»> i6: •K^'. ;«,'/.',» ,>,'sr»^.*?'. ts*-; >>;'? ^!S^ ^^. iWt','^.>= ADVENTURES IN RADIOISOTOPE RESEARCH tf ADVENTURES IN RADIOISOTOPE RESEARCH The Collected Papers of GEORGE HEVESY in Two Volumes Volume Two PERGAMON PRESS NEW YORK . OXFORD • LONDON • PARIS 1962 PERGAMON PRESS INC. 122 East 55th Street, New York 22, N.Y. 1404 Piew York Avenue N. W'., Washirtglon, 5 D.C. PERGAMON PRESS LTD. Headia^ton Hill Hall, Oxford 4 Si. 5 Fitzroy Square, London W. 1. PERGAMON PRESS S.A.R.L. 24 Rue des EcoUs, Paris V PERGAMON PRESS G.m. b.H. Kaiserstrasse 75, Frankfurt am Main Copyright © 1962 Pergamoa Press Ltd. Library of Congress Card A'o. 60 — 12557 Printed in Hungary hv The Printing House of the Hungarian Acad my of Sciences CONTENTS Volume Two Labelling of Red Corpuscles 51 A Method of Blood Volume Determination (with L. Hahn) 517 52 Determination of the Red Corpuscle Content (with K. Zerahn) 523 53 Thorium B Labelled Red Corpuscles 531 Clinical Investigations 54 Elimination of Water from the Human Body (with E. Hofer) 536 55 Excretion of Phosphorus (with L. Hahn and O. Rebbe) 540 56 Potassium Interchange in the Human Body 553 57 The Red Corpuscle Content of the Circulating Blood Determined by Labelling the Erythrocytes with Radio -Phosphorus (with K. H. Koster, G. S0rensen, E. Warburg and K. Zerahn) 561 58 AppHcation of ^^K Labelled Red Corpuscles in Blood Volume Measure- ments (with G. Nylin) 573 59 Apphcation of "Thorium B" Labelled Red Corpuscles in Blood Volume Studies (with G. Nylin) 580 60 Cancer Anaemia 597 Iron Metabolism 61 Effect of Adrenaline on the Interaction Between Plasma and Tissue Constituents (with G. Dal Santo) 610 62 Effect of Irradiation on Hemin Formation (with R. Bonnichsen) .... 624 63 Haemoglobin Present in the Nuclear Fraction of the Liver (with R. Bonnichsen, G. Ehrenstein and J. Schhack) 634 64 Apphcation of Isotopic Indicators in Haematology 639 65 Note on the Determination of Radioiron (with K. Agner and R. Bonnich- sen) 65 1 66 Embryonal Iron Turnover (with G. v. Ehrenstein) 655 Nucleic Acids 67 Rate of Formation of Nucleic Acid in the Organs of the Rat (with J. Ottesen) 663 81311 VI ADVENTURES IN RADIOISOTOPE RESEARCH 68 Rate of Renewal of Ribo- and Desoxyxibo Nucleic Acids (with E. Ham- marsten) 673 69 Turnover of Ribosenucleic Acid in the Jensen-Sarcoma of the Rat (with H. Euler and W. Solodkowska) 680 70 Life-Cj^cle of the Red Corpuscles of the Hen (with J. Ottesen) 688 Studies in Radiation Biology 71 Effect of X-Rays on Nucleic Acid Formation in the Jensen- Sarcoma (with H. Euler) 692 72 The Effect of X-Rays on Nucleic Acid Formation in the Organs Of The Rat (with L. Ahlstrom and H. Euler) 721 73 Turnover of Nucleic Acid in Retrogade Sarcomata (with L. Ahlstrom and H. Euler) 731 74 The Indirect Effect of X-Rays on the Jensen- Sarcoma (with L. Ahlstrom and H. Euler) 744 75 Attempts to Find Products Blocking Nucleic Acid Formation in the Circulation of an Irradiated Organism (with L. Ahlstrom, H. Euler and K. Zerahn) 758 76 Fate of The Nucleic Acid Introduced into the Circulation (with L. Ahlstrom and H. Euler) 761 77 Formation of Nucleic Acid in Sarcoma Slices (with L. Ahlstrom and H. Euler) 770 78 Application of Labelled Substrates in the Study of Enzymic Processes (with L. Ahlstrom and H. Euler) 783 79 Effect of X-Rays on the Incorporation of Carbon- 14 into Desoxy- ribonucleic Acid 791 80 Effect of X-Rays on the Incorporation of Carbon- 14 into Animal Tissue 793 81 Effect of X-Rays on the Incorporation of 14 C into Tissue Fractions of the Mouse (with G. Dreyfus) 795 82 Effect of Muscular Exercise and of Urethane Administration on the Incorporation of Carbon-14 into Animal Tissue 821 83 Effect of Irradiation by X-Rays on the Exhalation of Carbon Dioxide by the Mouse (with A. Forssberg) 825 84 Effect of X-Rays and Hormones on Resorption Rate of Injected Nai^HCOj, (with A. Forssberg) 828 85 Note on the Effect of X-Rays and Hormones on the Resorption Rate of Injected Na^HCOg (with A. Forssberg) 838 86 Effect of Irradiation with X-Rays on the Catabolism of Methyl- alcohol in the Mouse 841 CONTENTS VII 87 Effect of Irradiation with X-Rays on the Catabolism of Ethylalcohol in the Mouse 847 88 Radioactive Tracers in Radiobiological Studies. The Thirty-Sixth Silvanus Thompson Memorial Lecture 851 Botanical Studies 89 The Absorption and Translocation of Lead by Plants 876 90 Atomic Dynamics of Plant Growth (with K. Linderstrem-Lang and C. Olsen) 884 91 Exchange of Phosphorus Atoms in Plants and Seeds (with K. Linder- str0m-Lang and C. Olsen) 887 92 Interaction Between the Phosphorus Atoms of the Wheat Seedling and the Nutrient Solution 89 1 93 Exchange of Nitrogen Atoms in the Leaves of the Sunflower (with K. Linderstrem-Lang, A. S. Keston and C. Olsen) 905 94 Zinc Uptake by Neurospora (with I. Andersson-Kotto) 910 95 Ph osphorus Exchange in Yeast (with K. Linderstrom-Lang and N. Niel- sen) 916 96 Potassium Interchange in Yeast Cells (with N. Nielsen) 918 97 Note on the Number of Pollen Grains Identified in the Fruit of the Aspen (with C. Eklundh-Ehrenberg and H. Euler) 924 Lectures 98 Some Applications of Isotopic Indicators. Nobel Lecture 928 99 The Application of Radioactive Indicators in Biochemistry. Faraday Lecture 961 100 Historical Progress of the Isotopic Methodology and its Influences on the Biological Sciences. Read at the Turin Meeting of the Society of Nuclear Medicine 997 Index 1039 Originally published in Acta Physiol. Scand, 1, 1 (1940). 51. A METHOD OF BLOOD VOLUME DETERMINATION L. Hahn and G. Hevesy From the Institute of Theoretical Physics, Copenhagen The method usually applied in the determination of blood volume is that worked out by Rowntree and his colleagues (1929). The principle of the method is that a dyestuff is injected intravenously and its degree of dilution determined^^). As the dye only mixes with plasma, the volume of the plasma alone is thus measured. The relative volume of corpuscles and plasma is determined with the haematocrit. To arrive at the blood volume, the volume of the corpuscles is added to that of the plasma. Rowntree gives the following description of the method applied (comp. also Fleischer-Hansen, 1928). A 1.5 per cent solution of vital red in distilled water is prepared. Four centrifuge tubes are provided and 1 cc. of a 1.6 per cent solution of sodium oxalate is placed into each of them. A needle is inserted in the vein of one arm and 10 cc. of blood are removed. 5 cc. are placed into each of two centrifuge tubes for standard plasma colour. The dye is then injected. After 3 to 6 min, 10 cc. of blood are withdrawn from the vein of the other arm and 5 cc. placed into each of the two remaining centrifuge tubes. All four tubes are centrifuged and the relative volume of corpuscles and plasma measu- red. The second sample is compared with a known strength of the dye and the degree of dilution of the dye in the plasma is thus obtained. When considering the possible errors of this method, the main question at issue is whether, when the second sample is collected, the dye is uniformly mixed in the plasma and none has yet escaped into the tissue spaces or urine, a further possible source of error being the adsorption of a part of the dye by the enormous surface of the capillary wall. ^^' Instead of a dyestuff, diphteria antitoxin was used in some determinations (v. Behking, 1912; Madsen, 1934). 518 BLOOD VOLUME DETERMINATION DETERMINATION OF BLOOD VOLUME BASED ON THE DILUTION OF LABELLED CORPUSCLES In this note, we wish to describe a method of blood volume deter- mination based on an entirely different principle from that described above. We inject into the vein of a rabbit A a known volume of labelled corpuscles taken from another rabbit B and determine the extent to which these labelled corpuscles are diluted in the circulation of rabbit A. Labelled corpuscles of rabbit B are obtained in the following way. We administer by subcutaneous injection some labelled (radioactive) sodium phosphate to rabbit B. In the course of about a week, a substantial fraction of the phosphatide molecules of the bone marrow and other organs are renewed. As this renewal takes place in the presence of labelled phosphate, the newly formed phosphatide molecules will contain labelled P atoms. Corpuscles formed in a medium containing labelled phosphatide molecules will necessarily incorporate some of them. Labelled phosphatide molecules can also enter to some extent into the corpuscles by exchange of non-active phosphatide molecules with active phosphatide molecules present in the plasma. The various ways of incorporating labelledphospha- tide into corpuscles are described in detail in a paper which is in print (Hahn and Hevesy, 1940). Besides labelled phosphatides, labelled varieties of several acid-soluble organic phosphorus compounds as, for example, those of glycerophos- phate and adenosintriphosphate, are found in the corpuscles. Each of these compounds can be used as an indicator when determining the dilution of the corpuscles of rabbit B in the circulation of rabbit A. It is, however, more convenient to extract the total acid-soluble P and to use the mixture obtained as an indicator. DETERMINATION OF THE BLOOD VOLUME OF A RABBIT WEIGHING 2 kgm a) Making use of the labelled phosphatides of the corpuscles We administered radioactive sodium phosphate of negligible weight leaving the activity of about 0.001 milliCurie to rabbit B. After the lapse of a week, 1 cc. of blood of rabbit B containing 0.32 cc. cor- puscles was injected into the jugularis of rabbit A. After the lapse of 5 min, 50 cc. blood were collected and, after the addition of heparin, centrifuged. The haematocrit value of this sample was found to be 0.33. The phosphatides of the corpuscles were thoroughly extracted by Bloor's method. Their P was converted by wet ashing into phosphate. The phosphate was precipitated as ammonium magnesium salt. Before precipitation, sodium phosphate was added to the solution to obtain a precipitate of about 80 mgm. The activity of the precipitate was then determined by means of a Geiger counter. The comparison of the acti- ADVENTURES IX KADIOISOTOPE llESEARCH 519 vity of the samples is facilitated if they have practically the same weight and it is for this reason that we added to the original sample a compara- tively large amount of non-active phosphate. The corpuscles of 1 cc. of the blood of rabbit B were also extracted with ether-alcohol and the extract treated in the way described above. The activity of the sample thus obtained, was then compared with that of the corresponding sample of rabbit A. Let us denote the injected blood volume by Vj, the volume of the sample collected for analysis from rabbit B and rabbit A, respectively, by Vj and Vjj, and the activity of the two samples obtained by Aj and Ajj; then the blood volume to be determined (X) becomes ^^A,.V...V, A„.V, In some of our experiments, before injecting, for example, 1 cc. blood into the jugularis of rabbit A, we removed 1 cc. In that case, the second term of the equation becomes O. Operations involved in the determination of the total blood volume are thus: measurement of the volume of three samples, and the compa- rison of the radioactivity of two samples. In the above mentioned experiment the corpuscle phosphatides of 1 cc. blood of rabbit B contained 100 relative activity units; the activity of the corpuscle phosphatides extracted from 50 cc. blood of rabbit A was found to be 53.3. From these values it follows that the blood volume of rabbit B amounts to 93.8—1 cc. = 92.8. b) Making use of labelled acid-soluble compounds of the corpuscles We can check the result obtained above by another procedure in which, instead of the labelled phosphatides, the labelled acid-soluble phosphorus compounds are involved. After extraction of the phosphatides, the cor- puscles are extracted with 10 per cent trichloroacetic acid. The P of the filtrate obtained is converted, as described above, into ammonium magnesium phosphate. The activity of the sample obtained from rabbit A is compared with that of the sample from rabbit B, as described above. The figures obtained being 100 and 54.4 respectively, the total blood volume of rabbit A becomes Aii-Vi 54.4-1 The use of labelled acid-soluble P compounds leads thus to practically the same result as obtained with labelled phosphatides as indicators. The blood volume per kgm of rabbit weight was found, in the experi- ment described above, to be 46 cc. In another experiment the value of 38 cc. was found. 520 BLOOD VOLUME DETERMINATION DETERMINATION OF THE BLOOD VOLUME OF A CHICK WEIGHING 135 gm Labelled sodium phosphate was administered to chick B and, 24 hours later, 0.5 cc. blood of this chick was injected into the jugular vein of chick A. The haematocrit values were found to be 0.26 and 0.28, respec- tively. We found the activity of the phosphatides extracted from the corpuscles of 1 cc. blood of chick A to be 7.6, taking that of the phospha- tides secured from the corpuscles of 1 cc. blood of chick B to be 100. The total blood volume of chick A is thus X= 15^^!^ -0.5 = 6.1, 7.6-1 or 45 cc. per kgm weight. LOSS OF LABELLED P COMPOUNDS BY THE CORPUSCLES We wish now to discuss the possible loss of labelled P compounds by the corpuscles during the interval between the injection of labelled corpuscles of rabbit B into the circulation of rabbit A and the securing of blood samples of rabbit A. Such a loss would clearly involve a source of error by leading to too high values for the blood volume to be deter- mined. As to the loss of labelled phosphatides, we found that the labelled phosphatide content of the corpuscles of rabbit A, after the lapse of 1 hour, amounts to 90 per cent of that found after the lapse of 6 min. This result shows that the disappearance of labelled phosphatides from the corpuscles takes place at a slow rate. As to the disappearance of the labelled acid-soluble P molecules from the corpuscles, we find the following result. The labelled acid-soluble P content of 1 cc. corpuscle of rabbit A, 8.5 min after injecting the blood of rabbit B into rabbit A, is, as seen in Table 1 and Fig. 1, within the errors of experiment, identical with that found after 2.5 min. We have now to investigate if, during the lapse of 5 min or more, after which time blood samples in the experiments described in this paper were secured, a uniform mixing of the labelled corpuscles of rabbit B in the circulation of rabbit A took place. From the data contained in Table 1 and Fig. 1 we can conclude that, already after the lapse of 2.5 min., a uniform mixing actually took place. Such a result in the case of a small animal with fast circulation could be expected since, in experiments carried out on human subjects, a uniform mixing of dyestuffs injected into the plasma was found to take place in the course of 6 min. (Rowntree et alia, 1929; Graff et alia, 1931). ADVENTURES IN RADIOISOTOPE RESEARCH 521 Table 1. — Disappeakance of Labelled Acid- soluble P Compounds from the Cokpuscles Time elapsed after injecting the blood of rabbit B into rabbit A Per cent of labelled acid-soluble P injected, present in 1 cc. corpuscle of rabbit A 2.5 min 5 „ 6.5 ,, 8.5 „ 26.5 „ 60 „ 120 „ 210 „ 3.39 3.21 3.39 3.30 3.12 2.88 2.58 1.35 100 .2x> a. a> o a. _3 o s mm. Fig. 1 . Disappearance of labelled acid-soluble P from corpuscles, injected into the circulation of a rabbit. DISCUSSION The sole difference between the normal and the labelled corpuscles is that in some of the molecules present in the normal ones P (having the mass number 31) is replaced by radioactive P (having the mass number 32). In 1 cc. corpuscles of rabbit B, 0.06 mgm phosphatide P was present. Of these 0.06 mgm, lO^^^ mgm were radioactive ^^P atoms. As 1 cc. contains about 3 • 10^ corpuscles, one corpuscle contains on an average 10^^^ mgm 32p or only about one corpuscle in a hundred contained an active phos- phatide molecule. After injecting the blood sample of rabbit B into rabbit A, a strong dilution of the labelled phosphatides took place: only one in about three thousand corpuscles now contains a radioactive phospha- tide P atom. The replacement of a minute percentage of the ^ip atoms by 32p atoms in the P compounds of the corpuscles can hardly influence to any noticeable extent the chemical properties of the corpuscles and 522 BLOOD VOLUME DETERMINATION we can, therefore, claim that, when applying the method described in this note, no non-physiological component is introduced into the cir- culation. As to the i^ -radiation emitted by the ^^P atoms present in the corpuscle phosphatides, the number of /? -particles emitted per minute in the total circulation of rabbit A amounts to only about 1000, while the number emitted by the total acid-soluble fraction amounts to about 30 times that figure. How insignificant these figures are, can best be realized when we envisage that this radiation corresponds to that of only 10^^ and 10"^ gm radium, respectively. When carrying out experiments as those decribed above on human subjects, it may be advisable to make use of the acid-soluble P com- pounds of the corpuscles as indicators. Since, in this case, one may use less radioactive P, such experiments can be carried out on human sub- jects by administering by subcutaneous injection or by mouth to the blood donor sodium phosphate having a /5-radioactivity corresponding to that of about 0.01 milliCurie or even less. Summary A method of blood volume determination based on the determination of the dilution of labelled corpuscles is described. Radioactive sodium phosphate is administered to rabbit B; after the lapse of some days, a known blood sample of this rabbit is injected into the vein of rabbit A. A few minutes later, the cor- puscles of a Ivnown volume of the blood of rabbit A are secured, their phospha- tide content extracted, and its activity measured. Corpuscles of a known blood volume of rabbit B are treated in the same way. From the ratio of the labelled phosphatide P content of the corpuscles of rabbit A and rabbit B, the total blood volume of rabbit A is calculated. An alternative and often preferable determination is based on the comparison of the activity of the acid-soluble P secured from the corpuscle samples of rabbi t A and rabbit B. The blood volume per kgm of rabbit weight is found to be 42 cc, per kgm of chick weight 45 cc. Literature E. V. Behring (1912) Beitr. Z. exp. Ther. 12, 2. C. C Fleischer— Hansen (1928) Studier over Blodvolumen. Copenhagen. S. Graff, O. A. d'Esopo and A. J. B. Tillmann (1931) Arch. Int. Med. 48, 821. L. Hahn and G. Hevesy (1940) Kgl. Danske Vidensk. Selsk. Biol. Medd. (in print). E. Madsen (1934) Acta path, microbiol. Scand. 11, 376. L. G. RowNTREE, G. E. Brown and G. M. Roth (1929) The Volume of the Blood and Plasma in Health and Disease. Philadelphia. Originalh- communicated in Acta Physiol. Scand. 4, 375 (1942). 52. DETERMINATION OF THE RED CORPUSCLE CONTENT G. Hevesy and K. Zerahn From the Institute of Theoretical Physics, University of Copenhagen Some time ago, a method of determination of the red corpuscle content was described (Hahn and Hevesy, 1940). Corpuscles containing label- led phosphorus compounds are introduced into the circulation of the rabbit, for example, and the labelled phosphatide content, or the labelled acid- soluble phosphorus content of the corpuscles of samples, secured after the lapse of few minutes, is compared with the correspond- ing content of the corpuscles injected. Such a comparison indicates to what extent the labelled corpuscles introduced into the circulation are diluted by the corpuscles of the circulation, and it permits thus the calculation of the red corpuscle volume of the rabbit. In the present note, a simplified form of the above mentioned method is described. The modified method is based on the comparison of the total labelled phosphorus content of the corpuscles injected, with the total labelled phosphorus content of the corpuscles secured after the injection took place. The corpuscles are labelled in vitro. A blood sample of the rabbit is shaken in the thermostat for 1 to 2 hours in the presence of labelled sodium phosphate. By this procedure the corpuscles get labelled. The blood containing the labelled corpuscles is then reintroduced into the circulation of the rabbit. This modification of the previously described procedure was worked out in view of a possible clinical appli- cation of the method. LABELLING OF THE CORPUSCLES About 20 cm^ of blood are removed from the rabbit, placed in a flask the walls of which are coated with paraffin, labelled phosphate of neghgible weight (see below) is added, and the bipod is gently shaken in a thermostat for 2 houis at 370,(1) 10 cm^ of the blood are then reintroduced into the circulation of the rabbit. From the remaining blood, two standard samples, each of them weighing about 3 gm, are prepared. ^^' This temperature was chosen to accelerate the penetration of labelled phos- phate into the corpuscles. 524 ADVENTURES IX RADIOISOTOPE RESEARCH 3 minutes after the injection of the labelled blood into the jugular vein, a few cc. of blood are taken from the carotis. Further samples are taken at later times. The heparinized blood samples are centrifuged, the corpuscles are weighed and brought into solution by wet ashing; subsequently, 80 mgm of sodium phosphate are added, and the phosphate content of the solution is precipitated as ammonium magnesium phosphate. The standard samples are treated in a similar way. In view of the much larger activity of the standard samples, only ^j^^ of the solution obtained after ashing the sample is precipitated. Let us denote the amount of corpuscles injected into the rabbit by A, the ratio of the activity of 1 gm corpuscles of the blood injected and of the activity of 1 gm corpuscles secured from the circulation after the injection as B, then the total amount of the corpuscles present in the circulation (X) is given by X— A- B. In some of our experiments, we used ^^P prepared from carbon disulfide which was previously irradiated by a neutron beam. Should some of the ^^p present in the solution obtained by the extraction process be adsorbed by colloidal par- ticles or be present in the solution in another not properly dissolved state, this part of the ^-P will be found after centrifugiag the standard blood sample in the fraction containing the erythrocytes, while in the circulation this part may be taken up by the reticulo-endothehal cells. In order to avoid a possible error due to such an effect, we did not add the ^^P directly to the blood to be investigated but to a small blood sample which was centrifuged at once. The plasma of the last mentioned sample containing ^sp was added to the blood to be shaken in the thermostat. An alternative method is the following one. Labelled phosphate is administered to a rabbit, the blood of which thus becomes labelled. Few cc. of the labelled blood are introduced into the circulation of another rabbit after the lapse of several hours. By this procedure the activation in vitro can be avoided. Is the Label of the Corpuscles Properly Conserved? The method described above is based upon the assumption that the 32P introduced into the corpuscles while the blood containing labelled phosphate is shaken in the thermostat, is not given off during the experi- ment. One might expect that after the introduction of active blood into the inactive circulation, ^^P will leave the corpuscles and get replaced by 2iP atoms of the plasma. Such a process, if taking place at a suffi- cient rate, would clearly frustrate the application of the method. Since, however, a mixing of the blood introduced into the circulation of the rabbit with the circulating blood does not last more than some minutes or less, the blood sample can be secured, for example 3, 5, 7, and 9 minutes after the injection took place. The loss of activity of the corpuscles in experiments in which active corpuscles were shaken in the thermostat with inactive plasma for 12 min was found to amount to 1.5 per cent, only. (Cf. also Hahn and Hevesy 1940). That a possible loss of the labelling ^ap by the cor- puscles does not influence our results is also seen in Table 1, in which DETERMINATION OF IJED CORPUSCLE CONTENT 525 the average of the activity of 1 gm of corpuscles secured from 12 rabbits after 3, 5, 7, 9, and 15 min, respectively, is stated. The values obtained after 5, 7, and 9 min do not differ from each other within the error of experiments (zb 5 per cent). The value obtained after the lapse of 15 min, possibly indicates a slight loss of ^sp by the labelled corpuscles. The fact that, within a time amply sufficient to carry out determination of the corpuscle volun e, the label of the corpuscles remains conserved is due partly to the comparatively slow rate of passage of the phosphate ions 1 hrough the corp\iscle wall, and partly to the fact that the easily renewable (activated) acid soluble P content of the corpuscles is much higher than the corresponding fraction of the plasma. The major part of the ^^p Table 1. — Activity of 1 gj* Corpuscles Secured from Different Rabbits ai Different Times after the Injection of Labelled Blood (Corpuscles) Time in minutes 3 5 7 9 15 Rabbit 100 106 104 B. 2 100 102 95 96 B. 3 100 104 91 89 B. 6 Activity (taking the 100 105 106 97 B. 7 activity 100 107 105 92 B. 8 found after the lapse 100 95 96 110 B. 9 of 3 minutes to 100 102 99 B. 10 be = 100) 100 100 96 B. n 100 93 95 95 C. 1 100 100 97 C. 2 100 97 93 C. 3 100 104 95 - C. 4 Average activity 1100 : 11 1016 : 10 773 : 8 = 903 : 9 = 374 : 4 = =100 = 102 97 100 94 which entered the corpuscles while the blood was shaken in the thermo- stat is present as easily exchangeable organic acid soluble P. Now, the ^2P atoms have the same chance to leave the corpuscles as have the ^^P atoms present in the same state. The ratio of ^ap and ^ip atoms in the corpuscles is, however, much smaller than the corresponding ratio in the plasma. Correspondingly, the chance of a ^^p atom to leave the corpuscles is comparatively small. The corpuscles of 100 gm of rabbit blood contain about 20 mgm easily renewable acid soluble P atoms, while the plasma contains only about 2 mgm P atoms; the ^ap content of 1 gm corpuscles in the labelled blood injected is about the same as the ^sp content of 1 gm plasma. The ^^p atoms have thus a much greater chance to enter the corpuscles than to migrate in the opposite direction. Of course, if an exchange equilibrium of ^^p between plasma and corpuscles is reached, this statement is no longer valid, but — as follows from the 526 ADVENTURES IN RADIOISOTOPE RESEARCH above data — after shaking the blood at 37° for 2 hours, the ^sp : 3ip ratio of the plasma is still about 10 times the corresponding ratio in the corpuscles. Errors due to the Activity of the Plasma We do not inject active corpuscles but active blood into the circulation of the rabbit. The plasma of the rabbit becoming thus active, some active phosphate will penetrate into the corpuscles in the course of the experi- ment, increasing thus the ^^p content of the corpuscles. Such a process may entail a source of error. If, besides the corpuscles injected, labelled corpuscles are formed in the circulation, the value calculated for the erythron from the dilution figures will clearly be found too low. In order to estimate the error due to the above mentioned process the following experiment was carried out. Active plasma is injected into the blood of a rabbit and blood samples are secured at different times. The corpuscles are separated and their activity is determined. The figures obtained (cf. Table 2) show what percentage of the plasma activity enters the corpuscles during the experiment (3 to 12 min), similar values being obtained in further experiments. In our red corpuscle determination, the activity of 1 gm corpuscles injected was about the same as the activity of 1 gm plasma injected, i. e. in the relative units of Table 2 = 1000. Therefore the figures of the 4th column of Table 2 give almost exactly the percentage error of the red corpuscle determination due to the penetration of ^^P from the plasma into the corpuscles in the circul- ation of the rabbit. It is of interest to compare the rate of penetration of ^sp from the plasma into the corpuscles in experiments in vitro with the figures obtained in the above described experiment in vivo. Experiment in vitro Inactive blood was brought to 37° in the thermostat, active plasma of negligible weight was then added and the activity of the corpuscles was determined at different times. After the lapse of 3, 6 and 12 min, respectively, the corpuscles were found to contain 5.4%, 8.8%, and 12.9%) of the activity of the plasma. These figures were corrected for the activity due to adhesion of active plasma to the corpuscles which was found to make out 2%, of the plasma activity. In experiments in vitro in which the plasma activity does not much change during the experiment the activity penetrating into the corpuscles during 3 to 12 min is thus quite appreciable. In experiments in vivo, however, the plasma activity rapidly decreasing after the injection of the active blood, the amount of ^sp penetrating into the corpuscles is much smaller DETERMINATION OF RED CORPUSCLE CONTENT 527 (of. Table 2). If the plasma activity would disappear with the same speed in experiments in vitro as in experiments in vivo we would obtain the figures seen in the 5th column of Table 2. The following example shows how column 5 of Table 2 was calculated. Calculation of the value obtained after 2 min. The average activity of 1 gm plasma during the 3 first minutes can be estimated to be 500. As, after 3 min, in the experiment in vitro 1 gm corpuscles was found to contain 5.4% of the activity of 1 gm plasma, the figure registered in column 5 works out to be 2.7. The average activity between 3 and 6, and between 6 and 12 min, respectively, is estimated to be 200 and 130, respectively. When comparing the activity of the corpuscles with the activity of the plasma, we can calculate the increase in activity of the corpuscles and compare this activity increase with the activity of the corpuscles which we inject in our usual experiments in which blood is shaken with active phosphate in the thermostat before the injection. Table 2. — Penetration of ^^P of the Plasma into THE Corpuscles after Injecting Labelled Plasma into THE Rabbit Percentage of the plasma injected present in tlie Time in min Activity of 1 gm corpuscle Activity of 1 gm plasma corpuscles calculated from found experiments in vitro 1000 3 26 260 2.6 2.7 6 33 158 3.3 3.4 12 40 99 4.0 3.9 Adhesion of Active Plasma to the Corpuscles The activity of the plasma may also influence the results obtained in another way as mentioned above. Centrifuging of blood does not lead to corpuscles entirely free of plasma. After centrifuging blood for 25 min, in a centrifuge making 5000 revolutions per minute, we find the cor- puscles to contain 2% plasma (cf. also Hahn and Hevesy, 1942). In 1 gm corpuscles prepared from the blood to be injected, we shall therefore find only 0.98 gm corpuscles, the remainder being composed of plasma. In the blood to be injected, the