George de Hevesy

Ladies and Gentlemen, the fraction of the atoms of the ancestors which is conserved through the generations is determined by the fraction of the element in question which is transferred from the mother to the offspring, how many of these atoms in the animal body are replaced by atoms from food before the appearance of the second generation, and also what fraction of the element in question in the animal's body is involved at all in the development of the embryo. Let us first consider the example of sodium. Sodium is an element which displays relatively simple behaviour in the animal body. In a person of 80 kilos, about 55 grams of sodium circulate in the form of a roughly half-percent solution of common salt. If one introduces into this body with the food or by injection a small quantity of radioactively marked sodium chloride, which one could obtain for a few pence - from a uranium reactor for instance - then this radioactively marked common salt mixes with that in circulation, and all the circulating salt will be radioactively marked. Let us say that we have introduced a quantity of radioactive common salt which produces a pulse rate of 1000 counts a minute on a Geiger counter. We can show an activity in the urine collected on the first day of 40 pulses per minute, meaning that 4% of the eaten, and thus also of the circulating sodium chloride has been excreted. The excretion of sodium takes place mainly via the kidneys. We can also conclude from this that 4% of the total circulating quantity of sodium chloride has been excreted and replaced by common salt absorbed from food. So in about 14 days half of these sodium ions, which were initially present, will be excreted. And it is easy to calculate that after three years not a single sodium atom remains of those present at the beginning. So there is not much left for the descendants. However, the proportions are not quite so simple. Sodium is an element which is described as extracellular. That means an element which is chiefly present and circulating outside the cells, in the intercellular space. But there is also sodium in the cells. And these quantities of sodium, which are not so small - constituting around 10 g in such a person - even play a very important role in nerve and muscle excitation. In addition there are about 20 g of sodium in the body which is more or less tightly bound to the skeleton, the skeletal structure. Part of it so firmly that it does not reappear at all. From this observation it follows that the binding of the atoms in the skeleton is of the greatest significance for our problem. We must look into this question a little more deeply. That the atoms present in soft tissues are gradually replaced by atoms and molecules from nourishment, has been known for a very long time indeed. A discovery which was deepened greatly, especially in quantitative respects, after isotopic tracers became available. But we had no idea what happens in the skeleton, in the mineral framework of the skeleton. Whether the atoms are bound to the skeleton or take part in turnover and are replaced, we didn't know that at all earlier. It is quite understandable that directly after the discovery, the very significant discovery by Joliot-Curie of artificial radioactivity, where the production of radioactive isotopes of the components of the skeleton, primarily phosphorus, was achieved, that the first task attempted was to determine: What is really going on with the mineral structure of the skeleton? Can these atoms be exchanged or not? If, for example, one injects a very small quantity of radioactive phosphate into a rat, and checks after a few minutes whether the structure of the skeleton has become radioactive, then the answer is positive: radioactivity can be demonstrated in the skeleton within minutes, showing that radioactive phosphate has been transferred from the blood plasma to the skeleton, into the mineral components of the skeleton. In recent years we have... This is demonstrated with the Geiger counter. One isolates a small particle from the skeleton and places it under the Geiger tube. In recent years photographic methods have acquired greater and greater importance in such investigations. A tissue section is taken and placed on a photographic plate or inserted in emulsion, and if the section contains radioactive atoms then the film is darkened and the intensity of the darkening depends on the quantity of the radioactive substance. When dealing with weakly radioactive specimens, the specimen is left in contact with the photographic plate for days, weeks, months. And I would like to show you such a picture which was recently received from Leblanc, a much respected researcher in this field in Canada. Leblanc injected some radioactive phosphate into an adult rat, and after five minutes he investigated the leg bones, the tibia, the epiphyseal plate of the tibia to be precise, and very definite darkening is evident. And after only five minutes, this radioactive phosphate is demonstrable in the skeleton, that is phosphate from the circulation, and after two hours the effect is naturally much more pronounced, as we can see here. I can show you another picture from Leblanc: these are the incisors from a rat. That is a normal photograph and this one here is an autoradiograph, made by placing an incisor on the film. And the radioactive atoms, the emissions, cause a darkening. And that is also taken after five minutes. And here you can see in the pulpa, commonly known as the nerve when sitting in the dentist's chair, an accumulation of plasma phosphate can already be demonstrated. With the much more sensitive Geiger counter method one can even demonstrate radioactivity on the point of the incisor after as little as five minutes. The incisors, which keep growing in adult animals, they are continually being worn down by chewing - the material is worn down and newly formed, so that one can track the growth of bone, the rapid growth even in fully grown