George de Hevesy (1952) - The application of radioactive indicators in the investigation of physiological processes in animals (German presentation)

However, we never hoped to experience that in our lifetimes. The discovery of induced radioactivity has suddenly made all elements, as it were, more available for use as indicators. We immediately began to utilise these synthetic radioactive substances as indicators. And without much hesitation, we turned to phosphorus. The reason we investigated radioactive phosphorus first was not only the importance, the biochemical importance of phosphorus, but instead because we had hoped to be able to produce phosphorous compounds with our modest means. We took ten litres of carbon disulphide and mixed them with our beryllium-radium alloy, about a half gram of radium, melted into platinum tubes. And after a week, we agitated our carbon disulphide with dilute acid and the radioactive phosphate that formed went almost completely into solution as weightless phosphate P-32 and could be utilised in our experiments. The first problem we attacked was investigating the extent to which the skeleton of a grown animal is renewed and whether an exchange of the mineral components in the bone structure takes place or not. We fed animals phosphate, our radioactive phosphate, and then examined the bones for radioactivity. [The next image please.] Let us assume that the bones were, that the bone apatite has its phosphate completely replaced, as well as water molecules in the cells and water molecules in the extracellular liquid. Then, after a time, one milligram of phosphorus that we isolate from the bone apatite should exhibit the same radioactivity as one milligram of phosphorus from blood plasma. That is far from the case, however. Here you see the result obtained with frogs: even in the muscles, where a relatively large exchange takes place, we only have a replacement of two per cent over the course of four hours and the replacement of phosphate in the bones amounted to zero parts per thousand. The phosphate is probably at the surface of the apatite crystals, which was replaced with phosphate from the lymphatic fluid and plasma. It is well known from earlier investigations by Paneth that if you introduce a lead sulphate crystal containing radioactive lead ions into a solution, an exchange of the lead ions takes place between the solid and liquid phases. And this takes place only in the upper-most molecular layer. These surface phenomena were investigated later in extraordinary detail at the institute of Professor Hahn with important findings. In contrast, if you observe the bones over a longer time, then a second per cent shows up, and a third ... what is known as a biological recrystallisation, which consists of individual parts of the bone apatite being dissolved and others forming. I am speaking of grown animals. In growing animals - I will speak about this later - the relationships are completely different. A quantitative interpretation of these results is difficult because, when I give phosphate to an animal, for example injecting it to begin with, this means that a milligram of plasma phosphate has an activity level of, say, 1000 units. However, the radioactive phosphate enters the organs, becoming caesium phosphate, which was in the organs. And the result is that now, after some time, one milligram of plasma phosphate no longer has a radioactivity of 1000, but say, 100 instead. Therefore, the sensitivity of the radioactive indicator changes, increases, making the calculation more difficult. The relationships are very complicated as well, since the bone structure is extraordinarily heterogeneous, of course. The hard bones exhibit a completely different behaviour than the soft ones. And we have all the possible transitions between diaphysis and epiphysis. To make the calculation easier, we injected rabbits daily throughout a 50-day period with phosphate, radioactive phosphate, to maintain the activity of the blood plasma at a constant level and facilitate the calculation. After 50 days, the radioactivity of one milligram of epiphyseal phosphorus, soft bone material, attained about 28% of the radioactivity of the plasma phosphorous. Therefore, 28% of the soft bones were replaced. The hard diaphysis bone material exhibits completely different behaviour: just 7% of the diaphysis has changed in the course of 50 days. And if we had extended the experiment, as you see - this curve runs almost parallel - not much else would have been attained. And thus we can conclude from these numbers that a large part of the phosphate in the animal corpus of a grown animal, the bone phosphate, was not replaced. The bone structure is actually the sole place in the body of a mature organism - animal or human - where the phosphate atoms are safe. All phosphorous atoms in the soft parts, whether present in phosphates, carbohydrates, fats, or nucleic acids, are continuously replaced. Even here in the bones, in the largest parts of the bones, the phosphor atoms have safe accumulation areas where they can remain undisturbed. The next problem that we investigated was the replacement of phosphorylated carbohydrates. These kinds of compounds - the others are triphosphoric acid, the unstable phosphate - are replaced extraordinarily quickly. Half of the unstable phosphorus atoms of the adenosine triphosphoric acid in the muscle are replaced in 50 seconds. Adenosine triphosphoric acid is the most important phosphate donor in the organism and after this compound has given up phosphorus, it must reorganise itself again. And with reorganisation, other phosphate atoms enter the molecule, tagged ones. Other creatine phosphoric acids also are replaced very quickly and other carbohydrates containing phosphor too. Fats containing phosphorous, like lecithin, cephalin, need several hours to have half replaced in the liver and more in other organs. Deoxyribonucleic acid even needs days in maturing animals. That is a large study; there are many hundreds of treatises, some from highly competent researchers. And this study has contributed significantly to advancing our knowledge about the metabolism of carbohydrates and fats. Unfortunately, I cannot go into these results. [The next image please.] I would like to show you one additional image in connection with this investigation of bones. This is the incisor of a rat administered radioactive phosphate. Such an incisor is sectioned into pieces and it can then be determined where the newly deposited phosphate actually is. It is primarily in the vicinity of the pulpa, but even outside the area of the pulpa, as you see, you can detect newly deposited phosphate. [The next image please.] The replacement of these phosphorylated compounds varies not only from compound to compound, but also from organ to organ. And even within the cell, the various cellular components exhibit different replacement speeds. Lecithin