THE USE OF ARTIFICIALLY RADIOACTIVE SUBSTANCES IN BIOLOGY AND MEDICINE1

G. M. Frank, Moscow

The interest shown by biologists and physicians in the use of artificially radioactive substances is by no means accidental. The use of radioactive isotopes opens up fundamentally new ways of studying the processes of movement, distribution, and metabolism of substances in the organism. On the other hand, fundamentally new possibilities are also emerging in the field of the action of these substances on the organism.

Translating both of these into the language of medicine, we may say that, by using artificially radioactive substances, the physician obtains new methods of diagnosis and, in prospect, new methods of therapy as well.

How timely these questions are is evidenced by the literally avalanche-like increase, from year to year, in the number of works in which the investigator makes use of the method of radioactive indicators.

The first works were published as early as 1935 by Hevesy; in 1936 and 1937 these works were still counted only in units, while in 1938 and 1939 more than a hundred of them were published.

Fairness requires that it be pointed out that this was greatly promoted by very close cooperation between biologists and physicists, and by the assistance of physicists to biological and medical institutions in mastering a technique unusual for biologists and physicians. As a rule, the majority of published works appear on a joint basis from physical laboratories and institutes in a curious combination with biological and medical institutions of the most varied specialties and names.

On the other hand, hardly anyone’s name has appeared so often in the pages of the biological press as that of the physicist Prof. Ernest Lawrence. In the pages of American physiological journals, in dozens of papers his name appears as a coauthor, in the subtitle of articles as director, or in the acknowledgments at the end of the article.

The use of radioactive elements in biology and medicine is, of course, not a biological problem, but a method applied to the solution of various problems of physiology and patholo-

From this point of view, it also seems advisable to me, without enumerating everything that has been done in this field, to draw attention to a number of examples that could characterize the possibilities of the method itself.

A very curious picture emerges if one distributes the existing works by elements, i.e., according to the radioactive isotope used in the investigation. It then turns out that the absolute champion is radiophosphorus—it is the subject of more than half of all the existing works. This is not accidental: here we have a very fortunate combination of a fourteen-day half-life convenient for the investigator and the exceptional biological importance of this element.

Phosphorus-containing organic complexes not only form part of the tissues of the organism, but also take part in the chemical transformations that underlie the most important biological processes, such as, for example, the work of muscles, and so on. As is known, apatite-like inorganic calcium phosphate compounds form the basis of the bones of the skeleton.

All this makes it possible, in a separate report by D. E. Grodzensky, to consider specially certain aspects of the question of the significance of radiophosphorus for biochemical investigations.

After phosphorus come sodium and potassium. About ten investigations are devoted to each of these elements. Finally, only a few works have been carried out with radioactive bromine, chlorine, iodine, sulfur, iron, and so on.

Irrespective of what biological problem is being solved and which radioactive isotope is being used, it seems to me that one may note three main features of this method as applied to the study of metabolism.

1. Radioactive isotopes—“labeled” atoms—when introduced into the organism, can be traced along the entire path of their distribution, accumulation, and, finally, elimination from the organism, and, most importantly, can be distinguished from elements of the same name present in the organism. This makes it possible to detect the presence of, at times, very rapidly occurring exchange processes where these processes are masked by the counter-movement of chemical elements of the same name. In the latter cases, from the point of view of ordinary chemical analysis, a very “calm” constancy of composition is observed, which does not make it possible to assess the real rate of “chemical renewal” of organs and tissues. The same also applies to the rate of passage of a substance through the organism.

Let us take a roughly schematized example. Sodium chloride enters the organism daily and is excreted from it daily in approximately equal amounts. Without any additional information, one may suppose that the table salt introduced today is excreted on the same day, or, equally probably, that the salt introduced a year ago is excreted (having gone into the construction of certain chemical complexes in living tissue), or perhaps not a year but ten years ago. This problem is solved unambiguously only by using radioactive

indicators, and an understanding of certain fundamental aspects of the chemical mechanisms underlying vital phenomena depends on the solution of this problem.

Let us point out, for example, that in experiments on animals it was possible to establish (E. Anderson and M. Joseph¹) that after approximately \(2^{1}/_{2}\) days, \(50\%\) of the administered sodium chloride had been excreted. This passage of common salt through the human organism, apparently, may be even slower. Thus, according to Hamilton², over two days one of the subjects he examined excreted only about \(10\%\) of the absorbed salt. Hence one should expect that one half will be eliminated in the course of approximately 12 days. To be sure, these figures undergo substantial changes. The same Hamilton showed that 80–90% of radioiodine is eliminated in humans in 48 hours.