animals, by studying the incisors. So these investigations indicate that a rapid interchange takes place between a part of the skeleton and the blood plasma. But the question what fraction of the skeleton participates in such a process, in the formation of new tissue, is not answered by these investigations. Two rooms are connected so that people in one room can go across to the other room and vice versa, and one room contains dark people and the other fair people. We want to know whether complete mixing has taken place between the populations of the two rooms, so we determine the relation between light and dark, and when the relation is 1, then complete mixing has taken place. If we want to know whether all the atoms of the skeletal framework are in an interaction with the circulating phosphate in the blood plasma, meaning have been replaced, been newly formed, then we compare the radioactivity of 1 mg of phosphate taken from the circulation with 1 mg of phosphate taken from the bone. If such a comparison yields the relation 1, then we have complete exchange. If the relation is 0.5, we have 50% exchange, so 50% of the skeleton has participated in the renewal. But it is not so easy to carry out such a determination. For this reason, that the marked phosphate migrates very rapidly not only into the bones but also into the soft tissues - in the hundreds of different phosphorus compounds which exist there. Thus the activity reduces very rapidly at first, and later declines slowly, and we want to follow the effects for weeks or months, and the results we get are very imprecise and difficult to obtain. One way out, which we indeed took, is to inject the animals repeatedly with radioactive phosphate in the course of the day, in order to keep the level of radioactivity constant. Then after a month perhaps one can compare the radioactivity of 1 mg of phosphate from the blood plasma with 1 mg of phosphate from bone, and see whether this figure is the same or not. As you can see from the next diagram, the figure is not 1. After 50 days, equalisation has only reached about 30%. So the radioactivity of 1 mg of bone phosphate is only a third of the radioactivity of 1 mg of the blood plasma phosphate which was held constant. So only 30% of the bone has taken part in this exchange process. And this is the soft parts of the bone, known as the epiphyses. The same procedure carried out in the hard bone, in the diaphyses, yields a very modest equalization: only 7 or 8%. These results already show that only a fraction - it can't be much more than a third - of the bone participates in such a renewal process. Now there is a second way to answer this question. One can simply activate or mark a whole animal. So that, let us say, every millionth atom of the relevant type present in the animal becomes radioactive. Then the whole animal is marked. And then one can investigate: What happens after a month? How many are there of these marked atoms, how many of the radioactive atoms present in the skeleton have disappeared? Only this question is not so simple to answer, since, as we have just seen, a large part of the skeleton is not accessible. One cannot mark the whole skeleton of an adult animal since it is not renewed, does not allow the entry of the active atoms. One can do something else: one can raise animals which are wholly marked. One gives the mother radioactive atoms and then an offspring will be born which is naturally wholly radioactively marked from the radioactive mother. But radioactive phosphorus does not lend itself so well to such investigations since it has a half-life of only 14 days. One can track the results for perhaps 10 times 14 days, but not longer. Then the activity becomes so weak that one cannot measure it. The reason why initially we always used radioactive phosphorus in this and other studies is that we had no other radioactive material. The method for producing this radioactive material is the irradiation of sulphur, for example, with neutrons. Then the sulphur nuclei absorb a neutron, sulphur-32 turns into phosphorus-32, a kind of phosphorus which is heavier than the phosphorus found in nature, which has an atomic weight of 31. And because it is so heavy, it is unstable and does not really feel at home in this world, tries to turn itself back into sulphur. Luckily, this reverse transformation does not take place instantaneously, but in the course of 14 days as half-life. And we can use this time to employ these atoms as tracers. Now, initially neutrons could only be produced by mixing radium salts with some light element, for example beryllium. Neutrons are present in all atomic nuclei with the exception of hydrogen. And the neutrons of the light elements are the most weakly bound. So when an alpha particle from radium hits a beryllium nucleus, the particle knocks a neutron out. So you get neutrons. And if the neutrons go through sulphur, they are captured there and you get this radioactive phosphorus. Now these neutrons have a large useful range. We have very little... We had almost 1 g of radium available, not more - one needs a lot of sulphur, preferably from carbon disulphide, perhaps 10 litres. In the middle of a retort containing 10 litres of carbon disulphide, one hangs this source cast in platinum, consisting of radium chloride and beryllium. The resulting stream of neutrons produces the radioactive phosphorus which can easily be chemically separated from the sulphur. We were able to utilize this procedure with sulphur. But we could not apply it to most elements because the yield was too low. Then the cyclotron and later the uranium reactor were developed and radioactive isotopes of all - practically all - elements became available, including radioactive calcium. Radioactive calcium lives ten times as long as radioactive phosphorus. So one can work with this radioactive calcium for years and track for years what actually happens with these atoms in the animal body. This is why we have used radioactive calcium