activity of 1 gm corpuscles is about equal to the activity of 1 gm plasma, and the error due to the presence of plasma in the blood injected can thus be disregarded. However, other conditions prevail in the blood samples secured from the rabbits at different times. In the last mentioned samples, the adhering plasma is much less active 528 ADVENTURES IN RADIOISOTOPE RESEARCH than the corpuscles to which the plasma adheres. The activity of 1 gm of the corpuscles secured from the active circulation is thus not strictly comparable with the activity of 1 gm of the corpuscles injected. Knowing the activity of the plasma at different times (cf. Table 2), we can correct for this discrepancy. The red corpuscle value calculated without taking regard to the above mentioned source of error will be 1.5%, 1.7% and 1.8%, respectively, too high. We could eliminate the above mentioned sources of error due to the activity of the plasma by removing the active plasma and replacing it by an inactive one. However, this step would complicate the procedure. Not only, however, remains the error due to the adherence of the plasma to the centrifuged corpuscles within the errors of the experi- ment, it is In fact to a large extent compensated by the error due to the penetration of active phosphate from the plasma into the corpuscles during the few minutes which elapse between the injection of the active blood and the collection of the samples. An estimate of the three errors of the experiment, — viz. (1) adhesion of plasma to the centrifuged corpuscles, (2) penetration of ^sp from the plasma into the corpuscles in the circulation of the rabbit (error due to the fact that we do not inject labelled corpuscles but labelled blood), (3) loss of 32p by the corpuscles during the experiment — is seen in Table 3. After the lapse of 3 min., the plasma adhering to the corpuscles is thus less active (per gm) than the corpuscles to which it adheres. Since we determine the weight of the corpuscles + adhering plasma, and the adhering plasma (per gm) is only l^ as active as the corpuscles, we find the activity of the corpuscles too low, the error being 2—2 • 1/4 = 1.5 per cent. Correspondingly, after 5 and 7 min, respectively, we commit an error of about 1.7 per cent. Table 3. — Estimate or Difeeeent Eeeoes of Expeeiment in the Deteemination of the Red Coepuscle Volume Time in min Percentage error due to adherence of the plasma to the cor- puscles 3 6 12 + 1.5 + 1.6 + 1.8 Percentage error due to intrusion of ^"P of the plasma into the corpuscles -2.7 -3.4 -3.9 Percentage error due to the loss of '*P by the corpuscles + 0.5 + 1 + 1.5 RESULTS As seen in Table 4, the corpuscle content of the rabbit per kgm of fresh weight varied between 20.5 and 26.5 gm. It is difficult to decide whether an erythrocyte reserve present in the spleen and some other organs DETERMINATION OF RED CORPUSCLE CONTENT 529 interchanges with the erythrocytes of the circulating blood and, corres- pondingly, to what extent it participates in the dilution of the labelled corpuscles. Since our experiments were carried out with narcoticized rabbits and, as shown by Barcroft (1934), in ether narcosis the blood reserves of the body are to a very large extent released, our results are independent of the above mentioned source of error and indicate the total erythrocyte content of the rabbits investigated. Table 4 Rabbit Weight in gm Weight of corpuscles injected Activity per gm corpuscles injected Activity per gm corpuscle sample*!' Total cor- puscle con- tent in gm Corpuscle content in gm per kgm rabbit weight B. 2 . B. 3 . B. 6 . B. 7 . B. 8 . B. 9 . B. 10 . B. 11(2) 3050 3.40 2006 108 63.1 2290 2.66 2470 140 46.9 2470 3.98 1660 115 57.5 2550 3.71 2160 153 52.4 3200 3.38 2040 83.3 82.9 1990 3.06 2450 181 41.4 3160 3.10 2102 99.7 65.4 2010 3.23 2160 240 29.1 20.7 20.5 23.3 20.6 25.9 20.8 20.7 14.5 ^1^ Average of samples secured between 3 and 9 min (comp. Table 1). ^2) Anemia following previous operation. Hematocrit value — 22.5. Radio-Phosphorus and Radio-Iron as Indicators Hahn, Balfour et al. (1941) determined the corpuscle volume of the dog, using radio-iron as an indicator. Within a few days after admi- nistration of radio-iron to dogs, most of the radio-iron present in the body is concentrated in the erythrocytes as a constituent of the hemo- globin molecules. Such erythrocytes labelled by the presence of radio- iron were used in the same way to determine the red corpuscle volume of the dogs as corpuscles labelled by the presence of radiophosphorus were applied by Hahn and Hevesy (1940) and by the present writers to determine the red corpuscle volume of the rabbit. In the determin- ation of the red corpuscle volume, the radio-phosphorus method has the following advantages. Blood samples can be activated in vitro, while such a procedure cannot be carried out when using radio-iron. Corpus- cles containing labelled haemoglobin can only be obtained in experi- ments in vivo. Furthermore, radio-phosphorus is very much easier to procure than radio-iron, since the preparation of radio-iron in sufficient quantities requires powerful tools in contrast to the preparation of radio-phosphorus. 34 Hevesy 530 ADVENTURES IN RADIOISOTOPE RESEARCH Summary A simplification of the method of determination of the red corpuscle content previously communicated is described. A blood sample taken from a rabbit is shaken in the thermostat at 37° for about 2 hours in the presence of labelled sodium phosphate and, then, it is rein- troduced into the circulation of the rabbit. A comparison of the total labelled phosphorus content (radioactivity) of the corpuscles injected with the total label- led phosphorus content of corpuscle samples secured, few minutes after the injection took place, leads to the value of the red corpuscles. The red corpuscle content of the rabbits investigated varied between 20.5 and 26.5 gm per kgm of rabbit weight. References J. Barcboft (1934) Features in the Architecture of Physiological Functio7i. London. L. Hahn and G. Hevesy (1940) Acta Physiol. Scand. 1, 1. L. Hahn and G. Hevesy (1941) Ibid. 2, 347. L. Hahn and G. Hevesy (1942) Ihid. 3, 1£03. P. F. Hahn, W. M. Balfour, J. F. Ross, W. F. Bale and G. G. Whipple (1941) Science 93, 87. Originally published in Arkiv for Kemi 3, 425 (1951). 53. THORIUM B LABELLED RED CORPUSCLES G. Hevesy From the Institute for Research in Organic Chemistry, Stockholm The labelling of red corpuscles is an important application of radio- active indicators as labelled erythrocytes are applied among others in circulation studies and in the determination of blood corpuscle (blood) volume^^). Labelling of red corpuscles with ^^P is made possible by the presence of comparatively large amounts of labile acid soluble phosphorus in the erythrocytes. Much of the ^^P which intrudes from the plasma into the red corpuscles speedily participates in the glycolytic and other enzymatic processes taking place in the erythrocytes. This participation involves incorporation into labile organic phosphorus compounds and thus "dilution" of the intruded ^sp. As a result of this "dilution" if in the course of 1 hour 1 mgm intruding phosphorus carries 1 //curie into the corpuscles 1 mgm phosphorus moving from such corpuscles in the opposite direction carries into inactive plasma an appreciable smaller activity which amounts to about ^12 or ^^ss of 1 //curie only. Interchange of phos- phate between plasma and red corpuscles is a continuous process not influenced by the presence of added labelled phosphate, the latter having a negligible weight. Injected into the human circulation, such labelled red corpuscles will lose 8 per cent or less of their activity in the course of 1 hour. In the course of 20 minutes the loss is certainly less than 3 per cent. This time interval is in most cases sufficient to carry out circulation velocity or blood volume measurements. In a similar way, red corpuscles can be labelled by adding ^'-K to a blood sample and shaking it in a thermostat for an hour or two^^^. In this case — due to the much higher potassium content of the erythrocytes — the intruded ^^K becomes "diluted" by endogenous potassium in a similar way as intruded ^^P gets "diluted" by endogenous phosphorus and correspondingly the ^^K labelled corpuscles injected into the circulation lose less than 4 per cent of their activity in the course of 1 hour. ^1^ cf. G. Nylin, Svenska Vetenskapsakad. Arkiv Kemi A 20, No. 17 (1945). ^2^ G. Hevesy and G. Nylin, Acta Physiol. Scand. in print. 34* 532 ADVENTURES IN RADIOISOTOPE RESEARCH In some pathological cases with impaired circulation, mixing of the injected and circulating red corpuscles takes place at a much slower rate than under physiological conditions and accordingly it may be desirable to follow the path of the labelled erythrocytes in the circulation for many hours. Such observations can not be carried out by making use of ^sp or 42K labelled red corpuscles. Recently 5iCr was suggested as labelling agent of erythrocytes. Washed red corpuscles, labelled with Na./iCr04 in vitro, were injected intra- venously into clogs and were found to retain their activity without significant loss to the plasma for approximately 24 hours. The previously determined red corpuscle volume could be re-determinated within 5% for approximately 24 hr.^i^ Neither ^-particles nor positrons are emitted by ^^Cr but X-rays and y rays of low intensity. These can be measured very conveniently by making use of a crystal counter which, however, requires expert assistance, in contrast to the running of a Geiger counter. Future progress in the measuring technique may eUminate the diffi- culties encountered today by a clinical institution, when trying to apply red corpuscles labelled with ^^Cr in corpuscle volume measurements. In this note a labelling method of red corpuscles is described which permits to produce tagged erythrocytes which in the course of many hours lose only minute amounts of their radioactivity. The method is based upon the introduction of thorium emanation (thoron) into the blood sample. The emanation, which has a half-time of 55 sec, penetrates speedily into the red corpuscles, decays inside them producing the active deposit of thorium which remains even for many hours to a very large extent fixed in the red corpuscles. The sequence of the radioactive disintegration products of thorium emanation (Tn) is the following: Tn — ^ThA >ThB >ThC ^ThC 55 sec 0.16 sec 10.6 hr 60.5 min I ThC" 3.1 min The ThA decays with a half-time 0.16 sec and can thus be disregarded. ThB, however, which has a half-time of 10.6 hr, comes soon in exchange equilibrium with the following disintegration products. Consequently the activity of the erythrocytes decays with the disintegration period W S. J. Gray and K. Sterling, 1950 (A. E. C. U. - 1072); K. Sterling and S. J. Gray, 1950 (A. E. C. U. - 1026). THORIUM B LABELLED RED CORPUSCLES 533 of ThB. This radioactive body emits soft ^-rays, its disintegration pro- ducts hard y5-rays. One of these ThC" emits, furthermore, very hard y-rays. The approximate half- value thickness of the /5-rays emitted by ThB and its disintegration products in aluminium is the following: ThB (lead isotope) 1.6 xlO"! ^m ThC (wismuth isotope) 1.2 mm ThC" (thallium isotope) 7.7xlO"i mm The half value thickness of the y-ray emitted by ThC" is 17 cm. As the ^-rays emitted by ThC have almost the same hardness as those emitted by ^^p^ they are very conveniently measurable. In our experiment, a stream of oxygen after having brushed over the surface of a racliothorium preparation of about 1 mC activity was led through 4 ml of rabbit blood. Plasma and red corpuscles were then separated, washed 2 to 3 times with inactive plasma and then shaken with inactive plasma of the same rabbit for 2 hr at 37° in the ther- mostat. Corpuscle and plasma samples were dried at 60° and 50 mgm of each sample placed in an aluminium dish; their /5-activity was then determined with the Geiger counter. We determined the water content of each cor- puscle and plasma sample and could thus calculate from the activity figures of the dry samples the activity of fresh plasma and fresh cor- puscles. The thorium emanation penetrates rapidly into the red corpuscles. It decays very rapidly as well and we soon find 1 gm of corpuscles to contain twice or more of thorium-active deposit than 1 gm of plasma. These figures depend on the velocity of the oxygen stream and on other experimental conditions. Through decay of thoron thorium B is formed in the plasma as well and a part of this thorium B is also taken up by the erythrocytes. We found that after 2 hr shaking of active corpuscles with inactive plasma the corpuscles contained 50,560 counts but the plasma contained only 170. Thus the red corpuscles gave off only 0.34% of their activity in the course of 2 hr. The maximum loss was shown by a corpuscle sample containing 87,800 counts, with 403 counts in the corresponding plasma. In this case the loss was 0.46%. All these activities were measured 24 hr after leading thoron into the blood, thus after more than 2 decay periods of ThB have already elapsed. Strong corpuscle activities can thus be obtained by leading thoron for a few minutes only through a blood sample. That the erythrocytes so well retain their content of ThB and its disintegration products is partly due to the fact that much of the active deposit of thorium, present in the red corpuscles, is in a colloidal or protein 534 ADVENTURES IN RADIOISOTOPE RESEARCH bound state. Much of the decay of thorium emanation leads to formation of such thorium B particles. That much of the activity of the red corpuscles was due to colloidal or protein bound particles was shown by making use of the fact<^) that on a zinc plate, dipped into a solution containing colloidal ThB (lead isotope) or ThC (wismuth isotope), much more activity accumulates when acidifying the solution. WhenO.Sgmof corpuscles were hemolyzed by adding the same volume of water and one half of that volume was shaken after immersion of a zinc plate (2x2 mm) for 5 minutes, the washed zinc plate had an activity of 3540 counts per min. When first adding 0.5 ml of 1 N HCl to the other half of the hemolysate and then repeating the experiment, the zinc plate had an activity about 4 times as large, namely 14,897 counts per min than in the first mentioned experiment. The addition of acid con- verted much of the colloidal and protein bound active deposit into the ionic state. It follows that about 3/4 of the active deposit present in the corpuscles was in a colloidal or protein bound state. The reults of blood volume determinations obtained by Nylin and the present writer in a clinical investigation in which thorium B labelled red corpuscles were applied, will be shortly published. Summary By leading thoron (thoi'ium emanation) through blood for a few minutes, red corpuscles labelled by the presence of the active deposit ot thorium are obtained. The red corpuscles of the rabbit conserve their activity for several hours, less than 0.5 per cent being lost when shaking the active erythrocytes with inactive plasma for 2 hr at 37°. ^i^G. B.EVESY, Sitzungsber. Wiener Akademie der Wiss. 127, Abt 2 a. (1918). THORIUM B LABELLED RED CORPUSCLES 535 Comment to papers 51— 53 In the first investigation (1939) on the labelling of red corpuscles with^^p^ labelled sodium orthophosphate was added to rabbits blood (paper 49). The penetration of 3-P into the red corpuscles was found to be a fairly slow process; most of the penetrated ^^P was, however, found to be present very shortlj^ after penetration in the labile acid soluble phosphorus fractions of the erythrocytes. This distribution of the penetrated ^^F, in a pool of phosphorus compounds much larger than repre- sented by the inorganic phosphate of corpuscles, make it possible to label red corpuscles fairly stably and apply these tagged erythrocytes in blood csorpuscle volume determinations. If after incubation for 1 — 2 hr the inorganic phosphate of the red corpuscles alone took up substantial amounts of ^^P, after injecting such a sample into an inactive circulation, the ^^p would soon be lost. As the con- centration of inorganic phosphate in the red corpuscles is lower than in the plasma, the exodus of ^^P would even take place in the inactive circulation at a more rapid rate than its incorporation in the course of its in vitro incubation. The partici- pation of a large labile, and to some extent of a non-labile acid-soluble pool of phosphorus compounds in the uptake of ^^P, brings down the ^^P loss after injection of the labelled erythrocytes into inactive circulation to from one-twentieth to one-thirtieth of the loss which we would observe in the absence of such a pool. Moie recent investigations by Goukley (1952) and by Gerlach (1954) lead to the result that a part of the labelled acid-soluble phosphorus is already synthetized under participation of plasma phosphate in the course of the entrance of ^^p into the erythrocytes. One day after subcutanous injection of labelled phosphate to human subjects, the acid-soluble phosphorus of the corpuscles was found to contain almost twice as much ^^p than the plasma, while the plasma phosphatides contained only about one-third and the red corpuscle phosphatides, which are renewed at a slow rate, only about one-fortieth. These were the first experiments (1939) on the labelling of human erythrocytes (paper 49). In mYro experiments in which human blood was incubated with sodium phosphate containing ^^p were first carried out shortly afterwards by Eisenman (1940). In the last mentioned experiments, a decrease of the incorporation of ^sp into the red corpuscles with decreasing temperature was observed. A very strong dependency of the rate of incorporation of ^^P into the erythrocytes of the rabbit was later ascertained (paper 50), the energy of acti- vation of this process being found as high as 15,000 cal. References A. I. Eisenman, L. Ott, P. K. Smith and A. Winkler (1940) J. Biol. Chem. 135, 165. E. Gerlach, A. Fleckenstein and K. J. Freundt (1954) Arch. Qes. Physiol. 263, 682. D. R. H. GouRLEY (1952) Arch. Biochem. Biophys. 40, 1. 536 First published in Nature, 134, 879 (1934). 54. ELIMINATION OF WATER FROM THE HUMAN BODY G. Hevesy, E. Hofer From the Institute for Physical Chemistry, University of Freiburg Shortly after the first application of radioactive isotopes as indicators, the late H. J. G. Moseley and one of the present writers discussed the prospect opened by the introduction of this method, when indulging in a cup of tea at the Manchester Physics Laboratory. The latter then expressed the wish that an indicator might be found which would allow one to determine the fate of the individual water molecules contained in the cup of tea consumed. Even a man of the vision and outlook of the late H. J. G. Moseley considered this hope to be a highly Utopian one. The recent work of Urey and his collaborators brought, however, the above-mentioned wish within the range of realisation. Although diplogen and hydrogen, unlike the atoms of radioactive isotopes, are not practically inseparable by chemical means, yet if we add to a cup of tea a slight amount of heavy water and then find, for example, one per cent of the latter in the water which has left the body, we can assume that about one per cent of the 'normal' water molecules taken in with the cup of tea has shared the same fate. That heavy water present in high dilution in the organism behaves like light water is borne out by the fact that the heavy water content of urine and other excreta is the same as that of ordinary tap water, within a limit of 1 : 100,000 as found by us and other experimenters. ^i^ If we slightly increase the heavy water content of the normal water we can assume that, with an accuracy sufficient for our purposes, the heavy water will show the same behaviour as the normal one. As a further argumxcnt in favour of this view, we may quote the results obtained when investigating the behaviour of highly diluted heavy water in the body of fishes. *^2) Our first step was to investigate, if water prepared from urine has the same density as the tap water drunk. The result was within 1 : 10^ in the affirmative. The preparation of water from urine was carried out by combined absorption and distillation processes. 55 samples of urine and other excreta were investigated and more than 1000 distillation ELIMINATIOX OF WATER FROM THE HUMAN BODY 537 Table 1. — Density of Water Prepaeed from Urine AFTER THE INTAKE OF DILUTED HEAVY WaTER Time eUipsed since the intake of water started in hours Urine (volume passed in cc.) Density difference bet- ween water prepared from urine and 'normal' (dis- tilled) water 5 130 190 230 210 230 290 160 80 120 130 290 320 140 210 820 1120 2100 6 X 10-« 10 0.8 1 15 1.2 21 1.5 23 1.8 25 9 21 •'> 5 20 4 18 8 20 10 18 17 20 23 19 24.5 18 42 19 67 17 92 17 244 10 340 8 processes carried out. One of us took then in one experiment 150 cc. and in another 250 cc. water containing 0.46 per cent heavy water showing a density difference against normal water of 480 X 10~^. As the increase in density of the urine obtained after the intake of these quantities was only a few units in a million, an experiment was made in which 2000 cc. were taken. The increase in the density of the water obtained was then up to 25 : 10^. Some of the results are seen from Table 1. From the above figures, it follows that after half an hour from the beginning of the intake of water, some of the water drunk is found in the urine, though only 0.2 per cent of the amount taken. The bulk of the water leaves the body at a slow rate and it takes 9 i 1 days before half of the water taken has left the body. We controlled the water balance during the experiments and found (in hot summer weather) that on an average 60 per cent of the water lost, left the body through transpiration and evaporation. In the posses- sion of these data, and as we find that the density of urine water and transpiration water is the same within the limits of our accuracy relevant for these considerations ( J:: 5 per cent of the density excess), we can calculate the time which elapses before half of the water taken left the 538 ADVENTURES IN RADIOISOTOPE RESEARCH body by an independent method. The result works out again as 9 J^ 1 days. By dividing the last figure by In 2, we get for the average time a water molecule spends in the body 13 zh 1-5 days. To explain this comparatively long time, we have to assume that most of the water taken becomes completely mixed with the water content of the body. This assumption can be tested by calculating the water content of the body of the experimenter from thea mount of diluted heavy water taken, and the density of the water prepared from urine any day except the first one. We arrive at a water content of 43 ± 3 litres, namely, 63 i 4 per cent in fair accordance with known data. References 1. H. J. Emeleus, F. W. James, A. King, T. G. Pearson, R. H. Purcell and H. V. A. Briscoe, J. Chem. Soc. 1204 (1934). 2. G. v. Hevesy and E. Hofer, Hoppe-Seyler Z. 225, 28 (1934); cf. G. N. Lewis, Science 19, 151 (1934); H. E. Erlenmeyer and H. Gartner, Helv. chim. Acta 17, 334 (1934). ELIMINATIOX OF WATER FROM THE HUMAN' BODY 539 Comment on paper 54 Shortly after the first application of radioactive isotopes as indicators, in the spring of 1913, the late H. J. G. Moseley and the present writer discussed the prospect opened by the introduction of this method, when indulyinq in a cup of tea at the Manchester Physics Laboratory. The latter then cxpicssed the wish that an indicator might be found which would allow us to determine the fate of the individual water molecules contained in the cup of tea consumed. Even a man of the vision and outlook of the late H. J. G. Moseley considoiod this hope to be a highly Utopian one. It was the discovery of heavy water by Urev, which biought the above-mentioned wish within the range of realization. From the dilution figure of a known volume of administered labelled water, the water content of the organism was evaluated . This was the first application of iso- topic tracers in clinical studies, and the first application of the device of isotope dilution in life sciences [paper 54 and Hevesy and Hofer, (1934) where a more detailed presentation of the results obtained is given]. At present, not heavy water but hyperheavy (tritiated) water is mostly used in such investigations. From the water content of the organism, conclusions can be drawn among others as to its fat content, as shown recently at the Donner Laboratory by Prentice et al. (1951). Assuming the lean body to contain 73 per cent of water, the fat content is calculated according to the formula: per cent body water = 0.73 (100 — per cent fat) . References G. Hevesy and E. Hofer (1934) Klin. Wochenschr. 13, 1524. T. C. Prentice, W. E. Siri, N. I. Berlin, G. M. Hyde, R. J. Parsons, E. F. Joiner and J. H. Lawrence (1952) J. Clin. Inv. 3, 412. Originally published in D. Kgl. Danske V idenskahernes Selskab. Biologiske Meddelelser. 14, 3 (1939). 55. EXCRETION OF PHOSPHORUS G. Hevesy, L. Hahn, O. Rebbe From the Institut of Theoretical Physics, University of Copenhagen Because of the great importance of phosphorus in the formation of bones and the functional significance of a great variety of phosphorus compounds the balance of phosphorus intake and excretion has been investigated in numerous cases. A vast literature on this subject is available in which often also the route of secretion is considered, that is, the ratio in which the excreted phosphorus is to be found in the urine and faeces of the human subject or animal investigated. What percentage of the phosphorus excreted in the faeces is due to non-absorbed material and how much to phosphorus, originating from the body proper is, however, not yet known. Neither is any statement to be found on the fate of the individual phosphorus atoms, for example the phosphorus taken up with the food on one certain day. By using radioactive phos- phorus as indicator we can follow the circulation of the phosphorus taken up at a certain date with food, the route it takes, and the rate at which it leaves the body. Some information on this subject has already been given. (i) In this paper we are communicating the results of investi- gations in which the excreta of human subjects, produced in the course of few months, were investigated both by radioactive and by chemical methods. Data are also given on the phosphorus excretion of rats. GENERAL EXPERIENCE AS TO PHOSPHORUS EXCRETION Ingested phosphates are excreted partly in the faeces and partly in the urine, the ordinary distribution in adult man being about two thirds in the urine and one third in the faeces. Conditions that diminish the solubility or promote the precipitation of phosphorus in the intestinal canal, tend to reduce the amount excreted in the urine and to increase ^1^ O. Chiewitz and G. Hevesy, Nature 136, 754 (1935); Kgl. Danske Vidensk, Selsk. Biol. Medd. 13, 9 (1937); L. Hahn, G. Hevesy and E. Lundsgaabd, Biochem. J. 31, 1706 (1937); W. E. Cohn and O. M. Gbeenberg, J. Biol. Chem. 123, 185 (1938). EXCRETION OF PHOSPHORUS 541 that in the faeces. Vice versa, anything that favours solubihty of phos- phate in the alimentary tract, augments absorption and increases urinary phosphorus at the expense of the faeces. Thus, diets high in calcium and low in phosphorus lead to high faecal output and phosphorus defi- ciency, probably because the phosphate forms an insoluble precipitate of calcium phosphate in the intestine. It has often been observed that fatty acids, by diverting calcium from phosphoric acid, may release the latter for absorption. Anything which tends to produce a more acid medium in the intestine, exerts a favourable influence on the phosphorus absorption. Thus the ingestion of hydrochloric acid increases the urinary phosphorus at the expense of the faeces. The daily excretion of phosphate in the urine of an adult in normal conditions varies from 0.3 to 2 gm of P. A careful determination of the average daily phosphorus intake^^^ of 25 college women has shown an intake of 1.40 gm, which is thus somewhat higher than required by the Sherman Standard (1.32 gm). In experiments^^), in which the subjects used, were students and an acid- forming diet containing 780 gm, milk was administered, the daily phos- phorus intake was found to be 1.98 gm. When as large an amount as 10.8 gm P was administered to a human subject, an output of 8.9 gm was found, 79% of the latter being present in the urine and 21 % in the faeces. About one fourth of the phosphate fed was stored^^\ In the early hours of the day the rate of excretion in the urine is minimal^'*), and then it rises during the course of the day, to reach a maximum at 4 or 5 in the afternoon. The level of excretion is then maintained for the remainder of the day and throughout the night. Within wide limits, there is no relationship between the amount of urinary phosphate and urinary volume. The rate of phosphate excretion is independent of the rate of water elimination even when, owing to copious diuresis, the urinary phosphate is below the level of the plasma phosphate<^). As to the phos- phorus excretion in animals, we wish only to mention the following data collected by us. Rats weighing about 230 gm excreted daily 28.7 mgm P; within 7 weeks the ratio urine P: faeces P varied between 1.3 and 2.4, the average being 1.6. ^1^ R. E. Havabd and G. A. Reay, Biochem. J. 20, 99 (1926). ^2> M. M. Kramer, M. T. Potter and J. Gillum, J. Nutrit. 4, 105 (1931). (3) \v. T. Salter, R. F. Farquharson and D. M. Tibbets, J. Clin. Inv. 11, 395(1932). (*) Comp. C. H. FisKE, J. Biol. Chetn. 49, 171 (1921); S. Bellac, J. Chaussin, H. Langer and T. Ransox, C. R. 207, 90 (1938). (5) R. E. Havabd and G. A. Reay, Biochem. J. 20, 99 (1926). 