Phosphorus behaves in a substantially different way. Even in experiments on animals, according to Hevesy, only on the third day can it be considered that half of the administered quantity has disappeared from the organism. This is explained above all by the circumstance that a considerable fraction of phosphorus enters the bone tissue of the skeleton, where the processes of exchange occur relatively more slowly.

1. The second point that I should like to note is the use of artificially radioactive elements for studying the distribution in the organism of medicinal, highly active substances and poisons. This category of substances allows the possibility of chemical determination; however, they usually have to be applied in quantities far from indifferent for the experimental subject himself. By introducing into the molecule of the substance under investigation a “labeled” atom, or, even better, in successive experiments two or three different atoms, one can make use of negligible quantities of pharmacologically active substances, so that in the course of the investigation no functional shifts are produced in the organism.

Thus, for example, for the study of certain questions of acid secretion in the stomach, one uses the introduction of bromine into the organism and its determination in the form of hydrobromic acid secreted by the gastric glands.

However, in the quantities in which bromine has to be introduced into the organism for chemical determination, it substantially changes gastric secretion. In experiments of which I shall speak later, we succeeded in determining the same thing by using the radioactive isotope of bromine, but introducing it in amounts a thousand times smaller than had been done up to now—milligrams instead of grams.

1. By measuring the \(\gamma\)-radiation of radioactive elements, it is possible in some cases to detect the “fate” of these elements without disturbing the integrity of the organism of the animal or human being.

The beginning of this method was laid as early as 1927 by the Americans Blumgart and Weiss, who used radium emanation introduced into the bloodstream to determine the rate of the circulation of the blood. When emanation is introduced, the possibilities of the method are limited to observing the purely mechanical distribution of the emanation. Last year this direction received its brilliant development in investigations

Hamilton’s^3 research on the γ-radiation of artificially radioactive substances.

Perhaps the most striking is the possibility shown by Hamilton (though so far only in isolated experiments) of registering the content of radioiodine in the human thyroid gland by applying a Geiger counter to the subject’s neck in the region of the thyroid gland.

Experiments of this kind mark the beginning of an entirely new era in the study of metabolism in the organism, one of exceptionally great practical importance for diagnosis. The investigator gains the possibility of following the movement of a substance in the organism during the very course of the process of movement and, most important, without violating the integrity of the organism. True, such a method will have limited application; nevertheless, its use even for individual particular cases is already sufficient to direct serious attention to this fundamentally new path of investigation opened up by artificially radioactive substances.

In reviewing the results obtained in the field of biological research through the introduction of artificially radioactive substances into the organism, the first thing that strikes the eye is the considerable speed with which not only their distribution in the organism or penetration into individual organs and tissues takes place, but also their entry into complex organic complexes.

Thus, for example, Hevesy^4 showed that phosphorus introduced into an animal’s blood very quickly penetrates into various organs, so that after 3 min. only 19% of the introduced amount remains in the blood. Even more striking are his observations that 20% of the phosphorus introduced into the organism is already, after half an hour, found in the composition of the calcium-phosphate basis of the bones of the skeleton.

Hahn^5 and his collaborators also discovered an unusually rapid entry of radioactive iron into the hemoglobin of the red blood corpuscles. Half an hour after radioactive iron in the form of its sulfate salt had been given to an animal with food, an appreciable fraction of it had time not only to be absorbed into the blood (to enter the blood plasma), but also, in perceptible quantities, could be detected in the red blood corpuscles as part of the complex substance—hemoglobin.

Four hours after feeding, the amount of radioactive iron in the blood plasma and in the red blood corpuscles was the same.

In further observations it was possible to establish that the level in the blood plasma gradually decreases, while in the erythrocytes it increases, so that by the end of the first day 90% of the iron that had entered the blood had already become part of the hemoglobin of the red blood corpuscles.

Very interesting observations on the accumulation of radioactive iodine by the thyroid gland were made by Hertz, Roberts, and Evans^6,7. Ten minutes after the administration of small quantities of radioactive iodine, in the form of sodium iodide, its content in the thyroid ...

gland reached a constant level; moreover, this constant level is normally, at least ten times—and in hyperfunction of the thyroid gland many tens of times—higher in concentration than the initial level in the blood.

Even more interesting, in the very formulation of the experiment, are Hamilton’s studies³ of the rate of absorption in the stomach of various salts. In this work Hamilton used the γ-radiations of sodium, iodine, bromine, chlorine, and potassium.