in further investigations, calcium with an atomic weight of 45. And we fed mice with radioactive calcium, that is, calcium which was radioactively marked. And from these mice further generations were then born which were thoroughly marked. Including the skeleton - completely! And such a mouse may have perhaps ten offspring. One mouse is investigated immediately after birth. The next after a month has passed. It is easy to show that the siblings - not to the same extent, but almost wholly [...] with calcium, with the marked calcium. The first mouse was, as stated, killed and investigated. The rest were given to an adoptive mother which was not radioactive. And after one month, after two, after five, after ten months the siblings were killed and investigated. And the result can be seen in the next diagram. Let's assume that the mouse examined at birth produced an activity of 100 under the Geiger counter. Then, this mouse, which was investigated after 30 days, had a not insignificantly lower value. Atoms from the mother were lost at first, but later very few. And you can see that after the course of 600 days, which is a period which corresponds approximately to the lifetime of a mouse, that half of the maternal calcium atoms are still present. And the preservation of so much of the mother's calcium in the offspring can be attributed primarily to the skeleton. Incidentally, 99% of the body's calcium is present in the skeleton. It should not be very different with humans, since the structure of the human skeleton does not differ much in principle from the structure of the mouse skeleton. And we must take with us into the grave about half the calcium atoms that we received at birth. And we have carried out experiments where the first mouse was not investigated directly after birth, but the newborn mice were left with the radioactive mother and received radioactive milk. And later, after they stopped drinking milk, radioactive food - that is, food containing radioactive calcium - until they were fully grown. After about 100 days or so. Then we investigated the first mouse. Later, after 100 days the second, after 400 days the third. And that was continued over about two years. And these experiments showed - the skeleton was always investigated - that around 67% +/- 2.4% of the calcium atoms which were present in the adult animal when it was 100 days old, were still present at the end of the animal's life, so that two-thirds of the calcium of the adult animal - and it should not be very different with humans - were retained throughout life. And that can be attributed to the fact that part of the skeleton, two thirds is partially protected. What happens in the skeleton is that when these apatite crystals, which constitute the mineral structure of the skeleton, come into contact with the blood plasma and the lymph, then some of the bone structure goes into solution and presumably a new structural part is formed in another location. And the apatite crystals underneath are protected by this and from any contact with the calcium of the blood fluids. In the course of its life there flows [...] flow about 4 g of calcium. In the course of life about 20 or 25 kg of calcium flow through the human circulation. So, large amounts. And a significant part of the calcium of the skeleton has no contact with this large, circulating calcium. Incidentally, the ability of the skeleton largely to retain atoms acquires very great significance. Let us take, for example, lead poisoning, which is a very unpleasant poisoning - and also chronic. What makes lead poisoning so chronic is mainly the fact that lead is incorporated into the skeleton. Imagine some painter who paints with lead paint and eats his breakfast with hands that have been contaminated with the lead paint. He eats lead. The lead is absorbed, enters the circulation and lead can displace calcium to some extent from the apatite, and the lead in the plasma is built into such apatite crystals. In the meantime, the lead disappears from the plasma. Lead does not remain very long in the blood fluids. It is either built into the bone apatite, incorporated in the blood corpuscles or it is excreted. So the blood plasma is soon practically lead-free. Now a lead-free calcium apatite layer is built up from this blood plasma and covers the lead-containing layer. And this lead can quietly remain there for a really long time. In the meantime - the phosphate content of the blood plasma certainly changes, the composition changes, and there are other factors too. So the calcium phosphate layer over the lead layer goes into solution, and suddenly this lead can escape, goes into circulation. And this happens again and again and the lead poisoning remains chronic in this way. Or if we consider radium: here we find even more extreme circumstances. Radium has a great affinity for phosphate, greater than that of calcium. And if radium gets into the circulation it is very easily built into the skeleton and can remain there. In earlier times people were very unwisely given water to drink that contained radium. A horrible thought, as we know today. And a significant part of this radium was built into the skeleton. The literature records a case where a woman was given some radium chloride water to drink, that was in '24. In '34 she was examined and 1/10th of a microcurie found in her skeleton. One can actually measure radium beyond the body, quantitatively measure it, from the penetrating radiation. And measured again ten years later, all the radium was still present in the woman's skeleton. So it is a cemetery for radium, the skeleton. Also a cemetery for other atoms including some very undesirable ones. Here I am just pointing out that this preservation of atoms has a very great importance for the skeleton. But to return to our problem of the preservation of the ancestors' atoms: one can arrive at no conclusions at all about how many are transferred from the mother to the first generation. Because the mother