in the cell nucleus is replaced more slowly than in the cytoplasm. In a liver cell, it is replaced about half again as fast in the cytoplasm as in the nucleus. Despite these wide-ranging variations - variations occur in addition from animal to animal - there are certain bounds within which the speeds of replacement vary. And if these bounds are exceeded, then a transition from physiological to pathological mechanisms occurs. And this transition can be traced very conveniently with the aid of these indicator isotopes. If we, for example, expose an organism to ionising radiation, that of radium, X-ray radiation, then histological changes occur that have been observed almost as long as X-rays themselves. These kinds of histological changes must, however, have been introduced of necessity by chemical changes. Detecting this is quite difficult though. These indicator isotopes have proven very useful in detecting such immediate chemical changes that occur after irradiation. Working with Dr. Forsberg in Stockholm, we recently injected a large number of mice with radioactive phosphate - these kinds of experiments have to be carried out with large amounts of animal material, as the individual variation is very high - and determined over the course of 15 minutes how this phosphate is distributed among the organs. Then we took another group of mice irradiated with a massive dose of X-rays and repeated the experiment. It became clear that the livers of the irradiated animals, even only very few minutes after irradiation - This larger volume of phosphates in the irradiated animals is probably connected with the larger volume of glycogen. It has been shown by various researchers that when hungry animals - rats or mice - are exposed to X-rays, the glycogen content of the liver increases. This sounds somewhat strange, that a hungry animal is able to increase the glycogen content of the liver. However, it is not so surprising. It stems from muscle glycogen being broken down under the influence of the radiation, releasing lactic acid that is fed into the liver and utilised to form hepatic glycogen. It is therefore a transfer of muscle glycogen to hepatic glycogen. This observation is also not new: 30, or 20 years ago, Cori had already shown that hungry animals exhibit a higher level of blood sugar and a higher level of hepatic glycogen after being injected with adrenaline - for the stated reasons: mobilisation of the muscle glycogen and formation of hepatic glycogen. And our phosphate experiments indicate that under the influence of the radiation, in addition to numerous other effects - there are indeed an extraordinary number after irradiation - a mobilisation of adrenaline, a release of adrenaline from the adrenal gland takes place, which is thus responsible for these effects. Incidentally, many years ago, we had already investigated with Prof. von Euler the effect of X-ray radiation on the uptake of radioactive phosphate by deoxyribonucleic acid in rat sarcomas. When rats are fed radioactive phosphate, after a very short time P-32 can be detected in the deoxyribonucleic acid of sarcomas. The incorporation is significantly smaller, however, if X-ray radiation precedes the analysis. A half-hour, even 20 minutes, is sufficient to detect this effect: The formation of deoxyribonucleic acid is reduced by 50% or more and the formation of this substance, so extraordinarily important for cell division, is thereby prevented. With the aid of carbon-14, the effect of X-ray radiation on the incorporation of radioactive carbon in the purine body can also be detected. I carried out these kinds of experiments a few years ago using tagged acetate carboxyl groups. Back then, there was no other kind of source substance available. Acetate is a very poor source substance for this purpose. Only a minimal fraction of the carbon in acetate is transformed into nucleic acid or purine. However, even these small quantities sufficed to show that the incorporation of carbon-14 into adenine and guanine was reduced by 50-60% when preceded by X-ray radiation. Recently, we have had glycine, folic acid and these kinds of tagged compounds, which are much better suited as precursors - source substances - for purine, and, with the help of this substance, carried out numerous such experiments and confirmed this result. Carbon-14, radioactive carbon, is a radioactive indicator of utmost importance. One could even say the most important indicator, although phosphorus is also of great importance, as are many others. Before I say a few words about the use of carbon-14, I would like to say a few words about the use of phosphorus-32 in botanical experiments. Immediately after we began using radioactive phosphorus, we also carried out several experiments in which we incorporated a known quantity of phosphate, having a known radioactivity, into the nutrient solution of wheat seedlings. After several days, we analysed the stems and leaves for their chemical composition and radioactivity. This kind of combined analysis reveals, for example, what fraction of the phosphorus in a leaf comes from the nutrient solution and what fraction was already present. For example, after 3.8 days, you can see that 31.8% of the phosphorus has immigrated into the leaf from the nutrient solution and the balance was already present. Experiments of this kind have been carried out on a very large scale, as they are of chemical interest for agriculture. Agriculture was interested in these kinds of experiments. One of the problems was to what extent this phosphorus, which was applied to the soil in artificial fertiliser, contributes to the phosphorous content of the plants, and to what extent phosphate already present in the soil contributes? These trials have been carried out with 100 million times greater radioactivity than our initial experiments you see here. Almost unlimited phosphorous radioactivity can be achieved. These are the Canadian trials carried out by Spikes and his staff. However, there have been many other trials carried out in the United States and Europe. The problem is: What fraction of the phosphorus detected in the wheat is from artificial fertiliser and what fraction is from the soil. Moreover, you primarily see that barley takes up more artificial phosphorous than wheat. It is ... More than 60% of the phosphorus can stem from artificial fertiliser under favourable circumstances, the balance from the soil. If the experiment is not carried out in June, but later, then the proportion of artificial fertiliser becomes smaller and smaller. That sounds somewhat strange, but the explanation is presumably that when the plants grow - the wheat grows - the roots grow also. And when the roots become stronger, they reach deeper and can extract more phosphorus from the soil, and the soil becomes a continuously more threatening competitor to the phosphorous in the artificial fertiliser. Therefore, the