The investigator himself and his collaborators, in a series of successive experiments, drank radioactive salt. A Geiger counter was held in the fist and recorded the rate of appearance of the salts absorbed in the stomach, carried by the bloodstream throughout the body and, consequently, reaching the hand as well.

Figure 1 presents graphs from Hamilton’s work, where, as can be seen from the inscriptions, the abscissa axis gives time in hours, and the ordinate axis gives the number of pulses recorded by the Geiger counter held in the subject’s hand. The number of pulses corresponds to the concentration of radioactive salt that had entered the blood.

Fig. 1

From the curves we see that between the 3rd and 6th minutes after the subject drank the radioactive salt, a quite appreciable amount of it is detected in his hand, reaching approximately 50% of the final value. After half an hour, half of the maximum amount has already been absorbed, and after about 1½ hours there is no further increase in the concentration of the ingested salts in the blood.

Only potassium behaves somewhat differently, its absorption taking place more slowly. Its first traces appear only approximately 15 minutes after administration; the entire subsequent period proceeds correspondingly as well.

These experiments of Hamilton’s are a further illustration of the new prospects opened up by the application of this method, of

which were already discussed above when considering the results of his other work—with direct registration of the content of radioactive iodine in the human thyroid gland.

The rate of absorption of various substances is a very significant indicator of the functioning of the gastrointestinal tract. Hamilton’s work is undoubtedly only the first step toward the use of radioactive indicators in this field. It is quite clear that, by introducing a radioactive atom into the molecule of any substance, one can study its absorption in exactly the same way as Hamilton did for the simplest salts.

No less important than absorption is the process of secretion, and above all the secretion of hydrochloric acid in the stomach. The mechanism of the entire process of hydrochloric-acid synthesis is not yet entirely clear, and therefore radioactive indicators may also be of great service in this direction.

Fig. 2

As already stated above, when bromine is introduced into the organism it partially replaces chlorine, being secreted in the form of hydrobromic acid by the glands of the stomach.

Figure 2 presents the results of our experiments^8 on recording the secretion, in the gastric juice, of radioactive bromine introduced into the blood.

As can be seen from the curve, between the 5th and 10th minutes after introduction into the blood, an already appreciable amount of radiobromine secreted into the gastric juice can be registered. After 30 min. this amount reaches approximately half of the final level, and after a little over an hour it reaches its maximum.

The arrangement of this experiment, as can be seen, is the reverse of what Hamilton did. The latter studied the rate at which salts introduced into the stomach entered the blood; here we observed the rate of penetration of radioactive bromine from the blood into the stomach.

It is curious that in both cases, for entirely different mechanisms, approximately the same time regularities are observed.

We also carried out another modification of this experiment, when radioactive bromine was introduced not into the blood but with food into the stomach, and then its secretion was observed in another part of the stomach isolated according to Pavlov. In this case bromine followed a more complex path. In the latter case the form of the experimental arrangement combined both that which we discussed, referring to Hamilton’s work, and that represented by the curve in Fig. 2.

In this diagram, the ordinate axis shows the amount of bromine in 1 cm³ of gastric juice as fractions of the introduced bromine. If one takes into account that in our experiments only from 10 to

100 mg of radioactive sodium bromide, and it was quite reliably possible to detect, in the first minutes after administration, one-millionth of the administered amount, the advantage of the method of radioactive indicators over ordinary chemical methods becomes obvious.

From the examples given we see that the factor of time enters in a new form into our information about metabolism in the organism. Processes taking place over the course of several minutes are directly recorded where, by means of chemical methods, it was either entirely impossible to discover this or it was necessary to judge it only on the basis of indirect considerations.

The regularities obtained for the “movement” of a substance in the organism under normal conditions served as the basis for the simultaneous study of disturbances in various kinds of diseases.

Thus, a disturbance of phosphorus metabolism in rickets was studied with the aid of radiophosphorus. It is curious that, simultaneously with the reduced phosphorus content in the bones characteristic of rachitic animals, the content of radiophosphorus in the first day after administration into the organism of a rachitic animal is considerably greater than normal. Thus, alongside the impoverishment of bones in phosphorus, revealed by ordinary chemical methods, the use of radiophosphorus shows a considerably greater rapidity of passage of phosphorus through bone tissue in rickets.

Fig. 3

Figure 3 gives photographic imprints of the femur bones of a chick to which radiophosphorus had been administered.^9 The radiation of the phosphorus accumulated in the bones acted directly on the photographic plate. From this figure it is evident that the content of P* is considerably greater in the epiphyses of the bone (the head) than in the diaphyses (the tubular part).