is not thoroughly activated. So one has to begin with the first generation and ask what fraction of the marked calcium atoms of the first generation are transferred to the second. And one finds that 1 in 300 of the calcium atoms present in the mother is transferred to the next generation. So few, because a large part is locked in the skeleton, immobilised. From the second to the third goes another 1 in 300, from the third to the fourth 1 in 300 again. And that is repeated. And it is easy to show that after the 12th generation not a single atom is still present in the mouse which stems from the ancestors. Not a single one. Still, this result does not apply to all parts of the body. The lifetime of the ancestors' calcium atoms is so long because calcium is preserved so well in the skeleton, one part completely immobilised, another part retained for a long time. If, for example, you look at water molecules: yes, one can inject some heavy water, say 10 mg, into a mouse. The heavy water has a density about 20% greater than that of normal water. A minute is enough for the injected water to mix with all the water circulating in the mouse. One can take a blood sample from the mouse after a few minutes and extract the water from it and measure the density of this water. If this 20% is now diluted 10,000 times, so this density increase, that heavy water shows, then it follows that a total of 10 ml of water was present in the mouse. The half-life determined in humans with heavy water is about 10 days. And while after 160 days one cannot show a single water molecule that was originally there with a mouse, with humans it takes 800 days. But that is also a relatively short time, so not much is transferred to the next generation. With mice - it depends when they reproduce. It can be the second, third or the fourth generation at the latest, when not even a single water molecule from the ancestors is transferred. In the beginning one has such determinations of water in the body, water content determinations. Incidentally, these are quite important determinations, since endocrinologists have lately developed an interest in the body's total water content after an operation, after the removal of both kidneys or the pituitary the water content changes and monitoring is very important. And they perform such determinations of the total water contents of the body by injecting some heavy water and measuring how strongly this heavy water is diluted. If a lot of water is present, the dilution is greater, if less - then less. But recently super-heavy water has become available. That is water with hydrogen atoms not of deuterium with a mass of 2, but of tritium with a mass of 3. So that is super-heavy water consisting of tritium with hydrogen atoms, the hydrogen isotope with a mass of 3, and the oxygen atom. Two hydrogen atoms and the oxygen atom. And this tritium water is radioactive. One can measure this radioactive water. And these radioactive measurements are much more sensitive than the density measurements used to identify heavy water. So one can dilute 20 mg of super-heavy water by a factor of 10^12 times, which is extraordinarily high. And one can still detect it by its radioactivity. With heavy water, say 20 mg, if one dilutes it a million times, then detecting it is already very difficult. So lately one often uses this super-heavy water for the determination of the total water contents of the body. Now I would like to show you a picture, just to show how rapidly this marked... You can see that it is evenly distributed after only 10 minutes. So you see how rapidly the injected water mixes with the total, extra-, as well as intra-cellular water. One should not take that literally; there is certainly water in the muscles which is very strongly bound and does not reach equilibrium so quickly. Also in the skeleton. But statistically these quantities play no role; you can see that after 10 minutes practically all the water that was injected is mixed throughout the body. Then when experiments were done with this super-heavy water, something very strange appeared. Namely: the marked water which a mouse received disappears with a half-life of 21/2 days and suddenly, as shown by this tritium, the radioactive tritium, disappears much more slowly. This data is from Thompson in the United States. Now, the explanation is as follows: a very minimal fraction of the hydrogen atoms of the water is very slowly built into organic compounds. Everything which is slowly built in also reappears slowly later. And after the main amount has disappeared - you see, a decline from 10,000 to 10 - then this tiny fraction becomes noticeable. And this organically bound hydrogen, originally from water, reappears and finds a new oxygen partner, forms water molecules again and these then disappear much more slowly. The speed here is determined by the splitting off of the hydrogen. So these are no longer the mother's water molecules, but the hydrogen atoms from the mother's water molecules, which now have a new oxygen partner. And they can be demonstrated in the fourth or fifth mouse generation. That not a single calcium atom from the ancestors can be shown after the 12th generation illustrates very nicely the known fact that inheritance, the inherited characteristics have nothing to do with having atoms in common. So one can resemble ones ancestors without possessing a single atom in common with them. That is nothing new, but it is illustrated very nicely. We know very well that inherited characteristics depend on the fact that the atoms and molecules can be repeatedly reconstructed in reproducible ways. And Prof. Staudinger has also indicated to us this large number of possibilities in his lecture. Ladies and gentlemen, it would be extraordinarily incorrect if I were to continue my lecture. I have been able to report to you about twelve generations of mice. You will hear from Prof. Hahn about a time period which is extensive, literally, literally! Thank you very much.