542 ADVEXTURES IN RADIOISOTOPE RESEARCH EXCRETION EXPERIMENTS The most suitable method of analysis of urine was found to be the following: Evaporate to dryness an aliquote, preUminarily treated with fuming nitric acid and determine its activity. Another smaller known fraction is digested in a Kjel- dahl flask and its P content determined by the method of Fiske and Subbarow. The method tried first, based on the precipitation of ammonium magnesium phosphate from the urine, was found to be unsatisfactory, as activity measure- ments have shown that a part of the inorganic P present in the urine, remains in solution after precipitation with magnesia or the magnesium citrate reagents. In view of the low activity of many of the faeces samples, we had to work up seve- ral gm of dry faeces. It was too troublesome to dissolve such a comparatively large amount; we have therefore chosen the following procedure: The sample was first treated with nitric acid and then dried on a sand bath below 300°, to avoid loss of phosphorus. The activity of the substance was then determined. Another known part of the faeces sample, in most cases weighing only 30 mgm, was dige- sted in a Kjeldahl flask and its P content determined by the colorimetric method. Table 1 Time after administration of labelled P Volume of urine in cc. Specific activity of urine P (% of the labelled P admi- nistered found in 1 mgm P) 3 5 7 10 11 22 27 34 44 3 6 8 13 16 26 32 43 hours days 130 0.0051 80 0.015 110 0.0109 156 0.0059 88 0.0052 348 0.0033 170 0.003S 140 0.0024 570 0.0017 0.0013 0.00056 0.00064 average 0.00052 950 0.0005 0.0006 0.00027 0.00016 Excretion of labelled phosphorus through the kidneys In the experiment described first, the urine of a 40 years old female patient suffering from diabetes was investigated. The labelled sodium phosphate of negli- gible weight was given with a glass of millc. We intended originally to investigate the excretion of the above mentioned patient after treatment with insulin as well. However, because the patient was soon discharged from the hospital, this investig- ation could not be carried out. EXCRETION OF PHOSPHORUS 543 The average daily P excretion in the urine was found to be 950 mgni, the maxi- mum of the specific activity of the urine P is reached after 5 hours (comp. Table 1 ). The rapid decrease of the specific activity of the urine P, in the later stage of the experiment, is due to the rapid decrease of the plasma P activity with time; the labelled phosphate ions of the plasma being replaced by unlabolled ones ahea(i\' present in the body, primarily in the skeleton and the muscles. The specific acti- vity of urine P, which is derived from the plasma inorg. P, first increases with time, due to the increased absorption of the labelled P administered into the circulation. Besides, by interaction with ti.ssue phosphate, the plasma inorganic phosphate also becomes "diluted" by unlabellcd P absorbed into the circ;ulation from the food taken. The latter will therefore also influence the specific acti\it\' of the urine P. In the experiment recorded in Table 2, the urine of a 22 years old male subject was investigated. The subject took only a minimal amount of food and drink. His daily urine excretion amounted to 610 cc, only containing 660 mgm P. Table 2 Time since administ of the labelled P in 'ation hours Volume of urine in cc. Specific activity of urine P 3 75 0.0158 5 52 0.0205 7 59 0.0103 9 38 0.0090 11 54 0.0063 18 180 0.0049 23 150 0.0035 25 46 0.0026 27 50 0.0027 30 115 0.0032 33 80 0.0026 39 145 0.0022 48 175 0.0015 51 110 0.00097 In the course of the first day 6.8%, in the course of the second day 2.3% of the labelled P was excreted through the kidneys. Higher figures were found in the experiment on p. 542, In the experiment recorded in Table 3, we wanted to ascertain the amount of labelled P excreted after a very short time. The male subject, 23 years old, excreted daily 750 cc. urine containing 825 mgm P. Table 3 Time since administration of the labelled P Volume of in cc. urine Specific activity of urine P 20 min 90 16 520 00076 45 min 0.008 18 hours 0.0073 544 ADVENTURES IN RADIOISOTOPE RESEARCH After the lapse of 20 minutes, an easily detectable part of the labelled phosphorus, was thus fovind in the urine amounting to 0.1 per cent of the labelled P administered. From the rate of urine production by the subject in question, we can conclude that only about 10 cc. urine were produced in the course of the experiment, 80 cc. being already present at the beginning of the experiment in the bladder. By taking account of the latter, the specific activity of the P found in the urine formed within the first 20 minutes, work out to be 0.0068, i. e. each mgm P found in the urine, produced within the first 20 min after drinking the labelled sodium phosphate solution, contained 0.0068 per cent of the phosphorus atoms present in the latter. Table 4 Time after administration of the labelled P in hours Volume of urine in cc. Specific activity of urine P 4 125 0.047 18.5 590 0.0109 25 100 0.0043 28 87 0.0037 30.5 70 0.0035 34 130 0.0028 35 57 0.0026 38 160 0.0032 45 150 0.0028 75 800 0.0018 The fourth male subject investigated, 50 years old, excreted daily 730 cc. xirine containing 790 mgm P. The daily excretion was high, namely 21.1% per cent in the course of the first and 6.4 per cent in the course of the second day. The specific activities are seen in Table 4. In another set of experiments 5 cc. of a physiological sodium chloride solution were injected into the veins of each of twelve human subjects. The solution con- tained 1 mgm P as sodium phosphate, and also ^sp showing an activity of 10~5 milliCurie. We carried out these experiments to find out if the percentage of the activity, excreted within the first 24 hours tlirough the kidneys, varies much Table 5a. — Labelled P Administered per Mouth. Age OF Female Subject 28 Years, Weight 65 kgm Time after adminis- tration of labelled P in days Total volume in liter Total P content in gm % of total activity administered, present in urine 0—15 15—39 39—52 52—76 14.98 25.25 12.98 25.58 7.787 14.400 8.239 9.093 10.3 5.3 3.6 1.5 Excretion of labelled P in the course of 76 days 20.8 per cent. Total P excretion in urine 39.519 gm. Daily P average excretion 0.520 gm. EXCRETION OF PHOSPHORUS 545 Table 5h. Faeces Time after adminis- tration of labelled P in days xVmount of dry faeces in gm Total P content in gm % of total activity administered, present in faeces 0— 9 265,7 3.600 4.44 0—15 640.2 4.840 5.46 15—33 900.5 7.185 0.98 33—41 333.1 2.135 0.14 41—53 438.6 2.744 0.08 Excretion of labelled P in the course of 53 days 6.7 per cent. Total P excretion in faeces 16.924 gm. Daily average P excretion 0.320 gm. Table 6a. — Labelled P Administered per Subcutaneous Injection. Age of Female Subject 20 Years, Weight 65 kgm at the beginning of the experiment and 70 kgm at THE End. Time after adminis- tration of labelled P in days Total volume in liter Total P content in gm % of total activity administered, present in urine 0—16 18.75 13.00 8.0 16—40 24.79 16.91 3.1 40—53 15.25 16.17 1.7 53—77 27.20 12.24 1.5 Excretion of labelled P in the course of 77 days 14.3 per.cent. Total P excretion in urine 52.32 gm. Daily P excretion 0.680 gm. Table 66. Faeces Time after adminis- tration of labelled P in days Amount of dry faeces in gm Total P content in gm % of total activity administered, present in faeces 0— 6 6—17 17—38 38—45 132.6 572.6 636.5 214.7 2. .585 9.360 7.070 2.815 0.60 0.84 0.12 0.10 Excretion of labelled P in the course of 45 days 1.7 per cent.. Total P excretion in faeces 19.245 gm. Daily P excretion 0.447 gm. from individual to individual. Though the human subjects were kept on the same diet, both the total P content and also the activity excreted within the first 24 hours varied markedly from individual to individual, as seen in Table 7. 35 Hevesy 546 ADVENTURES IX RADIOISOTOPE RESEARCH Table 7. — % or Labelled Sodium Phosphate, Administered PER Intravenous Injection to Human Subject, Excreted Within the First 24 Hours through the Kidneys Human subiect Weight in I Volume of urine in cc. Total P in mgm % of labelled P recovered Specific activity of faeces P A 75 425 630 14.5 0.023 B 75 1515 1370 20.0 0.015 C 50 716 792 19.0 0.024 D 66 926 864 12.4 0.015 E 73 850 994 23.0 0.023 F 56 833 373 9.9 0.027 G 62 790 839 8.0 0.096 H 71 1164 1061 8.5 0.0080 I 80 1100 561 4.0 0.0071 J 61 632 707 14.4 0.020 K 64 420 518 8.9 0.017 L 71 1405 703 12.3 0.018 Excretion of labelled phosphorus through the bowels and the kidneys In the case of 2 female subjects, we investigated the excretion in urine and faeces over a period of several weeks. The results are seen in Tables 5a and 5b, resp. 6a and 6b. In the first experiment (Table 5), the labelled phosphorus found in the faeces was partly non- absorbed P and partly such originating from the body proper. In the second experiment, registered in Tables 6a and 6b, the labelled P being not given by mouth, the labelled P present in the faeces must have originated solely from the body phosphorus and got through the digestive juices into the faeces. The lower total phosphorus excretion in the last mentioned case (Table 6), is presumably partly due to the remarkable increase in weight of the subject in question during the experiment. Comparison of excretion through the bowels and the kidneys From the labelled P administered by mouth, in the course of two months 6.7% were excreted in the faeces. When given by subcutaneous injection about 1.7% left through the bowels. The latter must have reached the intestinal tract with the digestive fluids. These carry labelled P just as well, when the latter was administered by mouth; we have, therefore, to assume that somewhat less than \ of the 6.7% labelled P found in the faeces originated from the body proper. The same ratio was found in our former experiments^i\ while the absolute amount excreted in the course of the first week was in those cases 2.5 times as high as found in the present cases. The labelled phosphate which left the body unabsorbed was, therefore, 6.7%— 1.7% — 5.0%. We will now turn our attention to the result ^1^ O. Chiewitz and G. Hevesy, loc. cit. EXCRETION OF pnospnoKus 547 of chemical analyses which indicalo the excretion of total P contained in the diet of the subjects. From the total P of the diet excreted 33% and 35% left the body through the bowels in two experiments, thus a decidedly higher figure than found for the excretion of the labelled sodium phosphate. It is also higher than found in a for- mer case for the amount of labelled P which left the body absorbed (13%). To account for this discrepancy two different explanations can be put forward. Accord- ing to one explanation, phosphorus present in some of the organic phosphorus compounds of the food, is less effectively resorbed than the labelled inorganic phos- phorus added to the food. Such P is only split off in the lower region of the intesti- nal tract, in which place it has more chance to form insoluble calcium phosphate, for example, than in the more acid tipper region. An alternative explanation is, that it is not the binding of the phosphorus in the compound which matters, but the mechanical protection of the phosphorus compounds present in the food. From solid undigested particles, the phosphorus particles will not be leached out properly. As to the resorption of phosphorus, in a recent work, carried out in Verzab's laboratory, Laskowski^^^ has shown that the phosphate radical present in sodium glycerophosphate, introduced artifically into the upper part of the small intestine, splits off rapidly. The effect of this fast process is that the phos- phate of the above mentioned compound is absorbed into the circulation as quickly as that of the sodium phosphate. When experimenting on rats an absorption of 68% of the P administered was ascertained, after the lapse of one hour, with either compound. In the case of sodium phytin 62%, in that of sodium diphospho- glycerinate only 42% of the P content was resorbed. When ths phosphorus com- pounds were introduced into the lower part of the small intestine, the percentage absorbed into the circulation was much smaller^^^ and amounted, in the case of sodium phosphate, to 38% of that introduced. The difference observed, is pre- sumably due partly to the greater activity of phosphatases in the upper part of the intestinal tract, partly to the greater acidity prevailing there. We mentioned already that low acidity is favourable to the formation of insoluble phosphorus compounds. In so far as some of the phosphorus compounds present in the food decompose or get leached out in lower parts of the intestine, the yield will be lower and this may explain the difference observed between the absorption of labelled sodium phosphate and the total phosphorus present in the diet of the human subjects in question. We have also to consider that a part of the phosphorus may be contained in undigested fractions of the diet taken, protected by mechanical obstruction from the leaching effect of the digestive juices. We can expect more information on these points by replacing the administration of labelled sodium phosphate by that of vegetables, grown on labelled soil and thus containing labelled phosphorus compounds. We can also feed labelled eggs, layed by hens to which labelled sodium phosphate was administered, or labelled meat. The tracing of to what extent the labelled P is absorbed from these foodstuffs is to be expected to supply us with important information as to their digestibility and seems to be a rational approach to the study of digestion, especially if foodstuffs containing other labelled elements beside phosphorus could be administered as well. We can, however, also obtain a knowledge as to the amount of unresorbed P present in the faeces by an easier method than that sketched above, a method which we will describe in the following. (i^M. Laskowsky, Biochem. Z. 292, 312, 1937. (2^Comp. also F. Verzar and H. Wirz, Biochem. Z. 292, 174 (1937). 37^ 548 ADVENTURES IX RADIOISOTOPE RESEARCH Origin of faeces phosphorus Let us assume that all phosphorus present in the food, is absorbed into the circulation. In this case, all labelled P found in the faeces must originate from the body proper. It is ultimately the plasma inorganic phosphorus which is respon- sible for the formation of the phosphorus compounds present in the digestive juices and, therefore, the specific activity (activity per mgm P) of the faeces P should, in the above mentioned case, be equal to that of the plasma P. The speci- fic activity of the inorganic plasma P being equal to that of the urine P, we shall expect to find the specific activity of the faeces P to be equal to that of the urine P. If the above assumption does not hold and a part of the faeces P is unabsorbed, inactive P originating from the undigested food, the specific activity of the faeces P will be found in that case to be lower than that of the urine P. The ratio specific activity of faeces P X 100 gives the percentage of P present in the faeces specific activity of urine P which originates from the body proper. If the food P is, for example, quantitati- vely absorbed, then the above ratio will work out to be 100. It is clear that diffe- rent objections can be raised against the above considerations. One may object on the grounds that the specific activity of the plasma P, after the active P was iidded to the food, will first increase and then decrease, its variation with the time being thus an intricate one. Another objection which can be raised is that the tissue P of the organs involved, will also participate in the formation of the phosphorus compounds present in the digestive juices. These objections will not, however, be valid if we, before comparing the specific activity of urine P and faeces P, wait a considerable time, after administering the labelled P, before collecting the urine and faeces samples; preferably;, samples should be collected for several days. After the lapse of a considerable time, most P present in the different com- pounds of the organs responsible for the production of the digestive juices will be in exchange equihbrium with the plasma P, these showing thus the same spec- ific activity^i% In Tables 5a and 5b the amount of P found in urine and faeces and also its total activity is stated, from which the specific activities could be evaluated. In view of the very long duration of the experiment in question and the comparatively low activities shown by many of the faeces samples, the accuracy Table 8. — Specific Activity of Urine P and Faeces P of a Female Subject 7 resp. 8 Days After Administration of Labelled Sodium Phosphate per Intravenous Injection Fraction Number of counts P content in mgm Specific activity (% of the activity administered per mg^ P) Urine P 107.4 118.9 53.9 9.01 9.80 18.60 11.9 Urine P Faeces P 12.1 2.9 ^^' A possible source of error may be found in the different rates of decrease of the specific activity of the inorganic P, and of some forms of organic P present in the body (comp. G. Hevesy and A. H. W. Aten, Kgl. Danske Vidensk. Selsk. Biol. Medd. 14, 5, 35 (1939). EXCRETIOX OF PHOSPHORUS 549 of these experiments did not suffice to carry out such a calculation. To enable us to determine with sufficient accuracj' the ratio of the specific; activity of the urine P and the faeces P, we administered labelled sodium phosphate having an activity of about Viooo milliCurie to a female subject and investigated the urine and faeces collected after the lapse of 7 and 8 days resp. As the faeces, collected after the lapse of 8 days, actually accumulated in the bowels at a somewhat earlier date, it is advisable not to compare urine and faeces collected the same day, but to compare the faeces with the urine collected one day previously. The result of this experiment is seen in Table 8. The specific activity of the total faeces P is only 24% of that of the urine P, the faeces P must therefore to a large extent originate from non-absorbed food, which is the only source of non-active P. It follows from the above figure that 76% of the P present in the faeces of the human subject in question is non-absorbed P, the rest originating from the body proper. This is, however, not to be interpreted as indicating a phosphorus absorption of the food taken amounting to only 24°o- When interpreting the above figure, we must take into account that the P excreted through tht kidneys amounts to about twice of that lost through the bowels, and the sum of both values represents the total P present in the food, if we assume that the subject in question is in P balance. We then find that only 25% of the total P present in the food was not absorbed into the circulation. Table 9. — Specific Activity of Urine P and Faeces P of a Female Subject 28 Days after Administration of Labelled Sodium Phosphate BY Subcutaneous Injection F r a c t o n Specific activity Urine P 8.07 Urine P 8 10 Faeces P(i) 1.77 '■' 18»/„ of the total P found in the faeces was residual P obtained after tlie removal of the acid- soluble P (mostly calcium piiospliate) and tlie traces of phosphatide P present. Tlie specific activites of the different P fractions differed only to a minor extent. Through the courtesy of Dr. Kjerulf-Jensen, who is investigating the P metabolism of a human subject by making use of radioactive P, we could investi- gate the faeces P and the urine P, collected 28 days after administration of labelled P. The results are seen in Table 9. From the ratio of the specific activities it follows that 20% of the P percent in the faeces was of endogenous origin and that of the total P present in the food 27% was not absorbed into the circulation. Excretion by rats We determined also the ratio of the specific activities of the uiine P and faeces P excreted by a rat to which labelled sodium phosphate was administered, by subcutaneous, injection, 98 days previously. The results are recorded in Table 10. The rate of the specific activities is 2.4, thus 59% of the P found in the faeces originates G. Hevesy and E. Lundsgaard, Nature 140, 275 (1937). 552 ADVENTURES IN RADIOISOTOPE RESEARCH Comments on paper 55 The balance of phosphorus intake and excretion has been investigated in nume- rous cases. A vast literatiiie on this subject is available in which the route of secretion is often considered, i. e. the ratio of urinary to faecal phosphorus of the human subject or the animal investigated. To what percentage the phosphorus excreted in the faeces is due to unabsorbed material and to phosphorus originat- ing from the body proper were, however, not known before the application of radioactive indicators. This application of ^-P is reported in paper 55. The suppo- sition that the specific activity of urinary phosphorus parallels that of digestive secretion, an assumption on which the method applied is based, was tested later by KjERULF — Jensen (1941). He was able to support the above supposition. Among others, he compared the specific activity of the total phosphorus in samples of bile-pancreatic juice with the specific activity of urinary phosphorus, and found that 1 week after administration of labelled phosphorus the specific activ- ity of the total phosphorus in bile -pancreatic juice samples was already very near the value of urinary phosphorus. When interpreting the figures obtained for endogenous and exogenous phosphorus content in faeces, we must envisage the possibility that some phosphate interchange takes place thiough the intestine wall and that, correspondingly, the endogenous phosphorus present in the faeces may be partly secreted and partly interchanged phosphorus. The method outlined in paper 55, can be used to determine what percentage of almost any element present in the faeces is of endogenous origin. Reference K. Kjerulf- Jensen (1941) Acta Physiol. Scand. 3, 1. Originally publishod in Acta I'hy-siol. Scand. 3, 123 (1942). 56. POTASSIUM INTERCHANGE IN THE HUMAN BODY G. Hevesy From the Institute of Theoretical Physics, University of Copenhagen Interchange between the potassium present in the cells and the potas- sium present in the extracellular fluifl of animals, can be determined by using radiopotassium (*"K) as an indicator. Two methods were applied. One method is based on the comparison of the ^-K content of the plasma (extracellular) potassium and the ^-K content of the tissue potassium. (Hahx et al., 1939, 1941; Fenn et al., 1941. Comp. also Joseph ef ciL, 1939.) The other method (Hahn et al., 1939, 1941) is based on the measure- ment of the amount of labelled potassium which disappeared in the course of the experiment from the plasma. Since the amount of potassium present in the tissue cells is many times larger than the amount of potas- sium present in the extracellular space, a rapid interchange between plasma (extracellular) potassium and cellular potassium will soon lead to a strong depletion of the plasma ^^K content. The rate of disappearance of 42j^ from the plasma (extracellular space) is, therefore, a very sensitive measure of the rate of interaction between plasma (extracellular) potas- sium and cellular potassium. This method has the disadvantage that assumptions have to be made concerning the potassium content of the tissue cells. The advantage of the method is its great simplicity. It suffices to analyse the plasma or, as we shall see below, the urine. In experiments on human subjects clearly the last mentioned alone can be applied. EXPERIMENTAL PROCEDURE The labelled potassium was prepared by bombarding 80 mgm Iv(T with a deuterium beam in the Copenhagen cyclotron. The activity obtain- ed was 1/500 milliCurie. We are much indebted to Professor J. U. Jacobsen and Mr. O. N. Lassen for preparing the radiopotassium. The sample obtained was purified and dissolved in 20 cc. water. It was taken 5 hours after the last meal by mouth by a male suljject weighing 554 ADVENTURES IN RADIOISOTOPE RESEARCH 70 kgm. Urine samples were collected at intervals, and the activity of 42K the samples was determined. As the ratio of can be total potassium assumed to be about the same in the plasma and the urine, the deter- mination of the activity of the urine potassium informs us on the ^^K content of the plasma potassium at the time of the formation of the urine. The urine samples were ashed below 400° and their activity was compared with the acitivity of a standard preparation. This was prepared by adding an aliquot part of the solution of labelled purified potassium chloride taken by mouth to non-active urine. The ash samples weighed about 300 mgm. They were placed under the Geiger counter in aluminium dishes of 1.2 cm diameter. The potassium content of the samples was kindly determined by Dr. L. Hahn, using Sohl and Bennet's method. We shall first consider the case that, in the course of the experiment, no interchange takes place between the extracellular and the cellular potassium. Then, the total ^-K absorbed from the intestine should be present in the extracellular fluid of the body except for the *2K excreted and the amount taken up by the corpuscles. The amount of labelled potassium excreted in the course of 24 hours was found to be about 7 per cent of the amount present in the body, while as was found in the case of the rabbit and the rat, the corpuscles can be assu- med to contain about as much ^^K as is present in the extracellular fluid. Assuming the potassium content of the plasma to be 20 mgm per cent of the ^-K administered should in the above case be present in the corpuscles, 1 mgm plasma (ex- 46.5 „ tracellular) potassium will contain about = 0.012 per cent ot ' ^ 3800 the amount of ^^j^ administered. Let us now consider the other extreme case, viz. full interchange in the course of 24 hours between extracellular and cellular potassium. To estimate the percentage of ^^K administered which will be present in 1 mgm plasma (urine) potassium, we have to estimate the potassium content of the tissue of the human subject in question. The major part of tissue potassium is found in the muscles. The average potassium content of human muscles is stated (Cuming 1939; Mangus and Myers 1940) to be 330 mgm per 100 gm fresh weight. The weight of the muscles was estimated, following a suggestion of Dr. Brandt Rehberg, from the amount of creatinine excreted through the kidneys in the course of 24 hours. Dr. Rehberg most kindly determined the creatinine content of the urine and found a daily excretion of 1300 mgm. Prior to these determinsitions, the subject was kept on a vegetarian diet. In the deter- POTASSIUM INTERCHANGE IN THE HUMAN BODY 555 mination of the weight of the muscles from the daily creatinine excretion it is assumed that the creatinine excreted has been formed in the body from creatine and the daily conversion of creatine to creatinine amounts to about 1.32 per cent of the weight of the creatine. From these figures the creatine content of the body can be estimated to be 99 gm. Assuming the creatine of the body to be located in the muscles and the creatine content of the muscles to amount to 0.39 per cent of the weight of the muscles, we arrive at the result that the weight of the muscles amounts to 25 kgm and that, correspondingly, the muscles contain about 82 gm potassium. About % of the potassium content of the mammalian body is found in the muscles. Assuming this to be the case in the human body, we arrive at the estimate that the body of the human subject in question contains 110 gm potassium of which 106 gm are located in the cells. The amount of extracellular potassium of the body is thus 3.6 per cent of the total potassium content of the organism. In the case of total interchange between extracellular and cellular potassium, only 3.6 per cent of the amount of labelled potassium absorbed into the circulation minus the ^^K excreted should be present in the extracellular space. In the case of total interchange, 1 mgm urine potassium should thus, after the lapse of 48 hours, contain only 0.0010 per cent of the labelled potas- sium administered. RESULTS In the first experiment, the results of which are seen in Table 1, urine was collected during the first 2 days after drinking the solution containing the labelled potassium chloride. The presence of easily demonstrable amounts of ^^K could be shown as early as 12 minutes after drinking the active solution, while a negative result was obtained after the lapse of 5 minutes. The total urine was found to contain 10.5 per cent of the *2K administered, the potassium content amounting to 3.4 gm. Thus, 1 mgm average potassium present in the urine contained 0.003 per cent of the ^^K administered. This is much less than the amount which was to be expected (0.012) assuming an absence of interaction between cellular and extracellular potassium (see p. 554). The results of this preliminary experiment thus lead to the conclusion that a very substantial part of the 42K absorbed into the circulation, must have found its way into the tissue cells while a substantially equal number ^1^ We found the excretion of ^^K through the bowels to make out roughly 15 per cent of the amount excreted through the kidneys, the total excretion of ''-K in the course of the experiment being about 11 + 0.15x11 13 — per cent. 556 ADVENTURES IN RADIOISOTOPE RESEARCH Table 1. — Excretion of Labelled Potassium Administered by Mouth Time Percentage of '^K recovered in tlie urine 12 30 3 16 lA 401/2 481/4 mm. hours 0.0053 0.048 0.46 0.65 4.75 3.00 1.56 Total 10.47 Volume of urine = 2.041 cc. of non-labelled potassium atoms migrated from the cells into the extra- cellular space. In the two following experiments, besides determining the percentage of ^^K administered which was excreted in the course of 65^ hours and the potassium content of the urine, urine samples were collected on three consecutive days between 9 and ll^^ hours and their activity and potas- sium content were determined. The results of these experiments are seen in Tables 2 and 3. While no pronounced difference in the specific activity of the potassium collected after 15, 39 and 63 hours is observed, in the Table 2. — ^-K Content of Urine Samples Samples collected between (reck- oned from the start of the Potassium content per cc. urine in mgm Percentage of "-K administered present in 1 mgm urine potassium experiment) Found Calculated assuming total interchange 15 and 171^ hours 39 and 4iy2 hours 63 and 65^ hours 3.77 3.85 3.05 0.0014 0.0011 0.0014 0.0010 0.0010 0.0010 and 6514 hours 1.75 0.0024 Total urine volume = 2.990 cc. Percentage of the i-K administered present in the total urine 12.7 per cent. early phases of the experiment potassium of higher specific activity was excreted than in the later phases. This follows from the higher value found for the specific activity of the average potassium present in the mine collected. 