proportion of phosphorous from artificial fertiliser in August is much smaller than in July, for example. It was shown that - different salts were compared - that ammonia salt proved most suited. If ammonium phosphate was used to fertilise, then the proportion of phosphorous from artificial fertiliser to total phosphorous was the greatest. I would like to show you the next image - two images: autoradiograms, as they are known, or X-ray images. For quantitative analysis of the distribution of the radioactive bodies, the Geiger counter is best suited. Photographic plates are less well suited for quantitative determinations; that has to do with the nature of the photographic plate. It is nevertheless very important when judging histological specimens in these kinds of X-ray images to be able to immediately identify where the radiating particle is located. Here you see an aspen leaf, radioactive phosphate was applied to an aspen branch and we see how the phosphate advances and gradually spreads in the leaf. (The next image). The next image is a photo taken in Berkeley by Mr. Arnon and shows tomatoes, green tomatoes, which have been immersed in radioactive phosphate. And in the growing pips - as you see - large quantities of phosphate accumulate. In contrast, in the mature red tomatoes, with no extensive changes taking place in them or their pips, you only see a very small accumulation of the phosphate. The lecture only allows me to say a few words about the utilisation of [next image, if I may] about the utilisation of carbon-14. There are even organic compounds available today, a large variety of tagged organic compounds. For example glucose that is biologically produced. Plants are allowed to grow in an atmosphere containing radioactive carbon dioxide, and then their accumulated radioactive glucose, augmented in all six basic atoms, is separated out. When this kind of glucose is fed to titmice, it can be unequivocally shown that over the course of 24 hours about 70% of the carbon in glucose appears in the carbon exhaled. You find 10, 15% in the fatty acids of the body, which store an important part, and little in glycogen, proteins, and similar substances. The image you see here shows a very interesting result. With the help of indicator isotopes, new facts have been discovered. However, the primary application does not lead to new facts. It mostly leads to describing known facts in somewhat greater detail. A landscape that was already discernible, but shrouded in mist, so to speak making it visible, often in all of its details. It has long been known that in organisms afflicted with diabetes no or only reduced conversion of carbohydrates into fat occurs. With the help of radioactive glucose, quantitative results can be produced. These are experiments carried out in Berkeley by Charkoff, who incubated hepatic sections of rats suffering from diabetes, of diabetic rats, in a bi-carbonate Ringer's solution containing radioactive glucose. And it was shown that these hepatic sections were hardly associated with radioactive carbon dioxide. Detectable carbon dioxide only contains a minimal quantity, about 1.1 and 0.9 ... By comparison, if rats are treated with insulin several days previously and the sections from these rats treated with insulin are examined, the carbon dioxide emitted contains 4.9 - 4.4 - 3.6 - 5.0% radioactive carbon dioxide. And you can see: The fatty acids receive 4.9 - 4.4 - 3.6, and so on, these are the fatty acids, here they are. Carbon dioxide is very important in the history of carbon-14, which nicely and also quantitatively demonstrates how insulin functions and how it even counteracts the inability of carbohydrates to be converted into fatty acids. I already mentioned our experiments that we had carried out with animals exposed to X-rays. At first, we thought perhaps that X-ray radiation influences the production of insulin. We found, however, that carbon-14 was just as vigorously incorporated into the fatty acids of our irradiated animals as with those not irradiated. That was absolute proof that our results have nothing to do with insulin deficiency. I would like to say a few words still about another application area of indicator isotopes, namely, their use in carrying out these kinds of investigative methods that were already well known, but which, through the use of indicator isotopes, could be carried out much more easily and quickly. I already mentioned that the water content of the body - the total water content as well as the extracellular water content - can be determined very simply with the help of indicator isotopes. Another quantity of great importance is the volume of blood in the body. And this fundamental quantity, yes, it was usually determined previously by injecting dye, the dye was diluted and the dilution was a measure of the total blood plasma circulating in the body. One could then calculate with the help of the haematocrit the quantity of blood from this reading. If you want a direct determination of the number of blood corpuscles present in the body, the easiest way is to tag the blood corpuscles, inject them, and establish the dilution of the radioactivity in the body. In our earlier investigations, very early investigations with Mr. Artom, we introduced radioactive phosphate into a blood sample and investigated how long was required before these radioactive phosphate radicals could be detected in blood corpuscles. The onset is quite slow. It takes about two hours, even more in the example of human blood, until about half of the phosphorous ions in the plasma were replaced by phosphate from blood corpuscles. However: when individual phosphate radicals penetrate into blood corpuscles, they are almost instantly incorporated into the unstable organic portions. The others include phosphoric acid and the like. This fact enables the blood corpuscles to be tagged with the help of radioactive phosphate. Using a blood sample from a patient, a small quantity of radioactive phosphate is added, for instance, and agitated at a controlled temperature for an hour. Then the blood plasma is briefly centrifuged, the non-radioactive blood plasma or salt solution is replaced, and the sample re-injected into the patient. A complete mixing occurs after ten to fifteen minutes, even sooner. Then another blood sample is taken and the radioactivity of the injected specimen and the new sample compared. The ratio depends exclusively on the quantity, the number of circulating red blood cells. If there is a lot of blood in a person, then the radioactivity is strongly diluted, while if there is little, it is slightly diluted. This method has found very broad application, in particular in the United States in recent years. It can be used not just for determining the quantity of blood, but also be used for the speed of circulation. while the fatty acids are formed primarily in the liver, other organs contribute also ...