Recently in our laboratory we have begun to study disturbances of phosphorus metabolism in the bones also in bone tuberculosis, using radiophosphorus. Just as in rickets, the content of P* in the first period after administration is noticeably greater in the diseased bone than in the healthy one.

Apparently, the use of radioactive iron for studying metabolic disturbances in various forms of anemia promises great prospects for diagnostic purposes. Already in the work of Hahn^5 and his collaborators cited by me, it was shown how different is the “fate” of iron introduced into normal and artificially anemized—

…to exsanguinated, decolorized animals. Whereas in a normal animal an utterly negligible percentage of iron introduced with food is absorbed, in artificially anemized animals this utilization proceeds with great avidity.

Four hours after the introduction of radioactive iron, only hundredths of a percent (of that introduced) can be detected in the red blood corpuscles of a normal dog, whereas in anemized dogs whole percentages—40–50 times greater—can be found. The same also applies to the final result of absorption of radioactive iron in the organism as a whole.

Fig. 4

Thus, one day after the introduction of considerable quantities of radioactive iron (up to one hundred milligrams), its absorption in the organism—in the red blood corpuscles, as well as in other organs and tissues—amounts in individual cases in “anemic” dogs to as much as 10–12%; in a normal dog Hahn and Bale did not observe absorption above 0.24%.

The same Hamilton³, whom we have cited repeatedly, gives very interesting differences in the absorption of radioactive salts in different individuals. In Fig. 4 are presented curves corresponding to the entry of radioactive sodium, in the form of sodium chloride, in eight different clinically healthy subjects. We see that these curves are highly individual, which indicates different functional characteristics of the gastric mucosa in a number of the subjects. It is quite beyond doubt that in various diseases of the gastrointestinal tract, disturbance of this absorptive function must be clearly detectable by means of the method used by Hamilton. The same applies also to the reverse process, i.e., gastric secretion. One need not be too bold to assert already now, with full confidence, that in the clinic of internal diseases the use of artificial radioactive substances will, in the very near future, take an honorable place among the various diagnostic methods.

I have already mentioned the works of Hertz and Roberts\(^{6,7}\), as well as of Hamilton\(^{10}\), on the recording of the accumulation of radioactive iodine by the thyroid gland. It has long been known that in various diseases of the thyroid gland this capacity for iodine accumulation increases sharply.

Fig. 5 presents curves for the level of accumulation of iodine by the thyroid gland in the normal state and in various disturbances of its function. For one case of so-called spontaneous goiter, shown in this diagram, it is seen that 30 min. after the introduction of radioactive iodine its content in the thyroid gland is approximately 15 times greater than normal.

The absorption by the thyroid gland of iodine, taken as a percentage of the amount introduced, decreases as the amount of the substance taken increases.

In fact, when the introduced radioactive iodine is increased from 10 to 100 mg, i.e., by a factor of 10, its accumulation in the thyroid gland increases only by a factor of 3.

These relationships are still more distinct with quite negligible amounts of radioactive iodine—fractions of a milligram—introduced into the organism. At the same time, the sharp difference between iodine accumulation in a normal gland and in a gland with increased functional activity can be detected only with certain, sufficiently small, absolute amounts of the introduced substance; when it is increased, these differences are smoothed out. The accumulation of iodine by the thyroid gland is a very important diagnostic sign. If one takes into account that the clearest results are obtained when only negligible amounts of radioactive iodine are introduced, it becomes clear what a powerful tool we give to clinical practice through the use of radioactive indicators. Especially attractive are the prospects of observing the process of iodine accumulation in the thyroid gland without carrying out any additional manipulations other than applying a Geiger counter to the patient’s neck.

Fig. 5

▲ — spontaneous goiter, △ — significant hyperfunction, + — moderate hyperfunction, × — pregnant female, ○ — normal

The results obtained in the field of studying the distribution of radioactive iodine in the organism and the accumulation of iodine by the thyroid gland directly point to new paths in radiotherapy that are opening up with the use of artificially radioactive substances.

The opinion is fairly widespread that artificially radioactive substances may prove to be a cheap substitute for scarce radium. Unfortunately, the difficulties of effective application of radiotherapy are far from limited to the shortage of radium, and a simple

its replacement by kilograms of radioactive Na, K, P, etc., which can be manufactured in the three dozen American cyclotrons, does not solve the problem of cancer therapy.