1 cc. urine collected between 9 a. m. and llYo a. m. was found to have an appreciably higher potassium content than 1 cc. of average POTASSir^[ IXTERCIIAXGE IX THE HUMAX BODY 557 virine. This result is in accordance with the general experience according to which urine collected between the early hours of the day and noon has the highest potassium concentration. Table 3 Samples collected between (reck- oned from the start of the Potassium content per cc. urine in mgm Percentage of ^-K administered present in 1 mgm urine potassium experiment) Found Calculated i 15 and 17^4 hours 39 and 41l^ hours 63 and 65% hours 2.24 2.70 2.30 0.0015 0.0011 0.0013 0.0010 0.0010 0.0010 and 65% hours 1.26 0.0025 0.001 Total urine volume = 2.880 cc. Percentage of the ^^K administered present in the total urine 9.1 per cent. DISCUSSION While the accuracy of the method applied does not suffice to deter- mine whether a full interchange between the potassium of the cells and the potassium of the extracellular fluid took place, the results obtained clearly indicate (see the 2 last columns of Tables 2 and 3) that the greatest part of the labelled potassium ions and, thus, the greatest part of all potassium ions taken with the food find their way within 16 hours or less into the tissue cells while potassium ions formerly located in the cells move simultaneously into the extracellular fluid. In the average urine sample collected during the experiment, the potassium was found to be markedly less active than in the samples collected after the lapse of 15 hours. This is due to a higher activity of the potassium excreted in the first phase of the experiment. This result indicates that the inter- change between cellular and extracellular potassium is not a very rapid process since it takes several hours before a large part of the extracellular potassium interchanges with the potassium of the cells. Apparent Volume of Distribution In connection with the experiments reported in this note, it is of interest to recall experiments by BouRDiLLOisr (1937) in which the apparent volume of distribution of potassium chloride taken by him by mouth was investigated. The apparent volume of distribution = 558 ADVENTURES IN RADIOISOTOPE RESEARCH amount absorbed into the circulation minus amount excreted increase in concentration in serum water If the potassium taken by mouth would remain in the extracellular fluid, the apparent volume of distribution should be 25 to 30 per cent of the body weight. Bourdillon found, in experiments on himself, an apparent volume of exogenous potassium corresponding to 75 per cent of the body weight and a similar result was obtained in experiments on dogs by Winkler and Smith (1938) and on cats by Fenn (1939). The apparent volume of distribution of the labelled potassium taken by mouth in the experiments described in this note, can be computed to bo about 400 litres or 570 per cent of the body weight. The striking difference in the results of Bourdillon and the results arrived at in this note illustrates the great difference between accumula- tion and interchange of potassium. In Bourdillon's experiment, addi- tional potassium had to accumulate in some or all tissue cells and, if we assume the concentration of the extracellular fluid to remain isotonic, such an accumulation necessitates the exodus of other cations from the cells. This can only take place on a very restricted scale. Not so the interchange of extracellular potassium with cellular potassium. No other elements have to leave the cells to make place for the labelled potassium; it suffices that non-labelled potassium atoms make place for labelled ones. Excretion of Sodium The sodium content of the extracellular fluid is about 15 times larger than its potassium content. Since the potassium content of the urine does not differ much from its sodium content, we should expect a much Table 4. — Excretion or ^*Na Administered Through THE Kidneys Urine sample secured after Volume in cc. Percentage of the ^*Na administered present in the urine sample 10 min 5 2.9 3.9 10 80 590 1700 1440 0.00046 20 min 0.0076 46 min 0.036 98 min 0.056 6 hours 19 hours 43 hours 67 hours 0.31 2.86 4.75 4.5 Total 3832 12.5 POTASSIUM INTERCHANGE IN THE HUMAN BODY 559- larger part of the potassium present at any moment in the extracelluhu' space to be excreted through the kidneys in the course of the first 24 hours, for example, than of the sodium simultaneously present in Ihe extracellular fluid. Thus, in the urine, a much larger part of "^-K admi- nistered than of 24Na administered should ])e present. This is, however,, not the case. As seen in Table 4, the percentage of daily excreted '^^Na does not much differ from the percentage of daily excreted ^^K. From this result it follows that most potassium ions present in the urine are such as were previously located in the cellular and not in the extra- cellular fluid; a conclusion which is in accordance with the result arrived at in the previous section. RESULTS Labelled potassium chloride was administered to a human subject and the potassium and the ^^K content of the urine samples collected within 65 hours were determined. After the lapse of 12 minutes, 5x 10^^ part of the ^^K administered was found to be present in the urine. In the course of 48 hours, about 10 per cent were excreted through the kidneys. Making the assumption that the ^^K content of 1 mgm potassium present in the urine is about the same as the ^^K content of 1 mgm potassium present in the plasma (extracellular space), from the ^^K content of the urine potassium the ^^K content of the extracellular fluid of the body can be computed. By this method, it was found that a very large part of the ^^K absorbed into the circulation found its way into the tissue cells in the course of 16 hours or less. While the accuracy of the method does not suffice to determine whether a full interchange between the potassium of the cells and the potassium of the extracellular fluid took place in the course of the experiment, it is clearly shown that a very substantial part of the labelled potassium and thus of all potassium taken with the food, interchanges with the potassium located in the tissue cells in the course of some hours. Labelled sodium was found to leave the body at a similar rate as labelled potassium. References J. BouRDiLLON (1937) Amer. J. Physiol. 120, 411. B. B. Brodie, E. Brand and S. Leshin (1939) J. Biol. Chem. 133, 555. I. N. Cuming (1939) Biochem. J. 33, 642. W. O. Fenn (1939) Amer. J. Physiol. 127, 356. L. Hahn, G. Hevesy and O. Rebbe (1939) 33, 1540. G. Hevesy and L. Hahn (1941) Kgl. Danske Vidensk. Selsk. Medd. 16, I. 560 ADVENTURES IX RADIOISOTOPE RESEARCH M. Joseph E. Cohn and D. M. Greenberg (1939) J. Biol. Chem. 33, 1540. N. L. Kaltreider, G. M. Meneely, I. R. Allen, S. N. van Voorhis and V. F. Downing (1940) J. din. Inv. 19, 769. G. H. Mangus and V. C. Myers (1940) J. biol. Chem. 135, 411. T. R. NooNAN, W. O. Fenn and L. Haege (1941) Amer. J. Physiol. 132, 444. T. Wechselbaum, M. Somogyi and H. Rusk (1940) J. hiol. Chem. 132, 343. A. W. Winkler and P. K. Smith (1938) J. hiol. Chem. 124, 589. Originally published in Acta Med. Scand., 116, 561 (1944). 57. THE RED CORPUSCLE CONTENT OF THE CIRCULATING BLOOD DETERMINED BY LABELLING THE ERYTHROCYTES WITH RADIO-PHOSPHORUS G. Hevesy, K. H. Koster, G. Sorensen, E. Warburg and K. Zerahn From the Institute of Theoretical Physics, University, Copenhagen, and the University Clinic of the Rigshospital, Medical Department B, Copenhagen The red corpuscle content of the circulating blood is usually calculated from the plasma volume determined by the dye method and the hemato- crit figure. Some time ago (Hahn and Hevesy, 1940; Hevesy and Zerahn, 1942), the red corpuscle content of the rabbit was determined by making use of corpuscles labelled with radiophosphorus. This method was used by us to determine the erythrocyte content of the circulating blood of human subjects. Simultaneously, the corpuscle content was determined by the CO method and, furthermore, in some experiments the plasma volume was determined by the dye method. EXPERIMENTAL About 25 cm^ blood is taken by venous puncture. After adding heparin and about 1 cm^ plasma^^) of the same subject which contains a minute amount of labelled sodium phosphate showing an activity of about 1 /^ Curie, the blood is placed in a glass bottle with paraffine coated Malls and is gently rotated in a thermostat at 37° for 2 hours. After the lapse of this time, the radio-phosphorus added to the blood is found to be about equally distributed between the corpuscles and the plasma. A minor part of the labelled blood is kept as a standard preparation, while the rest is reintroduced into the human subject. The labelled corpuscles introdued into the circulation get soon mixed with all the circulating corpuscles and, as a result of this mixing, the activity of 1 gm corpuscles secured will be much lower than the activity of 1 gm corpuscles introduced. If we reintroduce 1 gm labelled corpuscles containing 1000 activity units while the circulation contains 1000 gm corpuscles, 1 gm corpuscles secured after the mixing, i. e. after the lapse of few minutes, will show an activity of 1 unit. ^1^ The plasma is obtained by centrifuging blood containing labelled phosphate. 36 Hevesy 5G2 ADVENTURES IN IIADIOISOTOIE EESEAECH If we wish to determine the amount of circulating corpuscles we have to know (a) the amount of the injected labelled corpuscles, (b) the activity of 1 gm of these corpuscles, and (c) the activity of 1 gm corpuscles secured from the circulation after mixing, (a) The amount of injected corpuscles is obtained from the volume of the injected blood, the specific weight of the corpuscles (1.08), and the hematocrit value. (6) The activity of 1 gm of these corpuscles is found from measuring the activity of 1 gm corpuscles of our standard preparation mentioned above, (c) The activity of 1 gm corpuscles secured from the circulation after mixing is found when measuring the activity of 1 gm corpuscles secured 5—10 minutes after the injection. Let us denote the amount of corpuscles injected into the circulation by A, the ratio of the activity of 1 gm corpuscles of the injected blood and of the activity of 1 gm corpuscles secured from the circulation after the injection by B, then the total amount of the corpuscles present in the circulation (X) is given by X = A-B The blood sample is secured in the interval between 5 and 10 minutes, preferably after the lapse of 5 minutes, following the injection. 3 minutes after the injection, a mixing of the reinjected labelled blood with the non-labelled one might possibly not have occurred. On the other hand, the loss of activity by the corpuscles might become noticeable after the lapse of 10 minutes. Table 1 illustrates the above statement. Table 1 Per cent of corpuscles Time in min injected, present in 1 gm corpuscles 1.25 0.026 2.0 0.044 3.0 0.052 4.8 0.053 6.9 0.050 9.9 0.052 15.8 0.048 18 0.045 30 0.044 Sources of error Three main sources of error have to be considered. (A) The centrifuged corpuscles are not free from plasma. (B) We do not inject active corpuscles, but active blood, and some radio-phosphorus may enter the corpuscles during the interval between the injection of labelled blood and the RED CORPUSCLE CONTEXT OF THE CIRCULATING BLOOD 563 securing of the blood samples. (C) During the last mentioned interval, some radio-phosphorus may leave the corpuscles. (^1) Though the amount of plasma adherent to 1 gm corpuscles of llie standard preparation can be assumed to be equal to the amount of plasma adherent to 1 gm corpuscles secured after the injection, the incomplete separation of corpuscles and plasma involves an error which becomes obvious from the following example. 1 gm standard preparation is composed of 0.97 gm corpuscles and 0.03 gm plasma and has an activity = 1000. As the activity of 1 gm corpuscles is about equal to the activity of 1 gm plasma after 2 hours of rotation in the thermostat, 970 activity units are due to the corpuscles and 30 to the plasma. In a sample secured 3 minutes after the injection, the activity of 1 gm plasma declines to V^ of its previous value. If, now, the activity of 1 gm of the sample is again assumed to be 1000, the share of the adherent plasma activity is only 8, the corpuscle activity being 992. When comparing the activity of the two corpuscle samples, w^e get thus a value of the corpuscle content of the circulation which is 2.3 per cent too high. The activity of the plasma adherent to the sample secured after the lapse of 10 minutes is still lower than the activity of the plasma secured after 3 minutes, f/i. about i/io of ^^^^ activity of the plasma injected. When securing the blood samples after the lapse of 10 minutes, we overestimate the total corpuscle content of the circulation with about 3 per cent. The above result was obtained after centrifuging the blood sample with 5000 revolutions per minute for 10 minutes. {B) As the injected blood contains active plasma, some radio-phos- phorus will penetrate into the corpuscles during the experiment lasting 5 — 10 minutes. Phosphate penetrates at a much higher rate through the capillary wall (Hahn and Hevesy, 1941) than through the membrane of the erythrocytes. The bulk of the radio-phosphorus content of the plasma will, therefore, leave the plasma in the course of 5 minutes, much reducing the amount of radio-phosphorus which otherwise would penetrate into the corpuscles. Table 2 shows the rate of disappearance of radio-phosphorus from the plasma and the percentage of plasma Table 2. — Penetration of ^^P from the Plasma into THE Corpuscles after Injecting Labelled Plasma into the Circulation Percentage of tlie ^-1* Timeinmin Activity of 1 gm corpuscle Activity of 1 gm plasma content of the plasma injected, present in the corpuscles 100 2 3.0 35 3 9 5.0 15 5 2.5 6.2 10 6 30^ 564 ADVEXTURES IN KADIOISOTOPE RESEARCH activity which penetrates into the corpuscles in the course of 25 minutes. The penetration of radio-phosphorus in the corpuscles during the experi- ment increases the activity of the corpuscles and, thus, makes the total corpuscle content of the circulation appear too low. The error in an experiment lasting 10 minutes amounts to 5 per cent. (C) 1.5 per cent of the radio-phosphorus content of the corpuscles was found to be replaced by inactive phosphorus from the plasma in the course of 10 minutes. This loss ascertained in experiments in vitro makes the dilution figure and, thus, the corpuscle content of the body appear too high. That the loss of radio-phosphorus by corpuscles in the course of 10 minutes is markedly restricted, is not due exclusively to the fact that the phosphate ions penetrate only at a moderate rate through the corpuscle membrane, but also to the following. 1 gm corpuscles contains about -/g as much inorganic P as 1 gm plasma and, moreover, comparatively large amounts of readily exchangeable organic P present in adenosintriphosphate and also in hexosemonophosphate, and some other acid solul)le organic P compounds. The concentration of such readily exchangeable P atoms in the corpuscles is with an order of magnitude larger than the concentration of inorganic phosphorus. As soon as the active P atoms enter the corpuscles, they interchange with phosphorus atoms present in the organic compounds. After 2 hours shaking of the blood in the presence of labelled phosphate, a large part of radioactive P atoms present in the corpuscles will, thus, be found in the organic fraction and, consequently, the activity of the inorganic P of the corpuscles will be kept at an appreciably lower level than the activity of the inorganic P of the plasma. An estimate of the different experimental errors in the determination of the red corpuscle content is seen in Table 3. The data of this table reveal that the value of the red corpuscle content obtained in experiments lasting 10 minutes is 0.5 per cent too low, the error being smaller in •experiments of shorter duration. Plasma volume determination by the means of the dye method In a number of cases, the plasma volume was determined by means of the dye method described by Gibson and Evelyn (1938). In this Table 3. — Estimate of Diffeeent Errors of Experiment in the Determination of the Erythron Time in min Percentage error due to adherence of the plasma to the corpuscles Percentage error due to intrusion of '*P of the plasma into the corpuscles Percentage error due to the loss of '^P by the corpuscles 10 +3 —5 -fl.5 EED CORPUSCLE CONTENT OF THE CIRCULATING BLOOD 565 method, use is made of the blue dye T 1824, 10 mgm of which are dissol- ved in 5 cm^ of water. Before injecting the dye, a blood sample which is used to determine the hematocrit figure is secured by venous puncture. Through the same cannula with wdiich the blood sample is taken, T 1824 solution from a calibrated syringe is injected into the circulation. In order to remove the last traces of the dye present, the syringe is filled with blood which is also injected. This process is repeated three times. Blood samples of the patient are drawn 15, 30, 45, and 60 minutes, respectively, after injection of the dye. After the blood sample has coagulated, the serum is centrifuged off and its dye content is deter- mined by making use of a photoelectric colorimeter. The light absorption by the dye-free serum is previously ascertained in the same way. The dye content of the different sera secured from the same patient at different times is plotted against time and, from the curve obtained, the dye content present at zero time is extrapolated. This procedure is necessary because it lasts some minutes until dye and blood are properly mixed and, during this time, some dye leaves the plasma. 15 — 30 minutes after injection, some dye was found to be present in the lymph of the ductus thoracicus (Cardozo, 1941), and Koster observed some dye in the bile 30 minutes after administration. From the plasma volume obtained by means of the dye method and the hematocrit figure, the corpuscle content and the blood volume were calculated. The results are shown in Tables 6 and 7. Determination of the corpuscle volume carried out with the CO method We determined, furthermore, the corpuscle content applying the CO method. Carbon monoxide is prepared under the action of con- centrated sulphuric acid on sodium formiate. The CO obtained is led through a sodium hydroxide solution, in order to remove any carbon dioxide or sulphuric acid spray possibly present. The purified CO is stored in a gasometer constructed by connecting two flasks containing diluted sodium hydroxide. Before filling the gasometer with CO, a stream of carbon monoxide is led through the liquid for some time, so that all air is removed from the gasometer. The gas stored in the gaso- meter was found to contain 97 per cent CO. The CO is administered to the patients by a Krogh "basal metabolism apparatus" containing a known volume of CO (200 cm^) and a few litres of oxygen. For the transfer of the CO from the gasometer to the Krogh apparatus, use is made of a Luers glass syringe carefully kept at room temperature, the CO being injected in the tube leading from the oxygen flask to the spirometer and the tube washed with an oxygen current. The patient inhales the mixture of CO -f- Og in the course of 566 ADVENTURES IN RADIOISOTOPE RESEARCH about 15 minutes. During this time, the oxygen taken up by the patient is replaced in the gaseous mixture. Subsequently, a blood sample is secured. The determination of CO in the blood sample was carried out by making use of Wennesland's palladium chloride method (1940). Under the action of sulphuric acid, CO is given off by the blood sample placed in a flask. The CO released diffuses into another flask connected with the first one and containing a known amount of palladium chloride. Special precautions are taken to avoid a loss of CO while connecting the flasks. Some palladium chloride is reduced to palladium under the action of CO. When determining the amount of palladium chloride still present at the end of the experiment, we can calculate the amount of CO given off by the blood sample. The determination of the remaining amount of palla- dium chloride was carried out after rotating the blood-sulphuric acid mixture for 3 — 4 hours. The blood volume was calculated according to the formula -^, , , cm^ CO administered • 100 Blood volume =: volume per cent CO in the blood sample RESULTS The results from determinations of the corpuscle content by the ^-P method performed on the same subject at different dates is seen in Table 4, while in Table 5 is given a survey of all our determinations carried out with application of the ^^P method. The mean value of the corpuscle content per kgm body weight is found to be 36.0 gm. The corpuscle content obtained when applying the CO method is obvious from Table 6 which contains also data found when using the dye method. While the CO method is a direct method of determination of the corpuscle content, the dye method is an indirect one, the corpuscle content being calculated from the plasma volume and the hematocrit value. As seen in Table 6, the corpuscle content determined when applying the CO method is larger than the corpuscle content found when making use of the ^'^P method. This discrepancy is possibly due to the uptake of CO by other compounds than by the haemoglobin present in the corpuscles of the circulating blood (comp. Asmussen 1942). That the corpuscle content calculated from the plasma (dye) volume and the hematocrit value is larger than the corpuscle content determined when applying the^-P method, can be interpreted in two different ways. (a) The plasma volume technique gives a falsely high plasma volume, (b) the plasma ratio of the circulating blood is lower than the plasma RED CORPUSCLE CONTENT OF THE CIRCULATING RLOOD 567 ratio determined by the hematocrit reading of blood samples drawn from the body. Some dye is lost by the plasma during the experiment. However, this loss is taken into account by extrapolating the dilution values obtained at different times to zero time. These considerations make it improljable that the above mentioned discrepancy is due to an overestimation of the plasma content by the dye method and suggest the alternative denoted by {b) to explain the discrepancy. Table 4. — Corpuscle Content or THE Same Subject Determined at Different Dates No. Date gm corpuscle content 2 13/10 2370 4 22/10 2190 5 27/10 2930 6 3/11 2600 7 6/11 1940 9 13/11 1910 11 20/11 1920 14 27/11 1920 8 10/11 1940 10 16/11 1890 17 4/12 1940 12 23/11 2340 15 30/11 2100 20 11/12 2140 13 25/11 2280 16 2/12 2100 19 9/12 2090 18 7/12 2930 21 14/12 2810 22 21/12 2640 Already some years ago, it was suggested by Smith, Arnold and Whipple (1921) that the hematocrit reading does not give the true corpuscle-plasma ratio of the blood in the whole body. They found the red corpuscle volume determined by the CO method and the Welkcr method to be approximately 25 per cent lower than the red corpuscle 568 ADVENTURES IX RADIOISOTOPE RESEARCH volume calculated from the plasma volume and the hematocrit reading and they interpreted their result as an indication of their suggestion. Furthermore, Hooper, Smith and Whipple (1920) have shown that, after having lowered the hematocrit reading by haemorrhage, the measured red corpuscle volume (from the plasma volume and the hematocrit reading) did not agree with the red corpuscle volume predicted on the basis of the volume of the erythrocytes removed. If the red corpuscle volume before bleeding is equal to the red corpuscle volume after haemor- Table 5. — Corpuscle Content of Hximan Subjects Determined by Making Use of ^sp as an Indicator No. Corpuscles injected in gm Activity of 1 gm corpuscles injected Activity of 1 gm corpuscles secured Hematocrit Total corpuscle content in gm Body weight in kgm Corpuscle content per kgm body weight in gm 1* 10.7 30.000 62.6 63 5180 78.8 65.7 2 6.16 14,750 38.3 48.8 2370 61.8 38.3 3 6.98 14.500 36.2 49.6 2800 70.0 40.0 4 8.92 19.900 81.2 47.6 2190 61.8 35.4 5 8.59 15.600 45.7 45.8 2930 64.9 45.1 6 6.85 12.500 32.9 46.9 2600 67.0 38.8 7 8.30 15.000 64.1 42.0 1940 57.2 33.9 8 7.77 15.000 60.0 42.7 1940 57.8 33.6 9 7.49 15.000 58.9 38.2 1910 57.8 33.0 10 8.10 15.000 64.4 42.3 1890 59.1 32.0 11 7.06 15.000 55.1 37.9 1920 60.0 32.0 12 9.05 15.000 58.0 46.5 2340 69.5 33.7 13 8.96 15.000 58.9 46.5 2280 54.0 42.2 14 6.28 15.000 49.1 37.0 1920 60.0 32.0 15 7.97 15.000 57.0 44.9 2100 69.5 30.2 16 9.00 15.000 64.3 42.8 2100 54.0 38.9 17 7.18 15.000 55.5 40.5 1940 64.0 30.3 18 9.36 15.000 47.9 47.1 2930 72.0 40.7 19 8.66 15.000 62.2 44.4 2090 54.0 38.7 20 8.69 15.000 61.0 44.6 2140 69.5 30.8 21 8.88 15.000 47.5 43.3 2810 72.0 39.0 22 8.53 15.000 48.5 44.6 2640 72.0 36.7 Mean value** • No. 1 was a patient suffering from polycytemia. The corpuscle content of this patient is not included in the average. ** Mostly lean subjects were investigated. rhage, plus the volume of erythrocytes removed, the hematocrit reading gives the correct corpuscle— plasma ratio of the whole blood; if the corpuscle volume before haemorrhage is greater than the corpuscle volume after bleeding, plus the volume of red corpuscles removed, the hematocrit reading does not represent the corpuscle — plasma ratio of the blood of the whole body. RED CORPUSCLE CONTENT OF THE CIRCULATING BLOOD 569 Stead and Ebert (1941) avIio recently carried out such bleeding experiments found that, in normal human subjects, 72 hours after venesection, the red corpuscle volume always appears lower than the red corpuscle volume predicted from the pre-haemorrhage red corpuscle volume and the volume of red corpuscles removed. The changes in hema- tocrit reading are, however, relatively small. Later, Stead and Ebert experimented with dogs in which a marked drop in hematocrit reading was produced by massive bleeding. Tlu^ spleens were removed as, under certain conditions, the spleens of dogs discharge blood rich in corpuscles into the circulation. In experiments in which aljout half of the red cor- puscles were removed, it was found that, while from the hematocrit reading and the plasma volume a red corpuscle volume of 1 390 cm^ was calculated, the volume of red corpuscles removed, plus the red corpuscle volume after bleeding, constituted 1039 cm^, only. From this result, these experimenters conclude that, when the hematocrit reading is approximately 50, the red corpuscle volume calculated from the plasma volume and the hematocrit reading is approximately 25 per cent higher than the true red corpuscle volume. Table 6. — Coepttscle Content Obtained when Using Different Methods No gm corpuscle content obtained, when using CO method Dye method '"P method 4 5 6 7 8 9 10 11 2640 2850 3090 2700 2480 2850 3250 2560 3350 3350 2340 3020 2530 2520 2940 2130 2560 2800 2340 2240 2290 1960 1940 2740 1990 2340 2210 2190 2930 2600 1940 1940 1910 1890 1920 12 9340 13 14 15 16 2280 1920 2100 2100 17 18 1940 2930 19 20 21 99 2090 2140 2180 2640 Mean value of those cases in which all three methods were applied : 2830 I 2320 I 1990 570 ADVENTURES IX RADIOISOTOPE RESEARCH Our results, based on an entirely different method, support the conclu- sion drawn by Whipple and his colleagues and by Stead and Ebert. We can account for the discrepancy between the blood volume obtained from the corpuscle {^^F) volume and the plasma (dye) volume and the corpuscle (^^P) volume and the hematocrit value, respectively, by assum- ing that the red corpuscle content of the blood samples is about 18 per cent higher than the average corpuscle content of the circulating blood. Blood volume A correct figure of the blood volume is obtained if we add to the corpuscle volume supplied by the ^sp method, the plasma volume found with the dye method (cf. Table 7). This procedure is independent of the hematocrit figure though based on the assumption that the dye method supplies us with a correct value for the total plasma volume. Table 7. — Blood Volume No. Corpuscle volume determined by the "P method + plasma volume determined by the dye method Blood volume determiiied by the dye method Blood volume calculated from the corpuscle volume determined by the '^P method and the hema- tocrit figure Blood volume calculated from the corpuscle voliune determined by the CO method and the hema- tocrit figure 7 8 10 11 4930 4830 4420 4910 5110 5170 4810 5130 4910 5330 5340 4630 5120 5880 5370 5220 5460 5300 4300 4240 4140 4690 4540 4760 4330 4430 4430 5940 5380 7110 6480 13 6670 14 15 5860 6240 17 5760 Mean value 6180 If we determine the blood volume by the dye volume, i. e. if we cal- culate the blood volume from the plasma volume and the hematocrit figure, we get a value which obviously is too high. As the corpuscle content of the total circulating blood is lower than the corpuscle content of the blood sample used in obtaining the hematocrit figure, we overesti- mate the corpuscle volume and, consequently, also the blood volume. This may be seen from the following example. Let us assume the hema- tocrit figure to be 50. If we then add to the plasma volume, 50, an equal corpuscle volume, we obtain a blood volume figure = 100. In view of the fact that the corpuscle content of the circulating blood is about 18 per cent lower than the corpuscle content of the sample secured for hematocrit determination, we should add to 50 (plasma volume) 0.82x50 (corpuscle volume) and, thus, find 91 for the blood volume. Consequently, RED COKPUSCLE CONTEXT OF THE CIRCULATING BLOOD 571 the correct value of the blood volume makes out 91 per cent of the value determined by the dye method. On the other hand, when the blood volume is calculaled irom the corpuscle volume determined by the ^^P method and the hematocrit figure, we obviously underestimate the blood volume. Now, if we over- estimate the hematocrit value, \v(^ underestimate 1lu> slian^ of the plasma in building up the ))lood and, thus, we underestimate Ihe blood volume. If the CO method would supply us with a correct value for Ihe cor- puscle content of the circulation, the blood volume calculated from the corpuscle volume and the hematocrit figure would be too low. However, as the CO method provides us with a too high value for the corpuscle volume, the opposite is the case. The error due to the uptake of CO by other compounds than the hen:oglobin present in the circulating blood, overcompensates the error due to an underestimation of the plasma content of the blood. Determination of the red corpuscle volume by using radio-iron as an indicator While Hahn and Hevesy (1940) and Hevesy and Zerahn (1942) carried out a determination of the corpuscle content of the rabbit and the hen by labelling the corpuscles with radio-phosphorus, Hahn, Balfour, Ross, Bale and Whipple (1941) used radio-iron as an indicator in experiments with dogs. Radio-iron is more stably bound in the corpuscles than radio-phosphorus. This fact makes possible to carry out experiments of several days duration. Experiments lasting only a few minutes lead to the same results regarding the corpuscle content as experiments lasting a few days, which proves that the mixture of the corpuscles injected with those beforehand present in the circulation had occurred already in the course of a few minutes. In contradistinction to the labelling of corpuscles with radio-phospho- rus which can be carried out in in vitro experiments, the labelling of corpuscles with radio-iron can only be made in vivo. In experiments on human subjects, it is necessary to work with donors whose blood was labelled with radio-iron .By making use of this procedure in order to obtain corpuscles of sufficient activity, very substantial radio-iron activities had to be administered. However, the preparation of even moderate iron activities — in contradistinction to that of large phos- phorus activities — is a difficult task. This may be the reason why so far only animal experiments were carried out with radio-iron. When using radio-iron as an indicator in the experiments with dogs, Hahn and his colleagues found a smaller corpuscle content than that obtained by a calculation of the corpuscle content from llic plasma (dye) 572 ADVENTURES IN RADIOISOTOPE RESEARCH volume and the hematocrit figure, the corpuscle content determined by the radio-iron method being 77 per cent of that calculated when applying the plasma (dye) and the hematocrit values. This is a result similar to that obtained by us when using the ^^P method. Summary To a blood sample taken from a human subject, a minute amount of sodium phosphate containing the radioactive phosphorus isotope ^^p is added. Then, the sample is shaken in a thermostat for two hours at 37° C. A part of the blood sample thus containing labelled corpuscles is reintroduced into the circulation. After the lapse of about 5 minutes, a blood sample is secured and the radioactivity of the corpuscles of this sample is compared with the radioactivity of the reintroduced corpuscles of equal weight. The ratio of the radioactivity of the two samples is a measure of the amount of corpuscles present in the circulation. The mean value of the corpuscle content of the lean human subjects investi- gated was found to be 36.0 gm per kgm body weight. In a number of cases, the plasma volume was determined by means of the dye method and the corpuscle content of the circulating blood was calculated from the plasma volume and the hematocrit figure. The figures obtained in this way, were about 18 per cent higher than those found for the corpuscle content deter- mined according to the ^^P method. When adding to the plasma volume determined with appHcation of the dye method, the corpuscle volume found by means of the ^^p method, we obtain the blood volume. The value thus calculated is independent of the hematocrit figure. The values obtained by means of this direct method of determination of the blood volume are found to be about 9 per cent smaller than the values calculated from the plasma (dye) volume and the hematocrit figure. The difference between the determined and the calculated corpuscle content (making use of the hematocrit figure) respectively between the determined and the calculated blood volume, supports the conclusion drawn by Whipple and his colleagues that the hematocrit figure is no proper representative of the corpuscle content of the circulating blood, this content being smaller than the hematocrit figure. This conclusion is based on the assumption that the dye method supplies us with a correct value for the total plasma volume. Literature E. AsMussEN (1942) Acta Physiol. Scand. 