Comment

In the present talk, the Hungarian de Hevesy picks up the topic of his 1952 Lindau lecture: the use of radioactive isotopes for studying the distribution and turnover of different chemical elements in living organisms. Isotopes share the same number of protons and electrons, but vary in the number of neutrons. As a direct consequence, their chemical properties (which depend on the electron shell) are mostly identical, their nuclear stability, however, may be different. Unstable isotopes decay emitting radiation which may be measure with a Geiger counter. If an isotope of an essential element, such as sodium or calcium, can be made and if this isotope is radioactive, it is theoretically suited for metabolic studies. If consumed, the body will metabolize it just as its non-radioactive counterpart. It can then be quantified in different body parts or fluids (tissue, bones, blood etc.) by measuring the radioactivity emitted.

In general, the 1950s were a very dynamic times for radiochemists like de Hevesy, because a lot of new radioactive isotopes could be made in the frame of the commissioning of the first “huge” particle accelerators with energies in the GeV range. One of these elements, and a main subject of this talk, is calcium. Using the artificial, radioactive calcium isotope with the mass number 45, de Hevesy and his team studied the behaviour of calcium in the organism by means of a rat model. They came up with a range of interesting figures. One result, for example, was the insight that that we keep half of the calcium atoms, present in our body at birth, for our entire life. This means that only one half is exchanged for calcium obtained from food. Considering that the calcium in the body of a new born is obtained exclusively from the mother, this implies that a couple of calcium atoms remain in the same family for up to twelve generations.

Despite the obvious disadvantages of feeding or injecting radioactive material, the methods developed by de Hevesy are still used today for selected purposes. This is mainly due to the fact that they give rather reliable data for living organisms and that they allow the study of fluxes, i.e. metabolic turnover rates.

Still, there also have been new developments, which avoid the artificial contamination with radioactive material. In the frame of investigations done on workers involved in the incident at the nuclear power plant in Chernobyl in 1986, it was found, for example, that the potassium isotope with the mass number 40, naturally present in the human body, emits high energy gamma-radiation, which may be quantified with suitable detectors [1]. This allows for the facile quantification of the total amount of potassium present in a living organism. Other elements, which do not show this natural radioactivity, may be activated by exposing the body to neutron radiation first. This so-called neutron activation analysis allows for the in vivo quantification of virtually all the major elements in the human body, such as hydrogen, oxygen, carbon, nitrogen, calcium, phosphorus, sodium, and chlorine [1]. On the basis of results obtained with these new techniques, a couple of the figures mentioned by de Hevesy had to be corrected slightly. While he estimated, for example, that a human of 80 kg body weight contains 55 grams of sodium, today we know that the correct figure is approximately double of that.

David Siegel

[1] K. J. Ellis, Physiological Reviews 2000, Vol. 80, No. 2, p. 649.

Lindau Mediatheque

George de Hevesy