It is well known that X-ray and radiotherapy of malignant tumors is based on the fact that the cells of these tumors are less stable—more labile—with respect to strongly ionizing agents. This explains why, by irradiating a cancerous tumor, it is sometimes possible to weaken its growth without damaging healthy tissue. Unfortunately, these differences in the stability of cancerous and normal tissue are comparatively small, and therefore any even moderately successful results in tumor therapy can be obtained when the action is localized with sufficient precision, when the tumor is subjected to irradiation and the surrounding organs and tissues are, as far as possible, not affected. This explains why the so-called external tumors—of the lip, tongue, and skin—are treated quite well with radium and X-rays. As for cancer of hollow organs, the stomach, liver, etc., here radiation therapy most often serves as an auxiliary method for the surgeon, and in inoperable cases does not yield even moderately satisfactory results.

Thus, the principal task of radiotherapy is by no means new sources for obtaining radioactive substances in large quantities, but new methods of localizing the action, new methods of bringing the active principle to the organ, to the tissue that is to be irradiated. It is not without reason that we emphasize the results of experiments with the mobilization of radioiodine by the thyroid gland.

According to Hamilton’s data^10, from 80 to 90% of the iodine introduced is eliminated from the organism during the first two days. A noticeable fraction of the iodine (from 0.1% of the amount introduced, normally, to almost 17% in hyperfunction) remains for a long time in the thyroid gland. During the period from the second to the fifth day after introduction, this level of content of iodine in the thyroid gland practically does not change.

The concentration of radioactive iodine over a long period of time in the thyroid gland of normal subjects is at least 50 times greater, and in hyperfunction of the thyroid gland several thousand times greater, than the average concentration in other parts of the body. Thus, the thyroid gland, accumulating radioactive iodine, will irradiate itself. I shall not undertake to evaluate the prospects for radiotherapy of diseases of the thyroid gland, and have dwelt on this question as an example indicating new ways of localizing the action, when one or another organ, one or another tissue, moreover, one or another structural element of a tissue, by actively absorbing and accumulating specially selected substances, will thereby be irradiated many times more intensely than the surrounding organs and tissues. If we take into account that we have at our disposal a large selection of radioactive isotopes, and if we also take into account that, with the use of these isotopes, various complex substances can be synthesized that are capable of being selectively absorbed in the organism in its various parts, we shall come to the conclusion that what has been said is far from utopian. Cancerous tissue is characterized by a whole series of chemical and physico-

chemical properties, and therefore, from my point of view, the task of selecting substances that will be absorbed, to a sufficient degree selectively, precisely by the tissue of malignant tumors is in principle solvable.

Speaking of questions of therapy, one cannot fail to mention the action of neutrons. Neutrons produce a considerable biological effect, and, as John Lawrence^11 asserts, neutrons act more selectively on some tissues than on others, even in comparison with X-rays. According to Lawrence, the difference in resistance between normal and cancerous tissue is supposedly more clearly expressed under the action of neutrons than of X-rays. In any case, this provided grounds for the fact that cancer patients, as Lawrence writes, “are regularly irradiated on the new 60-inch medical cyclotron.”

In the near future, one must suppose, data will appear that will make it possible to assess more objectively the prospects of neutron therapy.

Despite the fact that approximately two hundred papers in the field of the application of radioactive and stable isotopes have already appeared in the scientific press, soberly evaluating the results obtained, we can say plainly that this is only the very beginning. I hope that from my report the prospects for the development of this field are to some extent emerging.

Especially great attention should be paid to the prospects for the therapeutic use of artificially radioactive substances and neutrons. Here there is an unploughed field of work, and in order to realize these prospects, friendly joint efforts by physicists, biologists, and physicians are necessary.

Literature

1. E. Anderson and M. Joseph, Proc. Soc. Exp. Biol. and Med., 40, 344, 1939.
2. J. Hamilton and Stone, ibid., 35, 595, 1937.
3. J. Hamilton, Amer. Journ. Physiology, 124, 667, 1938.
4. G. Hevesy, Enzimologia, 5, 138, 1938.
5. Hahn, Baile, Lawrence, Whipple, J. Exp. Med., 69, 739, 1939.
6. S. Hertz, A. Roberts and R. Evans, Proc. Soc. Exp. Biol. and Med., 38, 510, 1938.
7. S. Hertz, A. Roberts, J. Means and R. Evans, Amer. Journ. Physiology, 128, 565, 1940.
8. G. M. Frank (in press, Academy of Sciences of the USSR).
9. Dols, Jansen, Sizoo and Maas, Nature, No. 3604, 953, 1938.
10. J. Hamilton, Amer. Journ. Physiology, 127, 557, 1939.
11. J. Lawrence, Science, May 25, 1940.

1. Report at the 1940 Conference on the Atomic Nucleus; see p. 241 in this issue. ↩