3, 156. E. L. Gabdozo (1941) Arch. need. Physiol. 25, 410. I. G. Gibson Jr. and K. A. Evelyn (1938) J. Clin. Inv. 17, 153. L. Hahn and G. Hevesy (1940) Acta Physiol. Scand. 1, 3. L. Hahn and G. Hevesy (1942) Acta Physiol. Scand. 4, 376. P. F. Hahn, W. M. Balfour, J. F. Ross, W. F. Bale and G. H. Wipple (1940) Science 93, 87. C. W. Hooper, F. P. Smith and G. H. Whipple (1920) Amer. J. Physiol. 51, 205. F. P. Smith, H. R. Arnold and G. H. Whipple (1921) Amei-. J. Physiol. 56, 337. E. A. Stead and R. W. Ebert (1941) Amer. J. Phijsiol. 132, 411. R. Wennesland (1940) Acta Physiol. Scand. 1, 49. Originally published in Acta Physiol. Scand. 24, 285. (1951). 58. APPLICATION OF ^ K LABELLED RED CORPUSCLES IN BLOOD VOLUME MEASUREMENTS G. Hevesy and G. Nylin From the Cardiovascular Clinic, Soderjukhuset, Slockhohn Labelling of red corpuscles by making use of ^^p as an indicator is made possible by the following facts. (a) A constant fairly slow interchange of phosphate between plasma and erythrocytes takes place at body temperature. (b) The organic labile P content of the red corpuscles is much higher than that of the plasma. (c) The individual P atoms, and thus also the ^^P atoms intruded into the erythrocytes, participate in phosphorylation processes and reach rapidly an exchange equilibrium with a part of the labile P present in the red corpuscles. Due to these facts, some of the ^^P atoms M'hich when blood is shaken with labelled phosphate penetrate into the erythrocytes in the course of 1 hr, during the same time 1/10 — 1/20 only find their way into the plasma, after injection of the active red corpuscles into the inactive circulation. The potassium content of the red corpuscles is also much — about 20 times — higher than that of plasma of the same weight and, corre- spondingly, the lifetime of an individual potassium atom in the red corpuscles is about 20 times longer than in the plasma. When introducing *-K atoms into the erythrocytes, we can expect them to leave the red corpuscles at a slow rate only. The loss within 20 minutes, which amply suffice to obtain mixing between the injected and circulating blood, can be expected to be less than one per cent. This induced us to carry out determinations of the circulating blood corpuscle volume by using ^-K labelled red corpuscles. EXPERIMENTAL To 10 ml of freshly drawn heparinized blood 11— 15 mgm of KCl, previously bombarded in the cyclotron and having an activity of 10—60 juC, were added and the blood was shaken for 2 hrs at 37 °C. In some cases, a known aliquot of the active blood, in others red corpuscles once washed with inactive plasma were 574 ADVENTURES IX RADIOISOTOPE RESEARCH reinjected into the human subject under investigation. The labelled KCl was previously carefully purified from radioactive impurities, especially from ^i^a. Under the conditions prevailing in the Stockholm and Copenhagen cyclotrons, where the KCl applied was bombarded with deuterons, the bombardment of 1 mgm of sodium svipplies about 30 times as much ^^Na as *^K is formed by bombardment of 1 mgm potassium. (Personal communication by Mr. K. Zerahn, to whom and to Dr. Mel ANDEB we are much indebted for the purification of the active potassium chloride samples.) The presence of minute amounts of sodium in the KCl sample can thus become disturbing. The labelled KCl was added to blood in physiolo- gical concentration thus as 1.1% solution. The blood samples, secured from the vena basilica at various intervals after injecting a known aliquot of the activated blood were centrifuged, the corpuscles washed with saline and their activity compared with that of a known aliquot of the blood (corpuscles) injected. In our first experiments we compared the acti- vity of dried corpuscle and plasma samples, but, in view of the fact that ^^K decays with a half time of 12 his and the dr^•ing of the samples takes some time, we applied later Zerahn's (1948) cuvettes in which the activity of fresh samples is measured. These cuvettes weie also applied when determining the distribution coefficient of *2K between plasma and red corpuscles in vitro. In these experiments to about 10 ml of freshly drawn heparinized blood kept at 37 °C, 0.1 ml of a physiological sodium chloride solution containing about 0.05 mgm labelled KCl was added. After the lapse of 15 minutes shaking was interrupted and the blood centrifuged in ice- cold centrifuge tubes. Centrifugation is not to be prolonged, as corpuscles when kept at a low temperature lose some of their potassium content. RESULTS In Fig. 1 the activity of 1 gm red corpuscles is plotted against time after intravenous injection of labelled whole blood, while in Fig. 2 the results of experiments are seen in which washed labelled red corpuscles were injected intravenously. ^300 ■^200 ° 100 a. V) Case 2. Red blood corpuscles 2700qr 38,2qr/kq 2500CC 3S3cc/kq Plasma 3120 « 44,0 « 3060" 43,2 " Circulaf.bl. volume 5820 " 82,2 " 5560- 78,5 " 1 1 1 i 1 1 10 20 30 40 50 60 Minutes after injection 120 Fig. 1. Change in the ^-K content of red corpuscles after injection of labelled whole blood. "K LABELLED RED COKl'USCLES IX BLOOD VOLUME MEASUREMEXTS 575 As distribution coefficients of^-K between 1 gm fresh corpuscles and 1 gm fresh plasma at 37° C in three experiments, each taking 15 minutes, the values of 0.200, 0.209 and 0.205 were obtained. The mean value is thus 0.205. Levi (1945) found a distribution coefficient of 1 after llie lapse of 1 hr, while Mullins and assoc. (1941) determined the time necessary to reach a 30 per cent exchange between corpuscle potassium and plasma potassium to 8.2 hr. Since this paper went into press we became cognizant of a paper by IIakker et al. (1950) and of several papers by Sheppard ct al. (1950,, T 1 1 1 1 r 5 10 20 30 40 50 60 120 Minutes after injection Fig. 2. Change in ■'-K content of red corpuscles after injection of washed labelled corpuscles. Value obtained for the red blood corpu- scle volume 2220 g. 1951) dealing with the in vitro exchange of potassium between red cor- puscles and plasma. Rakker et al. succeeded in maintaining erythro- cytes in an essentially normal state for over 48 hours and in showing that after the lapse of that time the specific activity of plasma potassium and red corpuscle potassium becomes equal, thus a total interchange between plasma potassium and corpuscle potassium takes place within that interval. They arrive at the result that at 37 °C 1.6 per cent of the potassium of the erythrocytes exchange per hour. Numerous data are stated by Sheppard et al. for the extent of potassium interchange be- tw^een plasma and erythrocytes after different times and under different experimental conditions. Their data contain also figures for an incubation time of 17 — 18 min only. When calculating from their figures the ^-Iv loss by the red corpuscles under that time interval, we arrive to a figure of 1.0 — 1.3 per cent, a result which compares with the loss of 1.0 per cent observed by us in experiments taking 15 minutes. From their results obtained when incubating blood in the presence of ^H\. for 2 min only, one can conclude that during 2 min an ^^K loss as large as 0.4 to 1.1 per cent takes place. On the other hand from the experiments of Shep- 576 ADVEXTURES IX RADIOISOTOPE RESEARCH PARD et al. taking 60 min or more, it follows that the red corpuscles lose 2 per cent only of their ^^K content in the course of 1 hour. These and our results indicate the presence of a small rapidly interchanging potassium fraction in the blood corpuscles. These may partly or wholly be due to a rapid interchange between the potassium of the plasma and that of those corpuscles which accumulate in the buffy coat. For canine blood where the rapidly exchanging potassium fraction is very conspi- cuous, Sheppard et al. succeeded in showing that at least an appre- ciable part of their rapidly interchanging potassium is to be looked for in the buffy coat of the isolated blood corpuscles. In experiments of very short duration the amount of ^^K entering from the plasma into the corpuscles during centrifugation furthermore may not be negligible. Lesion of some erythrocytes during the labelling process may also lead to some 42K loss. A removal of the buffy coat which may reduce the ^^K loss of the erythrocytes observed shortly after their injection into the circulation, cannot be considered to be a practical proposition in routine blood volume determinations. DISCUSSION Different methods exist which permit to determine the rate of loss of ^2K by the red corpuscles. We can activate erythrocytes, and after washing them to remove the adhering plasma, shake the active red corpuscles with inactive plasma, for example for 1 hr, and subsequently determine the .activity of the last mentioned plasma. The figure obtained indicates the loss of *2K by the corpuscles in the course of 1 hr. This procedure has the disadvantage that washing may harm the mechanism responsible for the permeation of potassium into the erythrocytes. The permeability of potassium is known to be of a different type from that of phosphate. The strong concentration of potassium in the red corpuscles in among •others easily influenced by addition of glucose to the plasma or by temperature changes. Therefore we preferred another method which is based on the following consideration. If during a certain time a percentage of the ^SR of negligible weight Added to the blood sample penetrated into the corpuscles, we can assume that a corresponding fraction of all potassium ions present at the start of the experiment in the plasma moved into the corpuscles as well. As the potassium concentration of the red corpuscles remains constant during the experiment (a minor deviation of this assumption would not :significantly influence the result arrived at) a corresponding amount of potassium must have moved in the opposite direction, hence from the corpuscles into the plasma. We can thus determine the percentage of corpuscle potassium which moves from the corpuscles into the plasma "K LABELLED EED CORPUSCLES IN BLOOD VOLUME MEASUREMENTS 577 and also the percentage of ^-K which leaves the corpuscles during a given time. From the fact that after the lapse of 15 minutes 1 gm of red corpuscles was found to contain 0.205 times as much ^^K as did 1 gm of plasma and 100 gm of blood to contain 40.1 gm of red corpuscles follows that, out of 100 counts added to the plasma 12 penetrated into the erythrocytes. As the potassium content of the plasma amounted to 17.8 mgm % it follows that 12% of 10.7 mgm = 1.28 mgm of those potassium atoms, which were in the plasma at the start of the experiment, are found 15 minutes later in the corpuscles, and vice versa. When making this statement we failed to consider the increase in sensitivity of the radioactive indicator in the course of the experiment. The plasma activity decreased during the experiment, took 15 minutes from 100 to 88.0, and correspondingly its mean value during the experiment was 94.0. From these figures it follows that the amount of potassium which interchanges between plasma and red corpuscles in the course of 15 minutes is not 1.28 mgm 1.28 but = 1.36 mgm. 0.94 As the red corpuscles contained 325 mgm % potassium, thus those present in 100 ml of blood 130 mgm the 1.36 mgm of potassium, which move from the corpuscles into the plasma in the course of 15 minutes amounts to 1.0 % of the potassium content of the erythrocytes. If we inject labelled erythrocytes into the circulation we can thus expect that 1.0% of their ^^K content is given off to the plasma in the course of the first 15 minutes. The loss of *^K by the active corpuscles when introduced into an inactive circulation can thus be expected to be smaller than the loss of radiophosphorus by ^^p labelled corpuscles during the same time. Reeve (1949); Nylin (1951). This conclusion is borne out by the results of experiments in which 42K labelled blood or red corpuscles were injected Into the circulation and are demonstrated in Figs. 1 and 2. After injecting labelled blood into the circulation, the activity of the red corpuscles seems to remain unchanged during 1 hr within the error of the method, which amounts to about 3 %. Injection of labelled red corpuscles is followed by an initial loss of about 2 % of their ^^j^ content, followed by a further loss of 5 % in the course of the first 2 hours. As we found a loss of 43 % in the course of 24 hours, the mean loss of ^^K by the labelled erythrocytes per hour works out to be 2.1 %, a figure almost identical with that found by Sheppard and slightly larger only than the new figure found byRAKKER in in t^iYro experiments. That no perceptible loss of ^^K by the erythrocytes was observed following injection of labelled whole blood may be due to an entrance of ^^K from the labelled plasma into the unlabelled corpuscles, which may compensate slight losses of •*2K by the red corpuscles. 37 Hevesy 578 ADVENTURES IN RADIOISOTOPE RESEARCH If we wish to apply labelled red corpuscles as a clinical tool, it does not suffice that the erythrocytes conserve their label during the deter- mination to be carried out. It is of great importance as well that the labelling process should not take more than some minutes, that the radiation emitted by the labelled corpuscles is easily measurable and that time consuming operations as separation of the erythrocytes from plasma can be avoided. We found thorium B labelled red corpuscles to respond to all these requirements. Thorium B labelled erythrocytes can be prepared by leading a stream of oxygen containing thoron (thorium emanation) through a blood sample for a few minutes only. A large part of the thoron absorbed by the blood sample, as its half-life time amounts to 55 seconds only, decays within the sample. As a result of this disinte- gration radioactive ThB is formed which decays with a half time of 10.6 hr. Most of the ThB formed in the plasma is taken up by the red corpuscles. Due to this fact and also to the very speedy disappearance of ThB from the injected plasma the activity of the blood samples secured after injecting ThB labelled blood into the circulation is almost exclusively due to the activity of the red corpuscles. A separation of the red corpuscles from the plasma in the secured blood samples can thus be avoided. Within the first 2 hr loss of ThB by the labelled corpuscles is almost negligible and for the coming hours restricted. While thorium B emits soft /3-rays its daughter product thorium C with which it soon gets in exchange equilibrium emits |S-rays of similar penetrability as does ^^P. When measuring the radioactivity of thorium B labelled erythrocytes we are thus mainly measuring the ^-radiation of thorium C. That the activity of thorium B -}- C decays with a half-time of 10.6 hr has the advantage that the organism is exposed to radiation for a very restricted time only after the radioactive isotope is introduced into the circulation, less than 1/5 of the isotope being present after a lapse of a day. A more detailed description of the above outlined method and the results obtained by its application will be soon published. Summary Red corpuscles were labelled by adding ^^KCl to a blood sample kept at 37° C for 2 hr. When reinjecting the blood sample to the patient in the course of 1 hr, within the error of the blood volume determination which is 3%, no change in the activity of the red corpuscles could be observed. Washed labelled red corpuscles injected into the circulation lost in the average 3.5% of their *^K. content in the course of the first hr, while the mean loss per hr in the course of 24 hr was found to be 2.1%. "K LABELLED RED CORPUSCLES IN BLOOD VOLUME MEASUREMENTS 579 References K. Zerahn (1948) Acta Physiol. Scand. 16, 117. H. Levi (1945) Kgl. Danske Videnskahs Selskab. Mat. Fys. Medd. 23, No. 10. L. J. MuLLiNS, W. O. Fenn, T. R. Noonan and L. Haege (1941) Amer. J. Physiol. 135, 93. E. B. Reeve and N. Veal (1949) J. Physiol. 108, 12. G. Nylin and G. Wade (1951) Ark. Kemi 3, Nr. 45. G. W. Rakker, I. M. Taylor, J. M. Weller and A. B. Hastings (1950) J. Gen. Physiol. 33, 691. C. W. Sheppard and W. R. Martin (1950) J. Gen. Physiol. 33, 703. C. W. Shepp.ajid, W. R. Martin and G. Beyl (1951) J. Gen. Physiol. 34, 411. C. W. Shepp.ard and G. E. Beyl (1951) J. Gen. Physiol. 34, 091. C. W. Sheppard (1951) Science 114, 85. 37* Originally published in Circulation Research 1, 102 (1953). 59. APPLICATION OF 'THORIUM B" LABELLED RED CORPUSCLES IN BLOOD VOLUME STUDIES George Hevesy, and Gustav Nylin From the Cardiological Clinics, Sodersjukhuset, Stockholm A method for determining blood volume utilizing thorium B, which has advantages over the use of ^sp, is described in detail. The problems of protecting the patient and operator from radiation activity are discussed in an appendix. The successful application of radioactive indicators in physiology caused the early introduction of a radioactive component into the erythro- cytes and their use in blood volume determinations. Radio-iron labelled eryhrocytes found extended apphcation in animal experiments^^^ These are not suitable for determination of the blood volume of humans, since iron-labelled red corpuscles can only be obtained in vivo, and thus the application of this method depends on the availability of iron-labelled donors. On the other hand the use of in vitro with P^s labelled red corpuscles^^^ has found a very extended application in clinical blood volume cleter- minations^^' *). This method necessitates incubation of the blood sample with radioactive phosphate for an appreciable time. It also requires centrifuging of blood samples and washing of red corpuscles, which consume some time. An ideal radioactive method of clinical blood volume determination should fulfill the following conditions: (a) The radioactive source should be available at a moment's notice, and should not need replacement for some years. _ (b) The rays emitted by the radioactive indicator should be easily measurable. (c) No significant loss of the radioactive indicator by the red corpuscles should take place in the circulation in the course of the experiment. (d) The half-life of the radioactive indicator should be sufficiently long, amounting to at least several minutes, to enable a trained nurse to carry out the radioactive measurements without difficulty. The half- life should, however, be shorter, and preferably appreciably shorter than one day, since it may be necessary to repeat the blood volume determination after some time. (e) Centrifugation of the injected or secured blood samples should not be necessary. It should suffice to compare the radioactivity of a known aliquot of the injected labelled blood, poured into a glass "THORIUM B" IX BLOOD VOLUME STUDIES 581 cuvette, or in dry state, with that of a sample secured after injection, in order to arrive at a correct figure for the circulating ])lood volume of the patient. The present communication describes a method of blood volume determination which fulfills to some extent the above conditions. It is based on an observation^^^ that when oxygen carrying thoron is led through a blood sample, an appreciable part of the thoron decays within the sample, and its decay product thorium B (ThB) is almost entirely taken up by the corpuscles, from which it is released at a very slow rate.* METHOD Radio-thorium preparations are easily available. We applied a sample prepared according to the Hahn procedure and having the activity of 2 mgm of radium. It was obtained (price £7 per mihicurie) from Harwell. Such a preparation is a copious source of thoron gas (thorium emanation). It is placed in a small glass vessel of a weighing glass type. Through the stopcock of the vessel two narrow glass tubes are inserted: through one, oxygen is led into the vessel, through the other, the thoron-loaded oxygen is led (for example, for 10 minutes) into the blood sample to be activated. The narrow glass cylinder containing the blood sample is placed in a small wash bottle. Since rubber strongly absorbs thoron, rubber tubing is kept at a minimum. Thoron absorbed by the blood sample decays with a half-time of 55 seconds and is converted into ThB and its disintegration products. Within five minutes, all thoron absorbed by the blood decays, and the activity of the blood sample is now exclusively due to the presence of ThB and its disintegration products, the activity of ThB decaying with a half-time of 10.6 hours. While the larger part, 10 ml, for example, of the active blood sample is reinjected into the human subject, whose blood volume we wish to determine, a small fraction is applied to prepare a standard sample. In preparing such a sample, we add, for example, 0.02 gm of active blood to 2 gm of inactive blood (or saline), hemolyse the sample by adding a few grams of saponin, and pour the hemolysed blood into one of Zerahn's cuvettes^^\ or preferably dry the sample and pour 100 mgm of the dry sample into an aluminium dish 1.2 cm in diameter, or, if a larger blood sample is available, preferably into a dish of larger diame- ter. The activity of the standard sample is then compared with the activity of a blood sample secured, for example, 10 minutes after the * The uptake of ThB by red cells was studied at an early date by Behrens and Pachur [Arch. exp. Path. u. Pharmakol. 122, 319 (1927)]. 582 ADVENTURES IN RADIOISOTOPE RESEARCH injection of the active blood took place. This sample is hemolysed as well, and treated similarly to the standard sample. It is of the greatest importance to compare the activity of dry blood samples having the same corpuscle — plasma ratio. This is facilitated by preparing the sam- ples from hemolysed blood. Thorium B and its Disintegration Products Before describing the measurement of the activity of the ThB-labelled blood samples, it is appropriate to recapitulate our knowledge concerning the disintegration of the active deposit of thorium, and the radiation emitted which follows this disintegration. The sequence of the radioactive disintegration products of radio- thorium (RaTh) is shown in the following schema: RaTh -^ ThX -> Tn -^ ThA 1.90 years 3.64 days 56 sec 0.16 sec 1 208Pb ^ 65%ThC" ^ The ^ 60.5 i ThB 10.6 min I 35%The" 3.1 kr min 1 208IJb Thorium B emits, as seen from the above schema, soft /5-rays, which are half-absorbed by a blood layer of 0.4 mm thickness. The disintegration products of ThB, ThC andThC " emit 7.5 times and 4.8 times as penetrat- ing ^-rays as ThB. Furthermore, ThC and ThC" emit a-rays having a range of 31 //, and ThC with a range of 56 ju, in dry blood; y-rays are •emitted as well. To what extent these radiations participate in producing ions in the Geiger tube, depends on the thickness of the layer, that of the window of the counter and the volume of the counter. The complexity of the radiation emitted by ThB and its disintegration products is in no way disturbing, as we are solely interested in the comparison of the activity of blood samples emitting the same complex type of radiation. When we use thin window counters, as applied when measuring the activity of i^C, some of the strongly ionizing a-rays emitted by the dry blood sample penetrate into the counter and contribute to the total number of counts produced. When we use glass cuvettes, however, these and other soft rays are stopped by the window of the cuvette; furthermore, these, and also harder rays, are partly absorbed by the water content of fresh blood. If we wish to administer a very restricted "THORIUM B" IX BLOOD VOLUME STUDIF;S :83 dose, we prefer to compare the activity of dry blood samples placed in an aluminium dish, and use thin window counters as when measuring the activity of i^C. The approximate half-value thickness of the ^-rays emitted by ThB and its disintegration products in aluminium is as follows: ThB (lead isotope) 0.10 mm The (bismuth isotope) 1.2 mm The" (thaUium isotope) 0.77 mm Of the disintegration products of thoron, ThA decays in a half-time of 0.16 second and can thus be disregarded. ThB, which has a half-time of 10.6 hours, takes about 6 hours to come into exchange equilibrium with its disintegration products. This is a fact of importance in inter- preting the activity figures obtained for ThB-labelled blood samples. After leading thoron through the blood for 20 minutes, for example, the maximum ThC activity of the sample is not obtained until after the lapse of 200 minutes, as shown by Table 1. In the succeeding 100 minutes, the activity decays by 3 per cent; later a decay with a half- time of 10.6 hours sets in; thus after the lapse of 21 hours, the activity is reduced to Y^, after the lapse of 42 hours to 7i6 ^^^ after 63 hours to Yed- These figures indicate the maximum fraction of the injected ThB still present in the organism. The actual fraction present is, however, Table 1. — Change or the Activity of the Blood with Time after 20 Minutes Exposure to a Uniform Thoron Stream. (Only the Activity due to the Rays Emitted by Thorium C and Its Disintegration Products Is Considered) Time in minutes Activity Time in minutes Activity 1.0000 1.0960 1.4682 1.9073 100 6.3626 6.7777 7.1787 7.4534 1 120 150 5 10 200 20 2.7060 300 7.2408 30 3.4077 4.3009 400 6.6706 6.0705 45 500 60 5.0309 600 5.4315 90 6.0998 800 4.3729 lower than the figures stated, as some ThB is lost by excretion as well as by disintegration. Activation of Blood We obtained the best results by the following procedure: Through 10 ml of freshly drawn heparinized blood, an oxygen stream of about 30 ml per minute percolates for 20 minutes after it has passed through 584 ADVENTURES IN RADIOISOTOPE RESEARCH the vessel containing the radio-thorium preparation. The tube containing the blood is placed in a small wash bottle. After leaving the wash bottle, the oxygen stream passes through four more wash bottles containing vegetable oil, which absorb all or most of the thoron still present in the oxygen stream. The activated blood is then gently shaken at room tem- perature for 30 minutes; 9 ml are reinjected into the patient, a known aliquot of the rest being applied to prepare a standard sample as described above. We found it less advantageous to incubate blood at 37° C than at room temperature. Instead of leading thoron containing oxygen through blood, we can dissolve the "active deposit" of thorium collected on the surface of a platinum foil in blood. Most of the ThB introduced into the blood accu- mulates in the corpuscles, though not to such a large extent as after thoron activation. After 30 minutes incubation at room temperature, about 6 per cent of the ThB is still found to be present in the plasma. When choosing this method of labelling red corpuscles we have, there- fore, to replace the active by an inactive plasma before injecting the labelled blood. In the above experiments, the active deposit of thorium w^as collected* for 24 hours, on the surface of a platinum foil connected with the negative pole of a 220 volt (or preferably 300 volt) circuit. The platinum foil is then placed in the blood sample, which is gently shaken at room temperature for 10 minutes. The blood is then centri- fuged, and the active plasma replaced by inactive plasma, before the blood is injected into the circulation. Evaluation of the Activity Figures As already mentioned, we measure, besides the counts produced by the rays emitted by ThB, to a large extent those emitted by ThC and its disintegration products. Since practically all ThB is taken up by the red corpuscles, the plasma does not contain this radioelement; it contains, however, appreciable amounts of the bismuth isotope, ThC, which does not accumulate in erythrocytes. Some of the ThC present in the plasma of the injected blood escapes in the course of the experi- ment through the capillary wall, while the ThC present in the standard sample has no way of escape. The ratio between the activity of the stan- dard sample and a secured sample shortly after securing the latter does not, therefore, afford a correct measure of the circulating blood volume, as indicated by the formula stated below. We can correct for the loss of ThC from the circulation during the experiment, or we can avoid such * The collecting vessel used is decribed among others in W. Makoweb and H. Geiger, Practical Measurements in Radioactivity. London (1912) and in G. Hevesy and F. A. Paneth Manual of Radioactivity. London (1938). "THORIUM B" IN BLOOD VOLUME STUDIES 585 a correction by waiting for a few hours, until tlie TliC of the plasma, which has a half-life of one hour, has decayed. But even in this case, when comparing the activity of different samples, we have to bear in mind that a decay of half of the ThB present takes place in the course of 10.6 hours. In evaluating the results of the activity measurements, we can avoid correcting for the change in the activity with time by comparing Ihe activity of the sample secured with that of the standard sample, provided both change their activity with time in the same way. If the secured sample has an initial activity of 200 counts per minute, for example, we will register, in the course of 10 minutes, almost 2000 counts. In fact, as the activity has decreased in 10 minutes from 200 to 197.8 counts per minute, we will measure only 1987 counts in the course of 10 minutes or 198.7 per minute. We now measure for one minute the standard sample, having 2000 counts. The