Radioisotopes in Science and Industry

BIOSYNTHESIS OF COMPOUNDS

Many plants produce nitrogen-containing compounds known as alkaloids. Generally, the alkaloids have no known function in plants, but a number of them are physiologically active and useful as sedatives or tranquilizers, and in various other forms, for treatment of human and animal diseases. Drugs range from mild stimulants, such as caffeine, through more complex drugs, such as quinine, morphine, and rauwolfia.

Considerable work has been done using radioactive tracers to study mechanisms of biosynthesis by which plants manufacture alkaloids. One of the simplest alkaloids is nicotine. Nicotine is synthesized in the tobacco plant root and moves up and accumulates in the leaves. If a tobacco stem is grafted to a tomato root, no nicotine appears in the tobacco leaves. Thus it may be possible to produce entirely nicotinefree tobacco, or on the other hand, force plants other than tobacco plants to produce nicotine.

Since drugs are notoriously active at very low concentrations, it was almost impossible to study their metabolism and mechanism of action prior to the advent of radioactive forms produced by biosynthesis. Most alkaloids are made in higher plants as a byproduct of metabolic processes. In order to label them with radioactive carbon 14, it was necessary to build special "isotope farms" which are small greenhouse-like structures wherein the plant may be grown for long periods of time in a radioactive atmosphere. Plants grown in this atmosphere at Argonne National Laboratory have been used to produce carbon 14 labeled alkaloids such as nicotine, ergotinine, ergotamine, reserpine, and many others. These compounds have been isolated and made available to various researchers in the United States for studies on localization in tissues, and metabolic breakdown in the body.

Techniques applicable to producing alkaloids. also have been used to produce carbon 14labeled vitamins, starches, fats, oils, sugars, essential oils (such as those found in the mint plant), rubber, proteins, and radioactive virus

particles. These techniques now have been refined to the point that virtually any known biological compound can be produced and isolated.

FARM ANIMAL STUDIES

Nutrition of Farm Animals

By the use of radioisotopes, the metabolism of many elements in farm animals can be conveniently followed from their uptake in food in the form of elements and inorganic and organic compounds, through their uptake and incorporation into various regions of the body, and finally through their degradation and excretion. Tracer techniques are of extreme value in nutritional studies. For example, an element which occurs in amounts of only a few parts per 100,000,000 parts of feed can be traced from the feed, through the digestive tract, and finally to its location in the tissues of a 1,000pound animal. All this can be done without interference with the normal physiology of the animal.

Many elements are required in small amounts by farm animals. A lack of cobalt in the diet decreases the formation of vitamin B 12. Tracer techniques also showed that high levels of molybdenum in cattle feed inhibit synthesis of vitamin B 12. Vitamin B 12 labeled with cobalt 60 has been used for many types of investigation with farm animals. For example, vitamin B 12 is transferred from the body of the cow to its milk during lactation, and the vitamin B 12 content of milk may be used as an indication of the status of the cobalt nutrition. Vitamin B 12 also is transferred from the hen to the egg. The vitamin deposited in the egg actually serves as the principal source for the chick up to several weeks after hatching. Even 12 weeks after hatching the original vitamin in the egg represents an appreciable part of the total vitamin in the bird.

Sulfur is rapidly metabolized by animals. For example, the milk proteins of goats were found to contain radioactivity within three hours after the goats were fed sodium sulfate labeled with radioactive sulfur 35. Until re

cently it was thought that chickens could not use sulfur in the form of inorganic sulfate for synthesizing sulfur-containing organic compounds such as the amino acid cystine. Again, using radioactive sulfur, it has been possible to show that both hens and growing chicks can utilize inorganic sulfur. In fact, under certain conditions, the addition of sulfate to certain poultry feed increases the rate of chicken growth. The growth of cattle and sheep on a low-protein diet can be increased by feeding inorganic sulfur.

Isotope techniques have permitted important practical studies on the absorption and utilization of calcium from feed by farm animals. These studies are complicated by the loss of endogenous calcium from the blood through the intestinal wall into the feces. The availability of radioactive calcium 45 has made such studies much simpler. It has been shown, for example, that the level of endogenous calcium in feces changes appreciably with age, becoming progressively greater in older animals. Also, it has been shown that both calves and adult cattle can obtain large amounts of calcium from powdered limestone included in the feed. It was further found that calves could absorb and retain significantly more calcium from milk than from hay or grain. Other tracer studies permit livestock feeders to get more efficiency from feed by more careful control of the calcium-phosphorus ratio of the diet and by eliminating high concentrations of materials which adversely affect absorption of these elements.

As indicated in the selected examples, radioactive tracer techniques have been of great importance in studying the mineral nutrition of farm animals. Tracer methods have been of perhaps even greater importance in studying the uptake and metabolism of organic foodstuffs. In the past, most of the detailed nutritional studies on animals were carried out with small laboratory mammals. Large farm animals have not been widely used because of the expense of purified diets. The use of tracer techniques, however, has permitted nutritional studies in livestock as well as in the smaller

animals. Studies with tagged compounds have been carried out with vitamin A, the lack of which sometimes produces impaired vision in cattle. Other tracer studies have led to techniques for correcting deficiencies in the amino acid methionine in dry plant livestock feeds by the addition of compounds such as choline and betaine.