George de Hevesy (1952)

The application of radioactive indicators in the investigation of physiological processes in animals (German presentation)

George de Hevesy (1952)

The application of radioactive indicators in the investigation of physiological processes in animals (German presentation)

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In 1952, de Hevesy participated in the second Lindau Meeting of Nobel Laureates, which was the first one dedicated to chemistry. De Hevesy’s early openness to the idea of Nobel Laureate meetings on German soil is truly remarkable, considering that, due to his Jewish ancestors, he had to emigrate twice during the reign of the Nazi Regime in Germany. In 1934, this inter alia brought him from Freiburg to Copenhagen, where he worked in the laboratory of his friend Niels Bohr. The initial part of the present lecture fragment summarizes the results de Hevesy obtained during that time. They relate to the use of the radioactive phosphorous 32 isotope as a means to study phosphorous exchange in animal bones and organs. However, the scope of de Hevesy’s work was much broader and his ideas were frequently picked up by scientist all over the world. Hence, in his 1952 lecture, de Hevesy goes on to give a comprehensive review of his life’s work and the related contributions of others.
One of his central achievements was the use of radioactivity measurements as a quantitative tool that can replace classical methods like weighing or volumetric measurements. An application, impressive due to its simplicity, is described later on in the lecture. How would you determine the blood volume in a person’s body – quickly and easily? And without doing any harm? De Hevesy suggests to draw a small portion of the patient’s blood, enrich it with a known amount of radioactive phosphorous 32 and to re-inject it. After a short while, blood is drawn from the patient yet again and its radioactivity is measured. From the reduction of the radioactivity (i.e. the dilution factor), the patient’s blood volume may be calculated. Despite the obvious disadvantages of injecting radioactive material into the human body, similar methods are still used today.
How do you come up with such Nobel Prize worthy ideas? In the case of de Hevesy, there is a nice anecdote* suggesting that the troubles of everyday life may play their part. After he obtained his doctorate degree at the University of Freiburg, de Hevesy moved to the laboratory of Ernest Rutherford in Manchester (1911), where he witnessed great discoveries in the field of nuclear physics. The workgroup regularly had lunch at a nearby guesthouse. Unfortunately, the food was of miserable taste, which led the students to the suspicion that the hosts scraped the leftovers off the plates to use them for the next day’s meals. To convict the culprits, de Hevesy mixed a small amount of radioactive material, which he had obtained when trying to separate radium-D from lead, into his leftover food. And indeed, next day’s hash was slightly radioactive! Not to be repeated in your local canteen!
Until his death in 1966, de Hevesy remained a close friend of Lindau meetings. With a few exceptions, he visited every meeting in the years to come, even those dedicated to physics and medicine.

David Siegel

* Retold according to Chem. Unserer Zeit 3, 87 (1969).

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