ratio between the activity of the standard sample and that of the secured sample works out then as — = iq 07 198.7 If greater accuracy is wanted, we again measure for 10 minutes the secured sample which has now a mean activity of 196.4, and repeat this procedure. Thus the correct activity ratio between standard and secured sample works out as 2000 2000 ^^j2 198.7 + 196.4] 197.6 2 As the standard sample is prepared by diluting active with inactive blood, a strongly active standard sample can be easily obtained. The measurement of the activity of such samples takes, in contrast to the 200 H iOO ^ n 1 1 1 — 36 15 I 2 Ser.opHs Hours Fig. 1. Change in the activity of blood with time after injection of thorium B labelled whole blood. secured samples, only very few minutes. When applying an automatic sample changer, which shifts five secured samples and one standard sample at 10 minute intervals, and permits reading the counts on six independent telephone counters, the following correction for decay has 588 ADVENTURES IN RADIOISOTOPE RESEARCH to be made on the registered counts. Since counter 2 starts and finishes counting 10 minutes later than counter 1, and during this time 1.1 per cent of the ThB has decayed, the counts registered by counter 2 must be increased by 1.1 per cent of their value. Similarly the counts registered c E en o ^ 200- < —I 1 1 1 ' 1 1 1 r- 10 20 30 40 50 60 2 3 4 Minutes Hours Fig. 2. Change in the activity of blood with time after injection of thorium B labelled whole blood. by counters 3, 4, 5 and 6 have to be increased by 2.2 per cent, 3.3 per cent, 4.4 per cent and 5.5 per cent, respectively, of their value, to make them comj)arable with the counts registered by counter 1. Fig. 3. Change in the activity of the blood with time in a case of polycythemia after injection of thorium B labelled whole blood We found it very convenient to place five secured and one standard sample in an automatic sample chamber which shifts the samples every 10 minutes and registers — on six separate telephone counters— the counts produced during the night. Even blood samples of very restricted activity could be measured with satisfactory accuracy by this procedure, as seen in Table 2. "THORIUM B" IX BLOOD VOLUME STUDIES 587 The procedure cannot be used when the result is wanted urgently. In such cases we centrifuge the blood sample before injection, and replace the active by an inactive plasma, thereby removing the disturbing ThG present in plasma. Applying this procedure we can also obtain labelled red cells by dissolving ThB collected on the surface of a platinum foil, which is then placed into the blood sample. This type of activation of the blood sample takes only a few minutes. Decaying actinium supplies emanation (acton) as well, which in tuin produces in the blood sample AcB, which has the same chemical pro perties as ThB, and AcC which has the same chemical properties as ThC. While one-half of the ThC present decays in one hour, the half-life of AcC is only 2.2 minutes, and this product correspondingly disappears from the plasma of activated blood in a few minutes. We are at present investigating the possibility of applying AcB-labelled red corpuscles in circulation studies. Calculation of the Blood Volume If we inject G gm of labelled blood, express the activity of the secured sample by P, that of the standard sample by S, and the dilution figure of the standard sample by D, the blood content of the human subject SG in gm X works out as X . We then compare the activities of 100 mgm of dry blood of the secured and of the standard sample, or the activities of these samples after being poured into cuvettes. It is assumed that all ThB is concentrated in the corpuscles; in fact, about 1 to 2 per cent of the ThB content is located in the plasma. About four-fifths of this ThB leaves the circulation within a few minutes. Correspondingly, the above formula overestimates the blood content by about 1 per cent and should be replaced by ^ 0.99 SG X = PD If, for example, S = 2000, P = 200, G = 10 g and D = 0.02, X works out as 4950 gm. Should we wish to compare the activity of cuvettes, one of which contains hemolysed blood, while the other (the standard sample) contains a hemolysed suspension of red corpuscles in saline, owing to the somewhat lower absorbing power of the latter sample for /9-rays, we have to multiply the above formula by 0.95. While Zerahn's cuvettes are most convenient for use in determining the circulating blood volume, the high water content of the 1.5 cm 588 ADVENTURES IN RADIOISOTOPE RESEARCH of fresh blood placed in the cuvette absorbs much of the radiation of the sample. An activity five times as large is registered when measuring the activity of 100 mgm of dry blood as in the measurement of 1.5 ml of fresh blood placed in Zerahn's cuvette. EXPERIMENTAL RESULTS Figures 1, 2 and 3 demonstrate the change in the ThB content (activity) of the circulating blood with time. The third case investigated was one of polycythemia, in which the mixing of the injected and the circulating blood was probably slower than usual, as the maximum activity of the blood was reached after only five minutes. Figures 4, 5, 6 and 7 demon- strate the results of blood volume determinations carried out by using first ThB-labelled erythrocytes, then ^^P-labelled red corpuscles. When we carried out the experiments, the result of which is shown by Figures 1 to 7, we were not yet cognizant of the beneficial effect of a 30-minute incubation of the active blood previous to its injection. Nevertheless, the ThB label was found to be better preserved than the ^^P label. Results of the experiment in which blood incubated for 30 minutes was injected, are seen in Table 2. Table 2. — Change in the ThB Content of Circulating Blood with Time Time between injection and sampling in minutes 3 5 15 30 60 Standard sample Activity of dry samples secured from human subjects (Counts per minute) Relative activities 69.3 37.2 82.4 39.7 100 100 100 72.5 57.6 82.5 39.6 104.2 101 100.1 72.6 36.9 84.6 39.9 104.3 98.6 102.7 71.8 38.1 84.9 41.3 103.4 102.4 103.2 71.4 35.7 82.1 39.1 102.9 96.2 99.7 775 1783 4113 204 1120 4780 4990 100 99.7 100.3 104 98.4 513 In this experiment, in the course of 57 minutes, the ThB loss by the circulating blood was found to be —3.0 per cent, +4.3 per cent, -|-0.4 per cent, and 1.5 per cent, respectively, which compares with a loss of about +6 per cent in the case of ^^P-labelled red corpuscles. In the course of 24 hours, one-third to one-fourth of the ThB content of the red cells is given off, which compares with a 50 per cent loss of ^-P by the labelled erythrocytes.*^7) "THORIUM B" IN BLOOT) VOLUME STUDIES 589 1969 Jan.29*^ 1952 1 I 400 300 c E O) o o. > 200 100 min — I — 10 Dried samples Hemolised samples measured in cavettes Red blood corpuscles 2400 g 34,1 g /kg 2230 cc 3l,7cc/kg PiQsmo 3270 « 46,5 < 3180 « 45,2 « Circulaf blood volume 5670 « eo,6 ' 5410 '1 76,9 ' Age Weight Heart volume Hemoglobin Hematocrit Red blood corp. F1 years 70.5 kg 1290 cc :705/m» F1% 42% 4,2 mill. Red blood corpuscles 2280 g . 32,3 g /kg 21I0CC 30,0cc/kg Plasma 3100 " 44,0 " 3010" 42,7 « Circuiot biooa volume 5380 « 76.3 " 5120" 72,7 - — 1 — 20 30 — I — 40 50 — I — 60 Time offer injecrion -I 2 Hours Fig. 4. Comparisons of data obtained by injections of thorium B labelled blood cells siispended in inactive plasma (above) and P^^ labelled cells (below). l970Febr2'"'l952 700- 600 500- c 1 o a. *^ u < 400- 300 200 Red blood corpuscles I330g 28,3 g /kg 1230CC 26,2cc/kg Plasma I840> 39.2 - 1790 « 38,1 « Circ blood volume 3170" 67,5 ■' 3020 " 64,3 ■ oo ^ INJECTION OF WHOLE BLOOD LABELLED WITH ^sp Febr. 8'*'I952 Red blood corpuscles Ii70g 24,9 g /kg lOeJcc 23,1 cc/ kg Plasma 1885 " 402 » 1650" 39,4 " Circ. blood volume 3055" 65,1 " 2933 " 62,5 " Age 66 years \A,eigh1 47 kg hemcglcbin 79% Femotccrit 42 % Red b'ccd ccrp. 4 mill. min. —I — 10 20 30 — I — 40 50 60 Time after injection 2 Hours Fig. 5. Comparisons of data obtained by injections of thorium B labelled blood cells suspended in inactive plasma (above) and ^-P labelled cells (below). 590 ADVENTURES IN RADIOISOTOPE RESEARCH 1977 Dec. n*" 1951 c o > HOOD 10000 - 900O- Red blood corpuscles 2570g no g /kg 2380 cc 25,0cc/kg Plasma 2880 " 30,2 " 2820 " 29,G " Circulat blood volume 5450 « 57,2 1 5200 " 54,6 • INJECTION OF WHOLE BLOOD LABELLED WITH ^^p Red blood corpuscles 2550 g 26,8 9 /kg 2360cc 24,7 cc/ kg Plasma 2710 " 28,5 " 2660" 27,9 " Circulat. blood volume 5260 1' 55,3 " 5020" 52,6 !• Age 45 years Weight 95,3 kg Hemoglobin 91% Hematocrit 47% Red blood Corp. 4,6 mill. Heart volume 1090 cc 510 cc/m" 1 1 r— O 10 20 30 Hinutes ofter iniecMon 40 50 60 Fig. 6. Comparisons of data obtained by injections of thorium B labelled blood cells suspended in inactive plasma (above) and ^ap labelled cells (below). 1971 Dec 10*'' 1961 \ L 500 400 300 c o a. 2100- 2000 1900 Red biooa corpuscles 1080 g 29,0 g /kg lOOOcc 26,8cc/kg Plasma 1002 » 26,9 '■ 1000 " 26,8 " CircuiQt blood volume 2082 " 55,9 " 2000 " 536 • INJECTION OF WHOLE BLOOD LABELLED WITH 32p Dec 13*" 1951 Red blood corpuscles I060g 28,4 g /kg 983 CC 26,4 cc/kg Plasma 1020 " 27,3 « 1000 " 26,8 " Circulot blood volume 2080 " 55,7 " 1983 « 53,2 " Age Weigtit Hematocrit 19 years 37.3 kg 50% 1 \ 1 1 1 1 — 10 20 30 40 50 60 Minutes offer injection Fig. 7. Comparisons of data obtained by injections of thorium B labelled blood cells suspended in inactive plasma (above) and ^^p labelled cells (below). "THORIUM B" IN BLOOD VOLUME STUDIES 591 Table 3. — ThB Content of 1 gm of Fresh Plasma. Expressed in Percentage OF THE ThB Content of 1 gm of Fresh Corpuscles, after Incubation of the Active Blood for 20 Minutes at 37° C Time in minutes of tlie passage of the thoron stream before incubation Percentage of ThB present iu phisma 1.5 2.4 30 45 60 75 1.3 0.97 0.72 0.63 90 105 120 0.53 0.50 0.50 10 30 1.6 0.83 10 30 30 1.2 0.86 0.76 Distribution of ThB Between Red Corpuscles and Plasma Immediately after leading thoron-charged oxygen for 20 minutes through a blood sample, the ThB content of 1 gm of fresh plasma is found to contain 4 per cent of that of 1 gm of red corpuscles. If we (K 10 -C — °s c Ti50 01 ^40 a> c ^30 CO r. >- vo a> TT> •E'O O) o fc Q. o E tn «D° £^? ff.E , a, c y c i|U Red cells Plasma 12 3 4 Hours elapsed afrer injection of active plasmc Fig. 8. Uptake of thorium B by red cells following injection of labelled plasma. 592 ADVENTURES IN RADIOISOTOPE RESEARCH wish to avoid this restricted activity, we can replace the active plasma by inactive plasma, and inject the blood sample, the activity of which is now exclusively located in the erythrocytes. We can, however, reach almost the same result by incubating the active blood for about 20 minutes at 37° C. The percentage of ThB still present in the plasma after incubation is seen in Table 3. The ThB content of the plasma can be strongly reduced by incubating the blood for 20 minutes, as seen in Table 3. Table 4. — Loss of ThB by Injected Active Plasma (2 X 10^ Counts Injected. Plasma and CoBPixscLE Content of the Httman Subject = 3020 gm AND 1990 gm Respectively) Time in minutes after injection Per cent ThB still present in the circulating plasma Per cent ThB located in the circulating red corpuscles 5 1.25 18.5 30 0.61 18.9 60 0.30 20.8 120 0.21 20.8 24 hours 25.6 In incubating active corpuscles with inactive plasma in vitro for 1 hour, 1 to 2 per cent of the ThB of the corpuscles enter the plasma phase. Loss of ThB by Injected Active Plasma ThB present in the injected plasma is lost at a remarkable rate, as shown by the data of Table 4 and in Figure 8. Part of lost ThB pene- trates the capillary wall, and part intrudes into the blood corpuscles. In some of our experiments, we found the percentage incorporation of the injected plasma ThB in the circulating erythrocytes after the lapse of 3, 60 and 360 minutes, respectively, to be as high as 25, 40 and 45. These figures compare with 1, 5 and 9 for ^sp infusion into the red cor- puscles following injection of ^sp-labelled plasma*^^\ Summary 1. Thorium B (ThB) is a disintegration product of the radioactive gas thoron. The procedure for preparation of ThB is described. 2. The advantages of ThB for blood volume determinations are that it accumu- lates to 99 per cent in red corpuscles, is released more slowly than radiophosphorus (loss less than 4 per cent in one hour) and has a half-time of 10.6 hours, which permits repeated determinations of blood volume. The radiation emitted by ThB is measured as easily as that of radioactive phosphoinis. "THORIUM B" IN BLOOD VOLUME STUDIES 593 3. The procedure for calculating blood volume is described in detail. In prin- ciple it consists in comparing the radioactivity of original whole blood samples with that of a sample removed from the patient after dilution. It requires no calcu- lations of cell — plasma ratios; ot;ntrifugat ion is unnecessary. APPENDIX Radiation Protection and Radiation Exposure Among the components of the active deposit of thorium, produced through decay of thorium emanation, is found thorium C ' which emits hard y-rays. In view of the penetrating rays emitted by the radiothorium sample, the operator must be protected from the effect of this radiation. Placing the glass tube contain- ing the sample in the center of a lead block 4.5 by 4.5 by 4.5 cm., reduces the intensity of the y-rays emitted to about one-eighth, as 1.5 cm of lead cuts down the radiation by half. A still more effective precaution is to increase the distance between the sample and the operator. While the y-radiation of radiothorium having the activity of 1 mgm of radium produces, at 1 cm distance, a dose of 8.6 roentgen equivalent physicals (rep) per hour, at a distance of 100 cm, only 1/10,000 of that dose is produced. When passed through the blood sample, the oxygen still contains some thoron. Though, owing to the short hfe-time of thoron, the activity released into the atmos- phere is rather restricted, it is advisable to lead the oxygen stream which has already left the blood through an aggregate of wash bottles containing olive oil or some other vegetable oil, before releasing it into the atmosphere. While the distribution coefficient of thoron between water and air at 20° C amounts only to 0.26, the corresponding figure for ohve oil and air is as high as 28. Radiation dosage is always an important consideration when applying radio- active indicators. Owing to the short half-life of ThB, the patient is exposed to radiation for a much shorter time than in administering ^^F, for example. This difference is partly offset by the fact that the disintegration products of ThB emit a-rays, which are, in the mammalian tissue, more effective in producing radiation effects than the less densely ionizing /9- and y-rays. The maximum number of rep produced in the body of a human subject weighing 70 kgm by ThB and its disintegration products having the activity of 1 mgm of radium is the following : The emits a-particles having an energy of 9.42 X lO"^ erg. A dose of 1 roentgen equivalent physical (rep) corresponds to the absorption of 93 ergs/gm tissue. This dose is thus produced by 0.94x10^ a-particles; 1 microcurie emits 3.2x109 a-particles per day, producing 3.4x102 rep. Assuming the weight of the human subject to be 70 kgm, 4.8 X 10-^ rep per gram are produced. We stated above the number of a-particles emitted by 1 microcurie in the cours of a day. The effective half-hfe of ThB is, however, only 0.31 day. Furthermore, 35 per cent of the ThC atoms disintegrate only under emission of a-particles. The number of reps, produced by the a-particles of ThC in equihbrium with 1 micro- curie of ThB thus works out to 0.52 x lO^^ rep./gm. One microcurie of ThC, 65 per cent of which disintegrates under emission of a-particles has an energy of 1.46 x 10-^ erg, produces during its hfe-time 1.4 X 10"' rep/gm. The biologic effect of the densely ionizing a-radiation is appreciably larger than that of ^- or y-radiation producing the same number of ions. To account 38 Hevesy 594 ADVENTURES IX RADIOISOTOPE RESEARCH for this difference, the notion of roentgen equivalent man (rem) was introduced, replacing the notion of roentgen equivalent physical (rep). For a-rays 1 rem = = 20 rep (or less), while for /3- and y-rays 1 rem corresponds to 1 rep. In the course of the disintegration of 1 microcurie of ThB + ThC + ThC, thus the a-rays emitted produced not more than 4.0 X 10-2 lep. We arrive at this figure by assum- ing that the whole amount of ThB administered decays in the body. In fact, some ThB is excreted previous to its disintegration. The mean energy of the jS-rays emitted by ThB and its disintegration products is 0.42 Mevs. or 6.7 X 10"'^ ergs per particle. By a similar calculation, as described above, we conclude that the y5-particles of 1 microcurie of ThB produce during its life-time an aggregate dose of 1.1 X 10~* rep/gm. The upper limit of the radiation dose produced by the decay of the y-rays of 1 microcurie of ThB and its disintegration products is obtained by assuming all y-radiation emitted to be absorbed in the body. The mean energy of the y-radiation emitted being 1.1 Mev., the number of rep produced per day per gm body weight works out to 3.0x10—*. The upper limit of rem/gm produced by 1 microcurie of ThB + ThC + ThC. Decaying in the organism is thus 4.0 X 10— ^ -f- + 1.1x10-1 -f 3.0x10-* —4.4x10-2. The dose actuaUy produced lies quite appreciably below this figure. We applied in all our experiments less than 2 micro- curies of ThB. Thus the total maximum dose administered was below 8.8x10-2 rem. References 1. P. F. Hahn, W. F. Bale and W. M. Baxfour, Radioactive iron used to study red blood cells over long periods; constancy of total blood volume in dog. Am. J. Physiol. 135, 600 (1942). 2. L. Hahn and G. Hevesy, A method of blood volume determination. Acta Physiol. Scandinav. 1, 3 (1940). G. Hevesy and K. Zerahn, Determination of the blood corpuscle content. Acta. Physiol. Scandinav. 4, 376 (1942). R. S. Anderson, The use of radioactive phosphorus for determining circulat- ing erythrocyte volumes. Am. J. Physiol. 137, 539 (1942). 3. G. Hevesy, K. H. Koster, G. Sorensen, E. Warburg and K. Zehrahn, Acta Med. Scand. 116, 561 (1944). 4. G. Nylin, Blood volume determinations with radioactive phosphorus. Brit. Heart J. 7, 81 (1945). G. Nylin and S. Hedlund, Further studies of the circulation with radioactive erythrocytes. Am. Heart J. 37, 543 (1949). N. I. Berlin, G. M. Hyde, R. J. Parsons, J. H. Lawtience and S. Port, Blood volume of the normal female as determined with 32p labelled red cells. Proc. Soc. Exper. Biol. & Med. 76, 831 (1951). E. B. Reeve, Use of radioactive phosphorus for the measurement of red cell and blood volume. Brit. M. Bull. 8, 181 (1952). 5. G. Hevesy, Thorium B labelled led corpuscles. Arkiv. Kemi. 3, 425 (1951). E. Alexander, Thorium B labelled red corpuscles. Arkiv. Kemi- 4, 363 (1952). 6. K. Zerahn, A simphfied method for measurement of radioactive corpuscle samples for blood volume determination. Acta Physiol. 16, 117 (1948). 7. G. Nylin and G. Wade, The loss of activity from blood cells and plasma which have been labelled with radioactive phosphorus {^-V). Arkiv. Kemi. 3, 413 (1951). G. Nylin, J. C. Scott-Baker and J. Karnell, Studies on the polycythemic reaction in cyanotic congenital heart disease by means of erythrocytes labelled with 32p. Compt. Bend. Congres Cardiol, Vol. 3, Paris (1950). 595 Comment on papers 57 — 59 In ourfirst determination of the erythrocyte volume in 1940 we availed ourselves of the blood of a rabbit injected with ^-P a few days previously. A known aliquot of the blood of this rabbit was then injected into a receptor rabbit. At different times after injection, the activity of the total acid-soluble P extracted from an aliquot of the receptor blood was compared with the corresponding activity of the same aliquot of the donor blood. In some of the experiments not the activ- ities of the acid-soluble fractions but those of the phosphatide fractions were com- pared. The device described (paper 51) necessitates the availability of a donor and thus could not be applied in clinical red corpuscle volume determinations. We, therefore, replaced, in 1942 the above procedure by an improved one in which the acid- soluble phosphorus compounds present in the erythrocytes are tagged in the course of incubation of blood at body temperature for 1 — 2 hr, and a known volume of this blood containing tagged erythrocytes is then reinjected into the circulation (paper 58). Within the first 10 min after injection, which under physiological conditions amply suffices to carry out a determination, of the red corpuscle volume, the loss of 3-P by the injected human red corpuscles is insignificant ; after the lapse of 1 hr the loss is about G per cent. We injected in our studies labelled whole blood to compensate the small ^ap by a corresponding incorporation of plasma 32P into the circulating erythrocytes (paper 52). Later, mostly labelled red corpuscles suspended in inactive plasma or saline were injected in such deter- minations. Comparing the results obtained when injecting whole blood or suspended erythiocytes, no significant difference was found by Hans Bohr (1954). ^^p {^ vitro tagged erythrocytes found a very extended application in the forties and the early fifties. At present, radiochromate labelled erythrocytes are mostly applied in the determination of red corpuscle volume. The most extensive clinical application of ^-P tagged red corpuscles starting at a very early date is due to Nylin. ^-P labelled red corpuscles found also application in his recent studies on brain circulation In vivo labeUing of erythrocytes through incorporation of radio-iron into their haemoglobin was introduced by Hahn et al. (1941), thus soon after the first appli- cation of in vivo ^-P labsUed erythrocytes in blood volume determiiiations (paper 51). Radio-iron in vivo tagged erythrocytes proved to be very useful in animal experiments, but were not suitable for clinical application (since they necessitated a ^^Fe activated donor). Also *-K labelled erythrocytes found application in red corpuscle determinations (paper 58). ^-K labelling of red corpuscles in vitro takes place about as rapidly as that of ^^p labelling. ^'^K is retained better, and as its life-time is about thirty times shorter than that of ^^P, a hum^m subject is exposed, after injection of ^^K labelled erythrocytes, to a smaller radiation dose than after injection of ^^P label- led red corpuscles of the same activity. The disadvantage of the method is that fresh ^-K must be procured about every second day. An isotope with a still shorter life-time than ^^K is the lead isotope ThB. By letting an oxygen stream strike a radio-thorium sample, the former carries gaseous thoron given off by radio-thorium. The thoron having a half-life of 56 sec only decays to an appre- ciable extent when passing a blood stream producing ThB, which is almost quanti- tatively taken up by the erythrocytes (paper 53 and Alexander, 1953). Activation 38* 596 ADVENTURES IN RADIOISOTOPE RESEARCH of the erythrocytes takes only minutes, and the ThB is better retained by the red corpuscles than 32p or ^'-K. Since the plasma is practically inactive, it does not have to be removed before injection. This very convenient method (59) has two drawbacks. Emanating radio-thorium preparations which are precipitated with iron hydroxide lose with time much of their emanating power, as already noticed by Otto Hahn to whom this method of preparations is due. The dis- integration products of ThB, which are the ThC products, emit a -particles. These densely ionizing particles are several times as biologicaUy effective than f^- or y-rays. The increasing biological activity of the a-rays is compensated by the short hfe-time of ThB, and correspondingly the subject investigated is exposed to radia- tion for a short time only. The not entirely unjustified reluctance to achninister a-rays emitting radioactive substances to human subjects, is responsible for the fact that this method has not found an extensive appHcation. References H. BoHB (1954) Bestemmelse af Blodvolumet med radioaktivt Fosfat. Schuhz For- lag, Copenhagen. G. Nylin (1945) Ark. Kemi A 20, 1. O. Nylin (1953) Acta Med. Scand. 147, 275. P. F. Hahn, W. M. Balfour, J. F. Ross, W. F. Bale and G. H. Whipple (1941) Science 93, 87. E. Alexander (1953) Ark. Kemi 4, 304. Originally published in Naturwissenschaftliche Rundschau 13, 247 (1958). 60. CANCER ANAEMIA Paper read at the Lindau Conference of Nobel Laureates in 1957 Numerous data are to be found in the literature on the haemoglobin concentration in the blood of cancer patients; for example, Shen and HoMBURGER/i^found that more than 60 per cent of the cancer patients whom they studied showed a haemoglobin concentration in the blood which was 20 per cent or more below the normal. In the study of animals with cancer it was also found, even shortly after the inoculation of rats, mice, rabbits and other animals with cancer cells, that the haemoglobin concentration in the blood decreases. Five days after the inoculation of rats with Ehrlich's mouse carcinoma the number of red blood corpuscles fell from 8.55 X 10^ to 7.62 x 10^ per mm^ and the haemoglobin concentration in the blood from 96 per cent to 74 per cent. When 13 days had elapsed the haemoglobin concentration was only 46 per cent^^) . Seven days after the inoculation of mice with mammary-gland adenocarcinoma, a tumour was developed which had a diameter of 0.5 cm and the haemoglobin concentration had decreased by about 7 per cent; when the tumour diameter had reached 3 cm the haemoglobin concentration had fallen to half the normal value. <^) In so far as the blood volume is normal, a comparison of the haemo- globin concentrations and contents in cancer patients and healthy people yields the same result. The blood volume (plasma volume) of the can- cerous organism, however, is frequently not normal but enhanced. The enhanced plasma volume is significant according to experimental data which have been obtained in our lal)oratory (Fig. 1). They show, among other results, the haemoglobin concentration, blood volume and the haemoglobin content, calculated from these values, for forty-two nor- mal and cancerous mice, as determined 15 days after injection with Ehrlich Ascites cells. As a result of the 31 per cent enhanced plasma volume of the cancerous animals their blood volume is increased, and when this increase is taken into consideration it is found that the total haemoglobin content of the blood in the controls and in the cancerous animals shows no significant difference, whereas the haemoglobin con- centration in the blood of the cancerous animals is decreased by 18.9 598 ADVENTURES IX RADIOISOTOPE RESEARCH per cent. A similar result was an increased plasma volume in cancerous animals first observedby Furth andSoBEL^*^ and later by Ehrenstein, whose studies we shall discuss later. An increased plasma volume is especially evident with tumours producing oestrogens.^^) An increased plasma volume w'as also determined in numerous patients suffering from cancer. Berlin and co-workers(^^ studied 66 cases and found that more than one-quarter of them had a plasma volume greater than I 'be S s 03 bc I o CM n X) ; (u I o 'bi) O ;2r S =3 So i I i i : '- in - O r .in ■ QJ -t^ , -p ^ at S-, bc 0) •-! sma y we -^ >. O 'O (S ^ --H O — o -^ x> ft^ S bc 1 s i I 1^ o o Fig. 1. Haemoglobin concentration and total haemoglobin content in the blood of the mouse. Normal — normal. Krebs — cancerous. 