Biochemistry of Body Processes

In addition to more basic studies, radioisotope techniques have been of great benefit in studying biochemical processes in livestock.

Milk production. A study of the biochemical functions of micro-organisms which inhabit the stomachs of grazing animals may result in methods of controlling the chemical composition of milk. Grazing animals that chew their own cud (ruminants) depend for their existence on billions of bacteria and other microbes contained in the rumen (first compartment of the ruminant stomach). These microbes produce materials necessary to the life of ruminant animals. In this way, the tiny organisms are in effect a "fluid tissue" and are just as vital to the animal as the tissues of solid organs: i.e., the heart, liver, and kidneys.

The ruminant is dependent upon a supply of fatty acids for its existence. These acids are the fermentation end-products of carbohydrates taken into the animal system in feed such as hay. Some complex carbohydrates are manufactured and stored by the microbes in the rumen. The complex carbohydrates made by rumen microbes are called polysaccharides which constitute a reserve source of energy for the microbes. It was found that this storage polysaccharide is chemically identical regardless of which of the various species of microbes is involved in its production. And the same end-products, fatty acids, are formed from the storage polysaccharide as is formed from rapidly used external energy materials. This means the ruminant is assured of a continuous supply of the fatty acids upon which it depends.

deuterium, has been superseded by water with radioactive isotope of hydrogen, tritium, which is more easily measured.

In the formation of ascites (abnormal fluid in the peritoneal cavity) a large and rapid interchange of water between the ascitic fluid with the blood plasma has been demonstrated. Studies with radioiodinated albumin have shown that the protein in ascitic fluid is in equilibrium with plasma proteins.

Another approach to quantitative estimation of body composition is through activation analysis. Radioactivity is induced in certain elements of blood or other biological material by exposing them to thermal neutrons in a reactor. By analysis of the radioactivity induced, the amount of an element present can be measured. This method has been used to demonstrate trace amounts of gold in blood, to measure iodine in the thyroid, to analyze sodium and potassium in muscle, and to measure amounts of cobalt, iron and zinc in other human material. The sensitivity of this method is much greater than microchemical techniques for many elements.

Cardiac Output

Albumin labeled with iodine 131 has been adapted for measuring quantitatively the output of the heart. An important advantage of this method is the fact that albumin remains in the circulation for comparatively long periods of time as contrasted with the use of freely diffusible radioactive elements. The radioiodinated albumin method is possible through use of highly sensitive detection apparatus. Cardiac output is determined by establishing a general dilution curve after iodine 131 is first introduced into the vascular system. Agreement with other methods of direct measurements in man is within 10 percent.

Peripheral Cardiac Disorders

Radioactive sodium has been much used in studies of peripheral vascular flow in man to measure either circulation time or efficiency.

The tagged albumin method also is applicable to determination of peripheral flow. Liver blood flow can be determined by measuring rate of disappearance of colloidal chromic radiophosphate from the blood stream. Various radiocolloids, such as heat-denatured albumin and gold, are similarly used.

Thyroid Disorders

By far, the most widely used radioisotope in medical practice is iodine 131. Its principal diagnostic use is to measure the functional state of the thyroid gland. Numerous tests have been proposed, but all depend on the thyroid gland's uptake of iodine, or the fact that the iodine not concentrated in the gland is excreted by the kidneys. The simplest and most reliable tests are 6-, 8-, or 24-hour thyroid uptakes. Clear diagnosis may be obscured at times by overlapping of the range of values in hypothyroidism or hyperthyroidism and normal function of this gland. The true functional state is usually revealed by doing double tests.

Another radioisotope of iodine, iodine 132, is of interest in studying thyroid function. It has a shorter half-life than iodine 131 (2.33 hours as compared to 8 days) and a more energetic radiation. From the important standpoint of minimizing accumulated radiation dosage to the patient, the shorter half-life far outweighs the greater energy. When iodine 132 is used instead of iodine 131 at equivalent dose levels, thyroid radiation is reduced by a factor of 30, and the total body dose by a factor of at least 15. This is particularly important for pregnant women and in children.

Other Diagnostic Applications

Radioactive vitamin B 12 labeled with cobalt 60 is used in the diagnosis of pernicious anemia and other macrocytic anemias.

A test using radiovitamin B 12 is being developed for measuring liver cell damage in hepatitis.

Employment of tracer quantities of Rose Bengal or Diodrast labeled with radioiodine permits measurement of the functioning of the

Cancer Therapy. Radioisotopes administered by injection are used for treatment of cancer. In above photo, radio

active chlorine 38 is funneled into the glass container (upper right) for treatment of a patient at the Commission's Brookhaven National Laboratory medical center.