes. Quantitative analysis of natural isotopes that are part of the natural radioactive series of uranium and thorium and the quantitative determination of the isotope ¹⁴C in dead organisms make it possible to determine the age of rocks and archaeological finds.

The third use of isotope tracers is to elucidate the mechanisms of various processes and study the structures of chemical compounds. The introduction of isotope tags at a definite position in the molecule removes the chemical indistinguishability of atoms, making it possible to elucidate unambiguously the mechanism of a given reaction, for which ordinary chemical methods describe only the initial and final states.

All the indicated uses are widely represented in various fields of chemistry, biology, medicine, engineering, and agriculture. Some examples of their use are given below.

Biology. Isotope tracers are used to solve fundamental and applied problems in biology, the study of which by other methods would be difficult or impossible. The substantial advantage of the method of tagged atoms is that the use of isotope tracers does not destroy the integrity of the organism or its basic vital functions. Many important achievements in modern biology, which have determined the intense development of the biological sciences in the second half of the 20th century, are due to the use of isotope tracers. The complex and interconnected processes of the biosynthesis and decomposition of proteins, nucleic acids, carbohydrates, fats, and other biologically active compounds, as well as the chemical mechanisms of the transformations of these compounds in the living cell, were elucidated and studied in detail by means of the stable and radioactive isotopes of hydrogen (²H and ³H), carbon (¹³C and ¹⁴C), nitrogen (¹⁵N), oxygen (¹⁸0), phosphorus (³²P), sulfur (³⁵S), iron (⁵⁹Fe), and iodine (¹³¹). The use of isotope tracers has led to a reexamination of previous concepts of the nature of photosynthesis and the mechanisms of assimilation by plants of such inorganic substances as carbonates, nitrates, and phosphates.

Numerous investigations in the most diverse areas of biology and biochemistry have been conducted with the aid of isotope tracers. One of the areas includes studies of the dynamics and migrations of populations of the biosphere and of certain individuals within a given population, and migrations of microorganisms and certain compounds within an organism. By introducing a tracer into an organism by way of food or injection, it has been possible to study the rate and paths of migration of many insects (mosquitoes, flies, locusts), birds, rodents, and other small animals and to obtain data on their numbers. In the study of the physiology and biochemistry of plants a number of theoretical and applied problems have been solved by means of isotope tracers; for example, the paths of entry of mineral substances, liquids, and gases into plants and the role of various chemical elements, including trace elements, in plant life have been elucidated. Specifically, it has been shown that carbon enters plants not only through the leaves but also through the roots; the pathways and transport rates of various substances from the roots to the stems and leaves and from those organs to the roots have been established. In animal and human physiology and biochemistry the rate of penetration of various substances into the tissues has been studied (including the rate of inclusion of iron in hemoglobin, of phosphorus in nerve and muscle tissues, and of calcium in bone).

An important area of research includes investigations of the mechanisms of chemical reactions in the body. Thus, in many cases it has been possible to establish the connection between initial and newly formed molecules, to trace the “fate” of individual atoms and chemical groups in metabolic processes, and to elucidate the sequence and rate of these conversions. The data obtained have played a decisive role in constructing modern schemes of biosynthesis and metabolism (metabolic charts) and transformation pathways of food, medicinal preparations, and poisons in living organisms. This area of research includes the elucidation of the origin of the oxygen liberated in the process of photosynthesis: it was found that its source is water and not carbon dioxide. On the other hand, the use of ¹⁴CO₂ has made it possible to elucidate the means of the conversion of carbon dioxide in photosynthesis. The use of “labeled” food has led to new ideas of the rates of absorption and distribution of nutritive substances and their “fate” in the body and has aided in tracing the influence of internal and external factors (starvation, asphyxia, fatigue) on metabolism. The isotope tracer technique has permitted study of the processes of the reversible transfer of substances through biological membranes. It has been shown that the concentrations of substances on both sides of a membrane remain constant with the maintenance of gradients of concentration characteristic, for each of the media.