46 ml per kgm. They considered plasma volumes lying between 32 and 46 ml per kgm body weight to be normal. Kelly and his collaborators^'^ observed a plasma volume of 63 ml per kgm in thirty-three patients with advanced cancer. The total water volume of these patients was normal. A decrease in concentration of plasma proteins usually goes hand in hand with an increase in plasma volume. Fig. 2 shows the concentration of plasma protein in normal and cancerous mice as determined by Lock- ner in our laboratory. Bernfeld and Homburger^^^ found a 24.5 per cent decrease in albumen concentration in the plasma of mice with tumours. The increase in plasma volume is essentially due to entry of water and salts into the vascular volume. A reduced haemoglobin concentration in the blood of cancerpatients can, however, often be observed, which is not attributable only to an increase in the plasma volume. Ross and his co-workers^^°\ for instance, CANCER ANAEMIA 599 have been able to prove anaemia in three-quarters of the leukaemia or advanced cancer cases which they have studied, even when taking into account the blood volume. Since bleeding was non-existent in these patients there was no visible haemolytic anaemia or anaemia due to defective nourishment, and the anaemia was clearly attributable to curtailment of the life-time of the red blood corpuscles. It has repeatedly been demonstrated that the life-time of red blood corpuscles is often curtailed in cancer and leukaemia. Berlin and his co-workers^"^ observed 6.0 5.0 c- 4,0 E o ^ 2,0 1.0 • Cancer o Control _L. 15 20 25 Days after concor inoculation 30 Fig. 2. Concentration of plasma protein in normal and cancerous mice. a life-time of only 18 days for the red blood corpuscles in a patient suffer- ing from leukaemia instead of the 120 days in a healthy subject. In these studies the erythrocytes were labelled with i*C. Glycine labelled with 1^0 was given to the patient. For some days there was a decided incorpo- ration of i^(y' into the haemoglobin, but this later decreased to a very low value. In these investigations the erythrocytes formed in the course of the early days, were mainly labelled with i*C. This is the best method of labelling the red blood corpuscles, but it is only rarely applied in cli- nical studies because one is unwilling to introduce long-lived radioactive substances into the human organism. It is true that, after the addition of 100 jUG of glycocoll-2-i^C, which is sufficient for a determination of the life-time, the body is only exposed to a radiation dose of 37 mrep during the first 3 months and later to a steadily decreasing radiation^^^^ The incorporation of ^^C into skeleton is very small. In experiments with mice, in our laboratory, Zacharias found that only 1/20,000 of the injected glycine-2-i4C was present in the skeleton after 6 months had elapsed. The radiation dose given to the patient is therefore quite small. A far more serious clinical disadvantage of the method, however, is that blood samples must be taken from the patient through a period of several 600 ADVENTURES IN RADIOISOTOPE RESEARCH months because variations from normal behaviour are often observed only after such a long time. It is different when red blood corpuscles are labelled with radioactive chromate^^^^ The labeUing is far less stable than in the case of "C. One-half of the chromium leaves the healthy red blood corpuscles in a time of from 26 to 32 days. The labelling chromium disappears even earlier from the red blood corpuscles when the life- time of the erythrocytes is curtailed. This method is of widespread appli- cation in the study of the life-time of erythrocytes in cancer patients. It is sufficient, in this instance, to observe the patient for a few weeks. Ashby's serological method has also been applied repeatedly to deter- mining the life-time. In this method, the fate of normal blood corpuscles of the 0-group, transfused from another organism, is traced in the circu- latory system of the patient. Tracing the life-time of heterologous blood corpuscles in the circulatory system of the acceptor, however, may lead to erroneous results in certain conditions. Curtailment of the life-time of erythrocytes in cancer patients has often been observed with the aid of the two methods just mention- gj(i4, 15, 16, 19, 10) YoT example, the erythrocytes in a patient with severe cancer, labelled with ^iCr and investigated by Keiderling^^^^, had already lost half of the labelled atoms after 12 days. The studies mention- ed above were all performed on severe cases. We had the opportunity to study patients with cancer of the cervix, in the Gynaecological Divi- sion of the Radiumhemmet in Stockholm, which is directed by Dr. Kott- meier; the majority of these were exposed successfully to therapy after the study and later reached a stationary condition. A study by Dal Santo^^^^ indicated that in these instances there was a curtailment of the life-time in about one-half of the cases studied. The question now arising is how the frequently observed curtailment of the life-time of the red blood corpuscles comes about in the cancerous organism. The red corpuscles may be exposed to extracorpuscular effects in the cancerous organism and abnormal erythrocytes may also be developed in the can oez-o us organism. Red corpuscles have repeatedly been transfer- red from cancerous to normal subjects and these have not infrequently indicated a prolongation of the life-time^^^' 2^' ^^>. The transfer of nor- mal erythrocytes into the circulatory system of cancer patients not infre- quently resulted in curtailment of the life-time of the erythrocytes. Ross and Miller^-"^^^ mention that when normal blood corpuscles had circulated in the system of a neoplastic organism for 7 days and then been re-introduced into the normal organism they no longer exhibited the behaviour of normal blood corpuscles. From these results the conclu- sion can be made that blood corpuscles are subject to an injurious extra- corpuscular effect in the circulatory system of patients suffering from neoplasms. CANCER ANAEMIA 601 The question of whether the curtaihiicnt of the life-time of erythrocytes is attributable to an extracorpuscular effect or to their abnormal syn- thesis can easily be decided by the study of animals. The erythrocytes existing in the healthy organism can be labelled with ^H^ and after incorporation, which proceeds for only a few days at a noticeal)le rate^ the organism is inoculated with cancer cells. If the inoculation with cancer cells affects the life-time of the labelled erythrocytes, it is then certain that extracorpuscular agents are playing a part. Ehrenstein Cancer inoculated • Cancer o Control Fig. 3. Loss of ^*C by the haemin of red corpuscles of control mice and such inoculated with ascites cancer cells 5 days following injection of glycine-i'iC. in our laboratory has injected one hundred mice, each with 1 /uc of glycocoll-2-i^C, killed groups of ten mice at different times, isolated 20 mgm of haemin from the red corpuscles and compared the radioactivi- ties of the haemin samples obtained at various times. With another batch of one hundred mice the procedure was the same, except that these were injected with Ehrlich carcinoma cells 5 days after the beginning of the experiment. As shown in Fig. 3, the life-time of the erythrocytes in the tumorous mice (tumour weight 1 — 3 gm) was lowered to about one-half of the normal value. Because of the rapid disappearance of haemoglobin from the circulatory system of the cancerous animal, it would be expected that they would show a lowered haemoglobin content. This was not the case; the cancerous animals had the same total of 360 mgm haemoglobin content as the controls and there was a marked decrease in the haemoglobin content just before the death of the animals. This result can be explained only by a compensation of the more rapid decay of the blood corpuscles in the neoplastic organism by a more rapid formation of erythrocytes. Figure 4, which represents the results 602 ADVEJJiTURES IN JIADIOISOTOPE RESEARCH of other experiments by Eheenstein, in which the red corpuscles were labelled in animals with tumours but not in healthy animals, shows that this holds good. The incorporation of the i^C into the erythrocytes is increased fourfold in this case. The mice are able to compensate the curtailment of the life-time of red corpuscles in a quantitative manner by means of increased bone-marrow and spleen function. The cancer patient also usually shows a similar compensation. Mostly, of course, this is an incomplete compensation. We shall return to this question later. o Concer • Control Fig. 4. Life-time of red corpuscles labelled in a mouse with carcinoma and in a normal mouse. Figure 4 also shows that a portion of the erythrocytes formed in the animal having carcinoma decays rapidly, from which we may conclude that, apart from the extracorpuscular action to which the red corpuscles are exposed in the tumourous animal, abnormal erythrocytes are also formed to some extent in such an animal. The extracorpuscular damage to the erythrocytes must clearly be attributed primarily to a plasma factor or to activation of the reticulo- endothelial system. Haemolysing substances have often been observed to exist in tumours. It has also been found^^*^ that the volume of the individual erythrocyte undergoes an increase due to the action of the plasma of a cancer patient; a volume increase initiates haemolysis. A range of enzymes are present in enhanced concentration in the plasma of a cancerous organism. Warburg and Christian^^^' '^^' ^3) and others observed a marked enhancement of the aldolase content in plasma when large tumours existed. One in four of the cancer patients studied, was CANCER ANAEMIA 603 also observed to have an increased aldolase content in the plasma^"'*^ Phosphatases^"^^ and lactic acid dehydrogenase^^^^ have been Ibund, among others, at enhanced concentrations in cancerous plasma. Factors present in cancerous plasma may not exclusively be responsible for the curtailment of the life-time of red corpuscles. The R. E. system, which presumably plays only a more or less passive role in the uptake- of erythrocytes which have reached the physiological end of their exis- tence, can be activated in the neoplastic organism and can act upon the o Kontroll • Tumor • • 10 15 20 25 Tage Fig. 5. Liver weights of control and tumour-bearing mice. Kontrol — control; Tumor — tumour, Tage — days. red corpuscles. In healthy human beings red corpuscles have an approx- imately constant life-time of 100—120 days and when this has been attained they are taken up by the R.E. system. P. MiEscHER^^'^Mias sought for ^^Cr in the organs of rabbits into which ^^Cr-labelled red corpuscles had been transfused 25—35 days previously. He found the greatest concentration of ^UJr in the bone marrow, with the liver and spleen next in order. It might be argued against these results that the ^^Cr is not firmly bound in the red corpuscles, that it escapes from the intact erythrocytes, and therefore an enrichment of ^^Cr in an organ can be attributed to causes other than the decay of red corpuscles in the organ concerned. Experiments performed in our laboratory by Ehrenstein and LocKNER^^^\ with transfused erythrocytes which had been labelled with ^^Fe, showed a very similar result. Haemoglobin, so long as it is embedded in the intact erythrocyte, is among the most stable compounds present in the animal body and ^^Fe can escape from it only after damage to the erythrocyte. In the experiments mentioned above, the plasma of the acceptor-sister animal was inactive and ^^Fe was present only in the transfusing erythrocytes. The ^^Fe which they were able to detect 604 ADVENTURES IN RADIOISOTOPE RESEARCH in the deposited iron (in ferritin and haemosiderin of the bone marrow) , for example, 10 hr after transfusion, could only be derived from the erythrocytes degraded in this organ. Erythrocytes do not set free any ^^Fe as long as they are intact. This could be proved by performing experiments in which the labelled red corpuscles were first circulated Table 1. — Distribution of the »^Fe from the trans- fused RED CORPUSCLES, WHICH WERE NOT HAEMOLYSED IN THE ACCEPTOR RABBIT, BETWEEN THE FERRITIN PLUS FERRGSIDERIN FRACTIONS OF THE ORGANS (3 hr experiment) Organ Percentage distribution of ''Fe from the transfused red corpuscles in the ferritin plus ferrosiderin frac- tions of spleen, liver and bone marrow Spleen 8 Liver Bone marrow . . . 35 57 500 400 300 200 100 '^ Kontroll • Tumor • • 8 8 ° ° ° o ° 10 15 20 25 Toge FIg. 6. Spleen weights of control and tumour-bearing mice. Kontroll — control; Tumor — tumour; — Tage — days. for 2 days in a sister acceptor, in order to allow the escape of any possible mobile ^^Fe, and then adding these corpuscles to a third rabbit. These experiments yielded a result similar to that first discussed. The organ in which most of the erythrocytes end their physiological life is the bone marrow, with the liver occupying second place. One gm of spleen contained more Fe^^ than 1 gm of any other organ. Since the weight of the spleen is very small, especially in the rabbit, the whole spleen contained much less ^^Fe than either the whole of the bone marrow or the whole liver (cf. Table 1). CANCER ANAEMIA 605 In haemolytic anaemia and in a series of other ailments there is a quite different behaviour. In these illnesses the blood corpuscles do not attain nearly the same life-time but decay in accordance with a statistical law like that for radioactive atoms. The R. E. S. plays an active part in the death of these erythrocytes, and Mischer(^^^ has produced a series of arguments to support this. The cancerous mice involved in Ehren- stein's previously discussed experiments had greatly enlarged spleen and liver, with average weight increases for these organs amounting to 153 and 42 per cent (Figs. 5 and 6). A study of mice having spontaneous tumour yielded a similar result. Furthermore, the incorporation of ^^p into the desoxyribonucleic acid of the liver and spleen was found to be markedly increased in adult mice inoculated wdth breast cancer (Kelly and JoNES^^^' ^^^), Such incorporation into the liver and to some extent also to the spleen, must be attributed essentially to additional cell formation. An investigation by Berlin, Lawrence and Elmlinger^^^^ shows, among other data, that the erythrocytes in a diseased enlargement of the spleen frequently decay roughly in accordance with a statistical law, but that as soon as the spleen is removed all the red corpuscles attain approximately the same life-time. On the other hand, removal of the spleen from a healthy person, which has been done repeatedly after accidents causing a rupture of the spleen, does not noticeably affect the life-time of the erythrocytes^^^\ These results demonstrate quite clearly the great difference in the parts played by the spleen in physiolo- gical and pathological decay of the red corpuscles. METABOLISM OF IRON IN THE CANCEROUS ORGANISM The study of the metabolism of iron in the cancerous organism can lead up to valuable information concerning the curtailment of the life- time, the existence of hyperplasia of the bone marrow, and so on. In contrast to the determination of the life-time of red corpuscles, which has been discussed in the previous pages, such investigations require only a few hours, a fact to which great importance attaches in clinical studies. The iron content of the plasma is frequently decreased to a smaller extent than the iron concentration in the plasma, since indeed the plasma volume is sometimes increased (see p. 598). Even when this increase is taken into account, there is often a lower content of iron in the plasma of cancer patients. The magnitude of the iron content of the plasma is determined essentially by the amounts of iron discharging, chiefly to the bone marrow, and flowing in from the iron deposit. If the rate of forma- tion of red corpuscles is accelerated, as it is often the case with cancer 606 ADVENTURES IN RADIOISOTOPE RESEARCH patients, the iron metabolism in the plasma will be under greater strain. A lower level of plasma iron will be established if more iron is discharged from the plasma than is yielded by the supplying organs. The organism, however, is capable of compensating, within a wide range, for the lower level of iron by an accelerated output of iron. This is shown in Fig. 7, ivhich is taken from work performed by Dal Santo in our laboratory in which he studied women, suffering from cancer of the cervix, who were patients of the Kottmeier Clinic at the Radiumhemmet, Stockholm; c e K^ 40 60 80 100 120 140 160 180 200 ^g iron in 100 mL plasma Fig. 7. The decline of plasma iron, labelled with ^^Fe, as a function of the iron concentration in the plasma. the method applied was that due to Huff and co-workers^^®^ In several cases the iron moves too rapidly and the T ^/g values turn out too low. When more prolific bleeding can be excluded, such a result is a very plaus- ible indirect proof of the curtailment of the life-time of erythrocytes. In order to compensate for a curtailment of the life-time of the erythro- cytes by about one-fourth of their normal value, it would be necessary to add the same extra amount of iron as that which would compensate a chronic bleeding of about 10 ml/day. In a series of cases (Fig. 7), the iron moves too slowly and the T^j^- values are too high. All of these patients were anaemic. Ross and Mil- LER^-^^^ in a study of twenty-eight severe cases, found twelve with a super- normal and only three with a subnormal iron metabolism. In nearly half of the cases investigated there was a definitely detectable curtailment of the erythrocyte life-time. CAXCER ANAEMIA 607 METABOLISM OF IRON BEFORE AND AFTER TREATMENT WITH RADIATION Fourteen of the cervical cancer patients, studied by Dal Santo, who had been found clinically healed after single radiation treatments by the chief of the clinic Kottmeier, were investigated by Lockner about 1 year later. The high iron metabolism and also the increased plasma volume were fully or partly normalized. The iron transport was found lowered from 0.96 mgm/hr per litre R. B. C. to 0.76 mgm (7^ < ) rt CO 05 CD t^ -* o CD O m 00 d --I 00 00 o CO CO o CD O rH CO in in 05 00 in 00 T— ( d in CO (M in O rt CO CO CO 00 o ^ 00 CO CO 05 00 o o CO CO 05 CO 05 O O —I CD 05 in CD in CO CD -^ d ^ d c c a ^ ^ cd ^ c3 cd sag CO CO CO ^ (d o3 t+H 54-( *+-l ooo 13 'C o o d e G ^ .-( .r-( C © a o C fl cS 03 a a CO ,, CO CO oS o C^^ C4^ ^4^ '"^ OOO 03 St3 2 ® o o o G o o in CO CD 00 CO CO ^ t^ 05 CO GO ■— I o >— I CO o in o ^ in CO CO CO CO 00 O rt CJ5 in in G o o CO o O >— I ,— I CD 05 in o 00 O i-H f— t C35 C35 05 (^ 05 d O ^H ^H in O 1— I ^H CD 05 CD 05 O O i-H i-H GO O G o o o3 03 o3 Q) a 51 S n. a 'o Si © fcl CO ® CO ?I CO m c3 o ^ 3 > G O Ah J Ah m PM ^ h^ pq c3 03 o3 c3 03 c3 a s s ,-> a "o ^ ® i1 (/J OJ CO ?i CO M 5 J Ah pq Ph s ^ m ^ EFFECT OF ADRENALINE ON PLASMA AND TISSUE CONSTINTENTS 613 It may be due to a decreased circulation rate in the bone tissue that, under the action of adrenaline, incorporation of ^'-P into the skeleton is reduced. Furthermore, recrystallization of the bone apatite, which to a large extent is responsible for the ^^P incorporation, is presumably an enzymatic process and it is quite possible that adrenaline interferes with the latter. The markedly lower ^sp content of the plasma of adrenaline injected mice could be due to a lower resorption rate due to the effect of adrenaline or an increased rate of efflux from the circulation. It is hardly probable that the former is the case. Adrenaline was found (1954) to decrease the resorption rate of intraperitoneally injected bicarbonate, the differ- ence in the rate of resorption due to the presence of adrenaline manifests itself, however, in the course of the first few^ minutes only. We can expect phosphate to show a similar behaviour. To make sure that we are con- fronted with an enhanced rate of loss of ^^p by the plasma under the effect of adrenaline, we injected labelled phosphate intravenously into rabbits, eliminating thus the possible role of a resorption process. The decrease observed in the ^^P content of the plasma of the adrenaline injected mouse is due to an increased rate of interchange between plasma and extra vascular phosphate, and not to an increased exodus of plasma phosphate. This follows from the fact that, in our experiments with mice taking 15 min, the inorganic P content of the plasma is not influ- enced by the presence of adrenaline, as seen in Table 2. PiNCUS and assoc. (1933) investigated the effect of subcutaneously administered 80 microgram/kgm of adrenaline on the inorganic phosphate Table 2. — Inorganic P Content of the Plasma or Controls and of Adrenaline Injected Mice of 10 mice each Inorganic P mgm% Groups Controls Adrenaline injected 1 .... 5.40 5.27 2 5.08 5.03 3 .... 4.64 6.34 4 .... 7.10 6.38 5 6.48 — 6 5.88 6.23 7 6.38 5.50 8 5.52 * 6.41 9 .... 4.70 4.05 10 9.27 8.54 11 .... 6.73 5.87 5.85 12 6.50 Mean value .... 6.09±1.28 6.12±1.14 614 ADVENTURES IN RADIOISOTOPE RESEARCH content of rabbit plasma. After the lapse of 15 min, they found 1% decrease only, one of 12% after the lapse of 1 hr. The observation that the inorganic P content of the plasma is reduced several hours after administration of adrenaline was reported by Cori (1930) at an early date. Effect of adrenaline on the rate of extrusion of intravenously injected labelled phosphate from the circulation of the rabbit Figure 1 demonstrates the rate of disappearance of the intravenously injected ^^F, 0.2 ml saline containing 0.1 mgm P as labelled phosphate, 7000H o 6300 5600 4900 E 4200 3 o E a 3500- ^ 2800 E < 2100 1400- 700- ■0-- c\f2 Mean value : 0.298 ± 0.158 EFFECT OF IRRADIATION OX HEMIX FOKMATIOX 629 Delay of the effect of irradiation on the incorporation of labelled iron into hemoglobin Red corpuscles are very radioresistant. We need very massive doses to achieve hemolysis and a dose of 4000 r for example as shown by Sheppard^^*^ does not influence the rate of intrusion of ''^K in the red corpuscle though it does to some extent weaken the mechanism respons- ible for the concentration of potassium in the red corpuscles. Dog blood exposed to a dose of 200,000 r doesnot hemolyse as observed by Nizet et alP^^ The radioresistance of circulating red corpuscles suggests that the not fully completed red corpuscles in the bone marrow are radiation resistant as well and continue even in the exposed animal to complete their hemo- globin content. In the bone marrow of swine exposed to radiation of the Bikini explosion often only fat cells and a few clumps of erythrocytes were found^^^\ The above conclusion is supported by the observation that the uptake of ^^Fe or ^^C by the hemoglobin of red corpuscles in vitro is not reduced by irradiating the animal before securing the blood sample. Reticulocytes and other types of incomplete red corpuscles can complete their hemo- globin content in vitro. Not only is the ^^C incorporation in vitro into the blood corpuscles of animals not smaller than into those of controls, it is even markedly enhanced as shown by Nizet et alS^^^ who investigated the uptake of "C by the dog. Rauntanan and one of us made similar observations when studying incorporation of ^^Fe in vitro into the red corpuscles of the hen which was found to be increased in extreme cases up to 300% if the hen was 18 hours previously exposed to a dose of 1000 r. Irradiation induces presumably the bone marrow to release some of the red corpuscles in an earlier stage of their matura- tion. Nizet et al. have furthermore shown that irradiation in vitro produces changes in the plasma which are favourable to ^^Fe incorpora- tion into the red corpuscles. These considerations support the conclusion that as far as red corpu- scles in the advanced stage of their maturation are present in the bone marrowy a completion of their hemoglobin content takes place even in the exposed organism and that some of the ^^Fe administered shortly after irradiation will be utilized by the bone marrow. With increasing lime the completed red corpuscles being discharged into the circulation or wiped out in the exposed organism, the utilization of ^^Fe by the bone marrow will diminish and after the lapse of about 1 day may almost cease. As the non-utilization of labelled plasma iron by the bone-marrow leads to a depressed turnover rate of the plasma iron, this depression is to be expected not to be shown to its full extent already shortly after irradiation but only at a later date. That this is the case is demonstrated by Fig. 1 and the data of Table 2. 630 ADVENTURES IN RADIOISOTOPE RESEARCH As seen in Fig. l,the effect of irradiation with such a high dose as 1500 r influences the rate of extrusion of 59Fe from the plasma, when measured 1 hour only after exposure, is much less than an irradiation with 800 r only, when the rate of extrusion is measured 2 days after irradiation. -■ 1 r 120 180 rime in minutes 240 30C Fig. 1, Effects of exposure to Eoentgen rays on the rate of extrusion of circulating labelled plasma iron from the circulation of rabbits. Table 3. — Incoeporation of Intekperitoneally Injected ^'Fe in the Course of 5 Hours into the Hemoglobin of Con- trols AND With 500 r Irradiated Rabbits. C = Control. R = Exposed Time in hours between exposure and injecting of "Fe Relative ^»Fe content Plasma Hemog lob in c IOC 100 100 100 100 R 100 118 124 223 447 c 100 100 100 100 100 R 100 68* 56 3.2 17.3 5 17 48 19** * For the bone marrow hemin the corresponding figure was 43. •* Data obtained by Huff et ai.("> When injecting rabbits weighing 600 gm intraperitoneally with labelled FeClg 48 hours after exposure to 500 r, we observed 5 hours after injection the 59Fe content of the plasma to be 120% higher than that of the control. In experiments in which 500 — 900 gm rabbits were injected intraperi- toneally within 1/2 hour after irradiation with 5 jugm labelled iron as FeClg and killed 3 hours later, the ^sFe content of the plasma was increased by 6% only. Further data on the effect of time which elapsed after EFFECT OF IRRADIATIOX ON HEMIX FORMATION 631 exposure to irradiation on the ^^Fe content of tlie plasma is seen from Table 3. This table also contains data on the incorporation of ^^Fe into hemoglobin, which is much more markedly depressed under the effect of irradiation after the lapse of 48 hours than after that of 1 hour. In these experiments 5 jug of iron as FeClg were interperitoneally injected to each rabbit. Similar observations on the effect of irradiation on the incorporation of ^^Fe into the red corpuscles were made by Hennessy and HurF^^\ These results support the conclusion arrived at that for some time after irradiation the completion of the hemoglobin content of the red corpuscles is still going on in the marrow. b) Muscle myoglobin In view of the fact that the chemical composition of myoglobin closely resembles that of hemoglobin — they are differing only as to their degree of polymerisation — it was of great interest to investigate the effect of exposure to irradiation on the incorporation of ^^Fe into the myo- globin of guinea pigs extracted from their muscles. The method of puri- fication was that described by Theorell and Akeson^^\ The myo- globin obtained was practically free from hemoglobin as revealed by a spectroscopic investigation of the CO-myoglobin. In 4 experiments in each of which 10 female guinea-pigs having a weight of 450 — 550 gm were injected 6 hours after exposure to 1400 r with 3 //gm of labelled iron as sodium iron citrate and killed 18 hours later and 10 controls were treated in the same way, the following rel. specific activity figures were obtained for the myoglobin and hemoglobin iron of controls and exposed animals. Under effect of exposure the ^^Fe incorporation into myoglobin was thus reduced to 1/2, that into hemoglobin to 1/4 of that of the controls. In the 2 last experiments we determined the effect of exposure on the Table 4. — Effect [or Irradiation on the Incorporation of ^^Fe INTO Myoglobin and Hemoglobin of Guinea-pigs. Control = C, Exposed = R No. of expt. Rel. spec, activity* of myoglobin ^ . Ratio T^— R Eel. spec, activity of hemoglobin „ . Ratio ^;r- R 1 2 3 4 c R C R 0.098 0.042 2.3 0.236 0.047 0.034 0.026 1.3 — 0.476 0.215 2.2 2.19 0.48 0.415 0.164 2.6 2.44 0.68 5.0 4.6 3.6 * Count per //gm Fe. 632 ADVENTURES IN RADIOISOTOPE RESEARCH specific activity of the total iron present in the muscles as well and found it, as to be expected increased under the effect of exposure, in the third experiment from 0.714 to 0.810, in the fourth one from 0.687 to 1.04. DISCUSSION Hemoglobin is one of the comparatively few molecular constituents of the adult organism that is formed in close connection with cell division. The latter being very susceptible to the effect of ionising radiation, exposure to radiation is bound to interfere with hemoglobin formation as well. Furthermore, marrow cells are radiosensitive and exposure to radiation may lead to destruction of marrow cells. Irradiation of rats with 400 r was found to lead to a decrease in the total number of marrow cells to almost one half of its normal value in the course of the first day following exposure (Brecher(^^>) and the chemical composition of the bone marrow was found markedly influenced. Lutwak-Mann^^'^ found 3 hours only after total body exposure to a dose of 1500 r the labile acid-soluble P of the bone marrow of rabbits to be reduced by 30o/o, that of DNA and RNA phosphorus by 20 and 16%. The total nucleic acid P of the marrow of the to 500 r exposed rat amounted to only half of that of controls. Manclel et al}^ report the bone-marrow of rats exposed to 500 r to show after the lapse of 26 hours a by 50% reduced PNA content, the DNA being reduced to 30% of that of the controls after the lapse of 2 days. Thus radiation anemia is at least partly due to the fact that hemoglobin is formed in close connection with the cell division and also that it takes place in the radiosensitive marrow cells. Altmann et al. (Richmond^^^^ Stokingek^^*^^) in their very extended studies found incorporation of i^C into globin and hemin to be influenced at a very different rate by irradiation. Incorporation of i*C into hemin of the exposed animals was greatly depressed, whereas globin was affected to a considerably smaller extent. From this finding it does not necessarily follow that it is not the milieu of its formation, the bone marrow, which is responsible for the radiation sensitivity of the formation of hemoglobin. We found no interference with the formation of cytochrome b in the liver of the strongly irradiated guinea-pig. In this case heme formation is thus not radiosensitive. We found, however, interference with forma- tion of myoglobin. EFFECT OF IRRADIATION ON HEMIN FORMATION 633 References 1. L. B. Flexnek, G. J. VosBURGH and D. B. Co^\^E, Amer. J. Physiol. 153, 503 (1948). 2. C. G. HoLMBERG and C B. Laxjrell, Acta Physiol. Scand. 10, 307 (1945). 3.C. B. Laurell, Acta Physiol. Scand. 14, Siippl. 4, 1 (1947). 4. F. WuHRMANN and B. Jasinski, Schweiz. med. Wochenschr. 83, 1 (1953). 5. R. L. Huff, W. F. Bethard, J. F. Garcia, B. S. Roberto and J. II. Law- rence, J. Lab. Clin. Med. 36, 40 (1950). 6.T. J. Hennessy and R. L. Huff, Proc. Sac. Exp. Biol. Med. 73, 436 (1950). 7. R. B. Lotfield and R. Bonnichsen, Acta Chem. Scand. In press. 8. H. Theorell and A. Akeson, Ann. Acad. Sci. Fennicae Ser. A, II, No. 00, 303. 9. K. Agner, R. Bonnichsen and G. Hevesy, Scand. J. Clin. Lab. Inc. 6, 261 (1954). 10. J. ScHUCK, Arch. Gynalol. 172, 52 (1942). U.S. LuDEWiG and A. Chanutin, Amer. J. Physiol. 166, 384 (1951). 12. P. J. Elmlinger, R. L. Huff, C. A. Tobias and J. Lawrence, Acta Haematol. 9, 73 (1953). 13. D. Haskins, a. R. Stevens Jr., S. Finch and C. A. Finch, J. Clin. Inr. 31, 542 (1952). 14. C. W. Sheppard and M. J. Stewart, J. Cell. Comp. Physiol. 39, Suppl. 2, 189(1952). 15. A. NizET, S. Lambert, A. Herve and Z. M. Bacq, Arch. Int. Physiol. 52, 129 (1954). 16. G. Brecher, K. M. Endicott H. Gump and H. P. Browner, Blood 3, 1259 (1948). 17. C. Lutwak-Mann, Biochem. J. 49, 300 (1950). 18. P. Mandel, p. Metais, C. Gros and R. Voegtlin, C. R. Acad Sci , Paris 233, 1685 (1951). 19. J. E. Richmond, K. I. Altman and K. Salomon, J. Biol. Chem. 190, 817 (1951). 20. H. E. Stokinger, K. I. Altman and K. Salomon, Biochim. et Biophyi. Acta 12, 439 (1953). 21. R. G. Thomas, K. I. Altman, J. N. Stannard and K. Salomon, Pad. Res. 1, 180 (1954). 22. A. NizET, Z. M. BAcq and A. Herve, Arch. Int. Physiol. 60, 449 (1952). 23. C. F. Strittmatter and E. G. Ball, Proc. Natl. Acad. Sci. U. S. 38, 55 (1952). Acta Chem. Scand. 5, 455 (1951). 24. P. F. Hahn, W. F. Bale and W. M. Balfour, Amer. J. Physiol. 135, 600 (1942). Originally published in Acta Chem. Scand. 11, 120—124 (1957). 