The isotope tracer technique has found application in the study of processes in which the transmission of information in the body plays a decisive role (conduction of nerve impulses, initiation and reception of stimuli). The effectiveness of the technique in studies of this kind is conditioned by the fact that the studies are conducted on integral, intact organisms, whose entire complex system of nerve and humoral connections is unimpaired. Finally, a certain series of works includes investigations of the static characteristics of biological structures, beginning with the molecular level (proteins, nucleic acids) and ending with supermolecular structures (ribosomes, chromosomes, and other organelles). For example, investigations of the relative stability of proteins and nucleic acids in ¹H₂O, ²H₂0, and H₂¹⁸O have made it possible to elucidate the nature of the forces that stabilize the structure of biopolymers, in particular, the role of hydrogen bonds in biological systems.

Of great significance in selecting an isotope is the problem of the sensitivity of the method of isotope analysis. Another important factor is the type of radioactive decay and energy of radiation. The advantage of stable isotopes (²H, ^l8O, ¹⁵N) is the absence of radiation that often produces side effects in the living organism being investigated. At the same time, the relatively low sensitivity of the methods of their determination (mass spectroscopy, densitometry) and the necessity of separating the labeled compound limit the use of stable isotopes in biology. The high sensitivity of recording gamma-emitting isotopes (⁵⁹Fe and ¹³¹’, for example) has made it possible to measure the rate of blood flow in the living organism. It also allows the determination of the quantity of blood and the time of its complete circulation and the investigation of the glands of internal secretion.

Medicine. The pathogeneses of a number of diseases have been revealed by means of isotope tracers; they are also used in the study of metabolism and the diagnosis of many diseases. Isotope tracers are introduced into the body in extremely small quantities, which are incapable of producing any pathological changes. The various chemical elements are unequally distributed in the body. The isotope tracers are distributed in a similar manner. The radiation that occurs during the decay of an isotope is recorded by radiometric instruments, scanning, and autoradiography. Thus, the state of the systemic and pulmonary circulatory systems, coronary circulation, and rate of blood flow are determined, and pictures of the cavities of the heart are obtained with the help of compounds that include ²⁴Na, ^{, 31}I, and “^MTc.

The isotopes 99^MTc and ¹³³Xe are used in studying pulmonary ventilation and diseases of the spinal cord. Macroaggregates of albumin of human serum with ^l3lI are used to diagnose various inflammatory processes in the lungs, as well as their tumors, and various diseases of the thyroid gland. The concentration and secretion functions of the liver are studied by means of the dye rose Bengal with ¹³¹ I and ¹⁹⁸Au; the function of the kidneys is studied through renography with ^13lI-Hippuran and scanning after the introduction of neohydrin tagged with ²⁰³Hg or “Tc. Pictures of the intestine and stomach are obtained by using “Tc, and of the spleen by using erythrocytes with ^{, 9}Tc or ^5, Cr. Diseases of the pancreas are diagnosed by means of ⁷⁵Se. The isotopes ^8SSr and ³⁴P also have diagnostic applications.

Agriculture. Such isotopes as ³H, ¹⁴C, ²²Na, ³²P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁶⁰Co, ⁶⁵Zn, and “Mo are widely used in agriculture to determine the physical properties of soil and its fertility and to study the interaction of soil and fertilizers, the processes of assimilation by plants of nutrients from mineral fertilizers, the entry of mineral food into plants through leaves, and other problems of soil science and agrochemistry.

Isotopes are used to elucidate the action of pesticides, particularly herbicides, on the plant organism, which helps in establishing the amounts to be used and the periods when plantings are to be treated. The most important biological properties of agricultural crops (in evaluating and selecting plant-breeding material)—yield, early ripening, and resistance to cold—are investigated using the isotope tracer technique. In animal husbandry, the physiological processes of animals are studied, and fodders are analyzed for their content of toxic substances (small amounts of which are difficult to determine by chemical methods) and trace elements. By means of isotope tracers automated agricultural processes are being developed, such as the separation of root and tuberous crops from stones and clumps of soil after harvesting with combines on rocky and thick soils.