63. HAEMOGLOBIN PRESENT IN THE NUCLEAR FRACTION OF THE LIVER R. BoNNicHSEN, G. Ehrenstein, G. Hevesy and J. Schliack From the Institute for Organic Chemistry and Biochemistry, University of Stockholm, Sweden The nuclear fraction of 1 gm of rat and rabbit Hver contains 0.34 and 0.58 mgm of in aqueous medium non-extractable haemoglobin. The corresponding figure for the nuclei of the red corpuscles of 1 ml of hen's blood is 0.9 mgm. The haemoglobin content of the nuclear fraction of 1 gm of foetal rabbit liver is 100 times as large as that of 1 gm of maternal liver. The ratio of their sspe content 11 to 17 h after labelling the maternal plasma iron, works out to be 100. One day or more after exposure of the rat to 500 r, incorporation of ^^Ye into the non-extractable haemoglobin of the hver nucleus fraction is strongly depressed. As mentioned in a previous note (Bonnichsen et alM^) the nuclear fraction of the liver of the guinea pig contains small amounts of haemo- globin not extractable by saline or other aqueous solutions. This paper contains data on the incorporation of ^aPe into this fraction isolated from the liver of adult rats and rabbits and of that of the rabbit embryo. This fraction was furthermore located in the nuclei of the erythrocytes of the hen. That in the basophilic erythroblasts of human and rat bone marrow the site of haemoglobin synthesis is primarily the nucleus, was repeatedly suggested. The methods applied in these investigations were ultraviolet absorption microspectroscopy and cytochemical staining procedures(2-9>. EXPERIMENTAL In 15 experiments with male rats an aggregate number of 173 animals, weigh- ing between 150 and 280 gm were injected intraperitoneally with 0.25 ml of a 3.8% ammonium-citrate solution containing 0.5—12 fxgm of with ^ ^Fe labelled iron of 0.1 — 12 [xC activity. Half the number of rats was exposed to 150 r — 500 r of X- rays. Injection took place 15 min to 5 days after exposure. The animals were kiUed from 2 to 48 hr after injection. The livers were perfused first with 0.145 M NaCl and then with 0.25 M sucrose containing 0.018 M CaClg. The weighed livers were homogenized in 9 vols, of 0.25 M sucrose — 0.0018 M CaClg. The further procedure was carried out according to Hogeboom et alS^^^ The nuclear fraction isolated by this procedure was cytologicaUy inhomogeneous. The fraction contained about 60—80% of the cell nuclei, it also contained erythro- cytes, connective tissue, residual intact cells and free mitochondria. HAEMOGLOBIN IN NUCLEAR FRACTION OF THE LIVER 635 In Older to hacmolyse the erythiocytcs, the fraction was lioniogcnized with 10 vols, distilled water and allowed to stand several hours. This last step was repeated at least three times more. After clearing the suspension of nuclei obtained in this way by adding 3% of deoxycholate, the haemoglobin bands were steadily visible in the handspectio- scope. The CO-band was that of haemoglobin. The pyridinc-haemoeliiomogen band was located at 567 m/u. The haemin was extracted with a mixture of acetone and HCl (10 ml 20% HCl in 1 1. of acetone). After filtration, the acetone was evaporated in vacuo. The haemin was crystallized twice from cone, acetic acid, and the crystals washed with 1 N HCl. The haemin was then combusted and the solution analyzed as de- scribed by BoNNiCHSEN et alS''-'^^ In order to know in which part of the above nuclear fraction the non-extract - able haemoglobin is located, we separated the fraction, prior to haemolysing the red cells with distilled water, in the counter-streaming centrifuge of Lindahl^i^) in five fractions containing particles of different size and different specific gravity. The fractions were examined with the phase-contrast microscope and with the handspectroscope. Thereafter, the specific activity of their haemin was determined. The result of one of these experiments is seen in Fig. 1. In five of the above experiments the liver nuclei have been isolated both in aqueovis medium and in organic solvents of low polarity according to the methods described by Dounce etalS^^^ and Axlfbey ef oZ.^"^The unclear preparations obtained in this way were treated and analysed as described above. No difference was found in the properties of the non-extract able haemoglobin of the nuclear fraction prepared either in aqueous or in organic medium. The liver ferritin was prepared as previously described by Loftfield et alS^^^ In experiments with 2 — 3 kgm rabbits 8 animals were investigated. Their plasma was labelled with ^^Fe as described by Ehrenstein et alS^^' and reinjected. The animals were killed 3 — 24 hr after injection. Simultaneously, in all our experiments haemin of the circulating haemoglobin was analysed as well. In two experiments with hens 100 ml of hen blood were incubated in vitro for 3 h at 37° C with 2 ml of 3.8% ammonium citrate solution containing 2 jugm labelled iron of 25 juC activity. The plasma was centrifuged off and the red cells washed four times with 5 vols, or more of isotonic saline. The erythrocyte nuclei were prepared according to Hogeboom et alS^''^ However, as the red cells are not sufficiently broken down by the homogenization procedure usually employed for the disintegration of other tissues, we haemolysed the cells by freezing at — 20° C and thawing them three times or more, suspensed in a mix- ture of 0.25 M sucrose —0.00018 M CaClg- The nuclei were then centrifuged down (International Refrigerated centrifuge head No. 269) for 10 min at 2000 r.p.m. Thereafter, the procedure of Hogeboom et alS^^^ was applied. Samples were taken from the whole washed red cells, the stroma-free haemolysate and from the final nucleus preparation. The haemin was extracted as described above. RESULTS AND DISCUSSION (a) Experiments with rats The amount of ^^Fe present in one jugm of iron of the nuclear haemo- globin fraction of the liver varied in experiments on rats between 0.6x10^4 and lOxlO" % of that injected. 636 ADVENTURES IN RADIOISOTOPE RESEARCH The liver of our rats contained 10 ^gm of nuclear haemin iron out of 2.5 mgm of total iron present in the liver. This fraction makes out 0.4% of its total iron content. In the course of the purification process in aqueous medium extract- able haemoglobin may have been removed. However, the non-extractable haemoglobin content of the nuclei was not found to be larger when the nuclei were isolated after lyophilisation in organic medium. In the haemo- globin content of the nuclear fraction of the red cells of the hen when 200- il60H o SI o £:-i20- o o 80- CO 40- ■ B 1 C I ■ D m Fractions of particles with increosing specific gravity and size Fig. 1. Results of separation of nuclear components of the liver cells of the rat in Lindahl's counter-streaming centrifuge. A — Total nuclear fraction, prepared according to Hogeboom. B — Impure mitochondria containing erythrocytes. C — Erythrocytes and small fibres of connective tissue. D — Cell membranes. E — SmaU nuclei. F — Large nuclei. these were isolated in an organic medium, Stern etalM"^^ found a haemo- globin-iron content of 19% of that of the total red corpuscle, while we find after isolation of the nuclei in an aqueous medium 1% of the total haemoglobin-iron content to be non-extractable. This 1% compares with 0.4% found by us in the liver nuclei of rats and 2% in those of the rabbit. We compared also the effect of irradiation with 500 r of X-rays on rats on the incorporation of ^^Fe into the liver nuclear haemoglobin fraction. As seen in Table 1 the X-ray effect is shortly after exposure a very restricted one for both haemoglobin fractions. The X-ray effect as seen in Fig. 2 doesn't seem as pronounced as that on the circulating haemoglobin. If we, however, take into account the enhanced activity of the liver of the exposed animal, this difference is strongly reduced. HAEMOGLOBIN' IX XUCLEAR FRACTIOX OF THE LIVER 637 (b) Experiments with rabbits The amount of ^^Fe present 4 hr after injection in one ^gm iron of the non-extractable haemoglobin of the nuclear fraction of the rabbit liver varied between 2x10"* and 6x10"*% of that injected. The iron content of the non-extractable liver nucleus fraction haemo- globin varied between 0.5 and 3.8 /Ligm with a mean value of 1.9 //gm per one g liver. I O (/I -• a o Fig. 2. Effect of exposure of the rat to whole body irradiation on the incorporation of intraperitoneally injected ^^Fe as citrate into Uver fractions and the circulating haemoglobin. For the nuclear haemoglobin content of 1 gm of rat and rabbit liver the corresponding figures are 0.38 mgm and 0.56 mgm. We also carried out experiments with pregnant rabbits 21 days after mating. A plasma sample of the mother was labelled as described by Ehrenstein e^a^(i^) The livers of the mothers and foetuses were investi- gated 11 to 17 hr after labelling the maternal plasma. The results are seen in Table 2. Table 1. — Ratio or Specific Activities of Iron Fractions OF to 500 r Exposed and Control Rats Injected with *'Fe shortly after e.xposure Injected with "Fe 1 day or more after exposure Liver nucleus fraction Circulating Total liver Liver nuc- leus fraction haemoglobin Oirculattng Total liver haemogolobln haemoglobin iron haemoglobin iron 1.16 0.53 1.60 0.915 0.161 1.15 0.97 1.04 1.41 0.503 0.063 2.48 1.15 0.84 0.93 0.570 0.238 1.72 0.78 0.46 1.27 0.727 0.072 1.92 0.318 0.029 2.44 Mean value 1.01 0.72 1.30 0.605 0.112 1.94 638 ADVENTURES IN RADIOISOTOPE RESEARCH Table 2 Mother Foetus 13.9 mgm 24 /. At the present time ^sFe is used almost exclusively for labelling iron; it has a half-life of 45.1 days and emits both easily mea- surable ^- (from 0.26 to 0.46 MeV) and y-radiation (1.1 to 1.3 MeV). Labelling of the plasma trans- ferrin is accomplished by incu- bating a plasma sample in the presence of ^sFe, e.g. as the cit- rate, at the body temperature for 20 min. If an amount of radio- active iron not exceeding 1 f^gm is added to 1 ml of plasma it w^U be combined quantitatively in transferrin. If the labelled plasma is now re-injected into the subject, the whole of his transferrin iron in the plasma will thereby be labelled. In animal experiments iron may also be added to a donor, possibly even in larger quantities and, e.g. labelled plasma may then be transferred from the donor to the acceptor after 1 hr has elapsed. Flexner and his co-workers^^^ were the first to label the plasma trans- ferrin of guinea-pigs by the injection of radioactive iron and to determine the rate at which the iron escapes from the blood fluid. Labelling the transferrin iron by injecting iron into the subject is frequently inapplic- 240 min Fig. 2. Loss of labelled iron from rabbit plasma after in- travenous injection of 2 /^gm of iron. 2 yMgm Fe injected as FeClg (caption on the graph). 41 Hevesy 642 ADVENTURES IN RADIOISOTOPE RESEARCH cable since a considerable part of the injected iron is transported into the organs, before it has the opportunity to be incorporated in the transferrin, and may complicate the experimental results by its presence in them . Even when the very small amount of 2 jugm of iron is injected as citrate or chloride into a rabbit, i.e. one-hundredth of the amount which the still available transferrin can combine with, this iron does not combine completely with the plas- ma transferrin, but a part of it leaves the plasma before having an oppor- tunity to combine with the protein. It is evident in Fig. 2, w^hich is ta- ken from a study performed in our laboratory by Giuliano, that this process is concluded after a period of only 4—5 min and that ^^Fe still present then disappears from the plasma of the rabbit with a half- life of about 2 hr, which is charac- teristic of the metabolism of physio - 7000 6300 - 5600 - 4900 4200 3500 - 2800 2100 1400 700 Konfroile Noch Adrenolm-Injektion ■ • . 0--0 10 20 30 mm 40 50 60 Fig. 3. The effect of adre- nalin on the rate of escape of labelled phosphate from the plasma. KontroUe = control test; Nach Adrenahn-Injektion = after injecting adrenalin; Aktivitat in 1 ml Plasma = activity per ml plasma. logical plasma iron. Vahlquist and co-workers have already observed the very rapid disappearance of 0.5 to 2.0 mgm iron, after injection into a rabbit, from the plasma^28)^ j^^t the radioactive method makes it possible also to fol- low the course of very small amounts of iron, 1 /ugm or less, and to measure both the unilateral loss of iron and the renewal rate of plasma iron. By making use of the ready accessibility of the radioisotopes of sodium, potassium, etc. it was shown at an early date that the rate of replacement of these ions may be extraordinarily high^is)^ j^ the course of 2 min about half of the sodium ions present at the beginning of the experiment in the plasma of a rabbit is replaced by extra vascular sodium ions. A rapid disappearance of injected iron salts such as that indicated in Fig. 2 is therefore not very surprising. The iron combined in the trans- ferrin, on the contrary, escapes relatively slowly with a half-life of from 70 to 120 min from the human circulatory system and still more slowly from the rabbit. Experiments with transferrin labelled with i^ij showed that this compound leaves the blood fluid with a half-life of a few days^i^). Iron to a very large extent escapes from the plasma not in a form com- ISOTOPIC INDICATOKS IN HAEMATOLOGY 643 2 Konfrollen Physiol. Adrenalin- Dosis '*-^. Phormokol. Adrenalin-Dosis'"''^ 60 120 min 180 240 Fig. 4. The effect of adrenalin on the rate of escape of iron com- bined in ^j globuUn from the plasma. Kontrolle = control test. Physiol. AdrenaHn-Dosis = physiological dosage of adrenahn. Pharmokol. Adrenalin-Dosis = pharmacological dosage of adrenalin. Aktivitat in 1 ml plasma = activity per ml plasma. bined with protein but only after it has been split off from the transferrin, and it is very probable that the process de- termining the rate of escape is the disso- ciation of iron transferrin since the iron ions or iron- containing radicals which are split off penetrate the capillary walls with the greatest ease. Plasma constituents such as phosphate, for instance, which transfer extraordi- narily quickly into the extracellular space, often return before they "finally" leave the plasma. This state is the more quickly established when the phosphate has the opportunity of finding an addi- tional way of entering into the cell. The reversion of phosphate ions into the plas- ma is thereby made more difficult and its ultimate disappearance from the blood fluid is thus facilitated. If the disappea- rance of phosphate from the plasma is accelerated, e.g. by the injection of adre- nalin, the effect must be ascribed in a high degree to an accelerated entry of the phosphate from the extracellular space • •Kon'i'oiie o_ o Noch Adrenal in- JfijeV'tion J 1 1 L. 4 6 mm Fig. 5. The effect of adrenalin on the rate of escape of labelled sodium from the plasma. Kontrolle = control test. Nach Adrenalin- In jek- tion = after injecting adrenahn. Aktivitat von 1 ml plasma = ac- tivity per ml plasma. 41^ 644 ADVENTURES IN RADIOISOTOPE RESEARCH into the tissue cells, owing to an enhanced metabolism taking place in the cells at this time. Figures 3 and 4 illustrate this type of accelerating effect of adrenahn on the escape of labelled phosphate and labelled iron from the plasma. The rate of escape of sodium ions (Fig. 5), on the contrary, is not affected by the injection of adrenahn. Sodium is es- sentially an extracellular element, although it is incorporated in not inconsiderable amounts into the skeleton and also into the cehs but this process of incorporation is not appreciable during the short experi- mental time of close to 10 min. In this instance, the exchange process takes place almost solely between the plasma and the extracellular space and its rate is therefore not accelerated by the injection of adrenaUn^i^^ The investigation by Flexner and co-workers on the rate of escape of labelled iron from the blood plasma was followed by numerous other studies by Lawrence and co-workers^is) and by many other workers. Huff and associates calculate the daily metabolism of plasma iron from the formula^^^^: Iron metabolism (mgm/day) = 0.693 X 24 hr /day X Fe mgm/ml x plasma volume (ml) half-life of the disappearing ^^Fe (hr) They find that the daily plasma-iron metabolism per kgm of body weight in a healthy person is equal to 0.4 — 0.45 mgm, whereas it is as low- as 0.205 mgm in pathological case such as a refractory anaemia and as high as 3.93 mgm in a case of haemolytic anaemia. By determining a fraction of the plasma-^sFe which has accumulated after a definite time in the various organ fractions and in the red blood corpuscles, an explanation is obtained of, among other things, that rather more than one-half of the metabolized iron in a healthy person is used for the synthesis of red blood corpuscles and that this value can fall to one-eighth in refractory anaemia. The metaboHsm of iron in the red blood corpuscles was calculated from the formuWi^). ^^Fe in the red blood corpuscles initial value of ^sFe content in the plasma iron metabolism in the plasma THE CONVEYANCE OF IRON FROM THE ORGANS INTO THE PLASMA The combining capacity of transferrin for iron is only about one-half utilized in healthy people and also in rabbits, and at first glance it appears remarkable that if indeed a better utilization of this combining capacity can be achieved by parenteral or oral administration the ISOTOPIC INDICATORS IN HAEMATOLOGY 645 enhancement attained is only temporary. A more permanent increase of the iron content in the plasma is spoiled by the fact that an increase of the iron level is accompanied by an increased rate of escape of iron from the plasma and, therefore, an increased rate of entry of iron from the organs is required to maintain the increased level of iron. By the oral administration of iron tartrate Laurell^**^ attained almost double the iron content in the plasma of a healthy person, but the normal plasma-iron level was re-established after 20 hr. After the injection of 10 mgm of iron, ToTTERMAN^"^ found the 2 hr value to be only about 15 per cent less than the 5 min value, indicating a partial compensation of the escaping plasma iron by the iron from the organs. We found the daily metabolism of plasma iron^^'^ in a rabbit weighing 2.6 kgm to be about 800 //gm; of this somewhat more than 400 jugm is used to replace the decayed blood corpuscles while only 80 ^gm is taken up by the liver. The 400 //gm of iron which is used daily for forming haemo- globin appears again after the decay of the blood corpuscles which contain this compound. Only small amounts of iron are absorbed daily by the digestive organs. The daily uptake of iron of the liver, which contains 7 mgm iron, from the plasma amounts to 80 //gm and this must be compensated hj a corre- sponding release to the plasma since, otherwise, the iron content of the liver would be doubled in the course of 3 months. The liver takes up these small quantities only from the transferrin of the plasma; in a period of 6 hr the liver absorbs almost one-half of 0.5 mgm of iron salt injected into a rabbit. Of 1.6 gm of "ferrivenin" injected, 84 per cent was ofund in the liver of an infected patient^24) The liver of a rabbit which had been injected for several months with iron (viviferrin) and which had an iron content of 35 mgm absorbed only 40 //gm daily from the plasma^i"\ The iron turnover in the plasma of such rabbits is greater than in normal animals. A 30 per cent higher content of haemoglobin, 12.9 gm % instead of 9.8 gm %, with an almost unchanged content of plasma iron, 151 //gm % instead of 145 //gm %, was essentially responsible for the increased metabolism from 800 to 920 //gm %. It is not likely that a liver burdened with iron will release less iron than a normal one, and since it absorbs less from the plasma than does the latter, some latitude becomes available for the release of its iron burden. An investigation by ANDERSON^i^Undicates that this does occur. While 18.4 mgm % iron was present 3 months after injecting iron into a liver burdened with iron, the content decreased to 16.1 mgm % after 6 months. Generally the liver is regarded as the main organ for storing iron, and the ferritin of this organ as the compound which yields iron to the plasma. Mazur and his co-workers^i^> have recently produced a proof that a small portion of the ferritin iron is present on the surface of the 646 ADVENTURES IN RADIOISOTOPE RESEARCH ferritin molecule as a mixture of ferrous and ferric compounds. The equilibrium between the ferric-disulphide— ferritin and the ferrosul- phydryl— ferritin is displaced in favour of the ferro-form (see also BiELiG and Baeyer^^;)) ^y venesection, which leads to anoxia, or by reducing agents such as glutathione, and the ferrous iron is conveyed into the plasma for formation of iron transferrin. a £ _a Q. 20 40 60 80 100 120 140 160 180 200 220 240 min e a Q. Fig. 6. Rate of escape of iron combined in ^^ globulin from the plasma of normal and infected rabbits. Gesund = healthy. Infiziert = infected. It is highly probable that the lack of iron in the plasma of infectious diseases is partly attributable to the disturbed transference of the ferritin iron into the plasma. In infectious diseases, of course, not only is there inhibition of the entry of iron into the plasma but there is also acceleration of the escape of iron from the plasma into the organs in which a more rapid metabolism now occurs. This is illustrated in Fig. 6, which presents experimental results obtained in our laboratory by Ehrenstein^-o\ who has compared the rate of escape of iron globulin, labeUed with iron, from the plasma of normal rabbits and rabbits infected with Pasteurella multicida. As has already been mentioned, an accelerated uptake of iron by the cells of the storage organs leads to an accelerated escape of iron ions from the plasma. Since, however, the level of iron in the infected plasma amounted only to about half of the normal, the amount of iron escaping from that plasma in unit time was not very much larger than that released from the plasma of normal animals. ISOTOPIC INDICATORS IN HAEMATOLOGY 647 The possibility must also be considered that conditions exist in infect- ious diseases which promote dissociation of the iron transferrin, a step which must precede the penetration of the protein-linked iron through the capillary wall, and thus these conditions accelerate the escape of iron from the blood fluid. The escape of iron from the plasma and the entry of iron into it are two independent processes which are presumably coupled by means of hormo- nes. Since the daily metabolism of iron amounts to about 30 mgm and the uptake from the gut constitutes only a small fraction of this quantity, the metabolism takes place essentially between the plasma, the intercellu- lar fluid, the organs and the red blood corpuscles. The rate of escape of iron from the plasma is promoted by an increasing haemopoiesis and also by an enhancement of the metabolism taking place in the storage organs. The two processes are possibly responsible for the 20 to 90 per cent increase of plasma-iron level observed in the early hours of the day. In animals which have been deprived of the suprarenal capsule, and still more powerfully in normal dogs, adrenalin leads to a temporary lowering of the iron level, wdiereas ACTH causes this effect only in normal animals. Cabtwright and co-workers^i even found that an intramuscular injection of physiological saline solution lowers the plasma iron content in dogs by from to 41 per cent, which presumably also is brought about by a hormonal effect. Oestrogen raises and androgen lowers the amount of iron stored in the liver of chickens^22) ^nd, therefore, these hormones also should affect the escape of iron from the plasma or its entry from the liver, or both of these processes. As Laurell^^' 25 has already found, it is highly probable that iron dissociated from the protein compound, and not iron transferrin, which is transferred from the plasma into the extra vascular extracellular space. With the aid of paper-electrophoretic methods, Ehrenstein of our laboratory has recently proved that the iron present in the lymph, and therefore the iron present in the spaces between the cells, is also combined with j5i globulin. He found that the iron content of the lymph of the rabbit is one-half to one-third the content in the plasma, which amounted to about 150 mgm %. He also found that the combining capacity of the lymph for iron, and therefore the transferrin content, is also about one- third that of the plasma. Laurell^^^^ pointed out that an importance attaches to the ratio (Fe transferrin content)/(Fe-free transferrin) = = K[Fe+"'" + ] in respect of the metabolism of iron, and it is not without interest that this ratio is found to be about the same in the plasma as in the lymph. Since the iron content of the intercellular space should be lower than that of the lymph, the values quoted represent an upper limit for the intercellular space and a lower limit for the space within the cehs. The volume of the intercellular space has been assumed to be four times the plasma volume. 648 ADVENTURES IN RADIOISOTOPE RESEARCH Figure 7 shows the transport of iron from the labelled transferrin iron of the plasma of a normal rabbit into the intercellular and cellular space. The iron disappearing from the plasma which is not found in the inter- cellular space has already been transferred into the cells. Normol-Koninchen JO o Infra celluldres Fe l~rT~r"T~ Fig. 7. Transport of iron from the labelled globulin iron of the plasma of a normal rabbit into the intercellular and cellular space. Normal Kaninchen = normal rabbit. Intracellulares Fe = intracellular iron. Plasma Fe = plasma iron. Lymphe Fe = lymph iron. Figure 8 shows the powerfully accelerated transport of globulin iron from the plasma of an infected rabbit; this acceleration is partly attri- butable to the fact that the turnover of red corpuscles (shorter life-time) is accelerated in the infected animal. Infekt Kaninchen Introcelljlares Fe I 20 4U 60 80 100 120 140 160 180 min Fig. 8. Transport of iron from the labelled globulin iron of the plasma of an infected rabbit. Infekt. Kaninchen ~ infected rabbit. Intracellulares Fe = intracellular iron. Plasma Fe = plasma iron. Lymphe Fe — lymph iron. ISOTOPIC INDICATORS IN HAEMATOLOGY 649 In contrast to the globulin iron, the non-physiological ly bound iron has been shown by electrophoretic studies to pass into the intercellular space essentially free from transferrin, as shown in Fig. 9; this diagram shows the distribution of non-physiologically bound iron introduced into the plasma, in the intercellular space and the cellular space of a normal rabbit. The problems mentioned in the introduction can be solved in other, though more tedious ways, e.g. by applying isotopic indicators, but a determination of the iron turnover cannot be considerate without the N E E Normal-Koninchen -pT-7 Introcelluldres Fe 1-r 1" 20 40 60 80 100 120 140 160 180 Fig. 9. Distribution of non-physiologicaly bound iron(lnigni) added to the plasma among the plasma, the intracellular space and the cellular space of a normal rabbit. Normal- Kaninchen = normal rabbit. Intracellulares Fe = intracellular iron. Lymphe Fe = lymph iron. Plasma Fe = plasmairon. 1 mgm citrate ^^Fe added as citrate. use of radioactive tracers. Use of these indicators provides a possibility of splitting up into components the dynamic equilibrium whose resultant is the plasma iron level. The result of splitting up this equilibrium directs our attention, with particular emphasis, to the importance of the de- ficient flow of iron from the storage organs into the plasma in infectious diseases and to some other pathological cases. In order to attain a per- manent increase in the plasma iron level in such diseases it would be necessary to increase the deficient flow into the plasma. That the lymph flow' plays a part in maintaining a normal plasma iron level is shown by the observation of Ehrenstein, viz. that the plasma iron level in a rabbit falls on the average from 191 to 146 //gm % 4 hr after removal of the ductus thoracicus and the truncus jugularis sinister, whereas the level remains constant in control rabbits which have been subjected to operation without removing the ductus thoracicus. 650 ADVENTURES IN RADIOISOTOPE RESEARCH References 1. G. FoNTES and L. Thivolle, C. R. Soc. Biol. (Paris) 93, 687 (1925). 2. G. Bakkan, Z. physiol. Chem. 171, 194 (1927). 3. V. Henriques and A. Roche, Bull. Soc. Chem. Biol. Fr. 9, 501 (1927). 4. O. Warburg, Biochejn. Z. 187, 255 (1927). 5. O. Warburg and H. Krebs, Biochem. Z. 190, 143 (1927). 6. L. Heilmeyer and K. Plotner, Das Serumeisen und die Eisenmangelkrank- heit. Jena (1937). 7. L. E. Totterman, Acta Med. Scand. Suppl. 230, 1 (1949). 8. C. B. Laurell, Acta physiol. Scand. Suppl. 46, 1 (1947). 9. C. G. HoLMBERG, C. B. Laurell, Acta Physiol. Scand. 10, 307 (1945). 10. R. L. Huff, P. J. Elmlinger, J. F. Garcia, J. M. Oda, M. C. Cockrell and J. H. Lawrence, J. Clin. Invest. 30, 1512 (1951). 11. L. B. Flexner, G. J. VosBURG and D. B. Cowie, Amer. J. Physiol. 153, 503 (1948). 12. L. Hahn and G. Hevesy, Acta Physiol. Scand. 2, 5 (1941). 13. P. J. Elmlinger, S. P. Masouredis, J. Soni, G. E. Fulton and S. L. Bel- knap, J. Clin. Invest. 33, 930 (1954). 14. G. Hevesy and G. Dal Santo, Acta Physiol. Scand. 32, 339 (1954). 15. J. H. Lawrence, P. J. Elmlinger and G. Fulton, Cardiologia 21, 337 (1952). 16. R. L. Huff, A. Tobias and J. H. Lawrence, Acta Haematol. 7, 129 (1952). 17. K. Agner, R. Bonnichsen, G. von Ehrenstein and G. Hevesy, In press. 18. N. S. E. Andersson, Acta Med. Scand. Suppl. 241, 1 (1950). 19. A. Mazur, S. Baez and E. Shore, J. Biol. Chem. 213, 147 (1955). 20. G. VON Ehrenstein, Acta Chem. Scand. 10, 703 (1956). 21. G. E. Cartwright, L. D. Hamilton, C. J. Gubler, W. M. Fellows, H. Aschenbrucker and M. M. Wintrobe, J. Clin. Invest. 30, 161 (1951). 22. D. G. Chapman, W. A. Max and R. H. Common, Sci. Agr. 30, 194 (1950). 23. C. B. Laurell, Blood 6, 183 (1951). 24. W. J. Kuhns, C. J. Gubler, G. Z. Cartwright and M. M. Wintrobe, /. Clin. Invest. 29, 1505 (1950). 25. C. B. Laurell, Pharmacol. Rev. 4, 371 (1952). 26. B. Jasinski and F. Wuhrmann, Verh. dtsch. Ges. inn. Med. 598 326 (1953). 27. H. J. BiELiG and E. Bayer, Naturwissenschaften, 42, 466 (1955). 28. G. Neander and B. Vahlquist, Acta Physiol. Scand. 17, 110 (1949). Originally published in J. Clin. Lab. Invest. 6, 261 (1954) 65. NOTE ON THE DETERMINATION OF RADIOIRON K. Agner, R. Bonnichsen and G. Hevesy From the Chemical Department, Seraf imerlasarettet ; Biochemical Department, Medical Nobel Institute, and Institute for Biochemistry, Stockholms Hogskola, Stockholm Shortly after radioiron became available, Hahn, Bale, Lawrence and Whipple (1939) applied this radioisotope with much success to the study of the absorption and distril)ution of iron in the animal body. An analytical method was devised by Hahn, Bale and Balfour (1942) which permits the determination of the iron content of a tissue fraction and its radioactivity. Following wet ashing of the tissue and after addition of a known amount of carrier the solution is electrolysed and the iron deposited on a copper planchet which is then placed under the Geiger counter. The yield of electroplating is tested by dissolving the electro- plated iron in acid and carrying out a colorimetric determination of the solution. Table 1. — Added and Recovered Radioiron ^®Fb as Ferrichloride was pipetted into test tubes, 500 micro- gram of carrier iron added, and THE Procedure described in the Text Carried out s'Fe added ^Te recovered (counts per min.) (counts per min.) 10 10.2 20 19.7 30 29.2 100 98.0 200 200.0 300 294.0 Though this analytical method proved to be of great importance in radioiron studies, we were induced to replace it by a much more rapid, simple, and certainly not less exact one since a long series of radioiron determinations in numerous tissue fractions was to be carried out. The new procedure is based on (a) colorimetric determination of the iron content of a solution of a tissue fraction after wet ashing, (b) addition 652 ADVENTURES IN EADIOISOTOPE KESEARCH of carrier iron as FeClg to bring up the iron content of the sample to a total amount of 500 microgram, (c) precipitation of the solution with HgS after neutralizing it with ammonia, (d) collection of the FeS obtained on a filter paper placed on the bottom of a perforated aluminum dish of 1.2 cm diameter and 2 mm depth (as used in the determination of the radioactivity of ammonium magnesium phosphate precipitates), (e) plac- Table 2. — Serum from Human Subjects Previ- ously Injected with Serum Containing ^^Fe 1 ml, Serum was Dried at 100° C, Wet ashed with 1 ml Sulfuric Acid and Some Perhydrol and its Activity Determined as described Relative active units per ml of serom Sample Combustion 1 Combustion 2 Combustion 2 1 269 230 234 2 194 171 3 137 133 141 4 108 98 100 5 79 77 79 6 62 62 63 7 1.9 1.9 3.3 8 2.8 2.0 2.5 9 263 267 275 10 35 35 37 11 19 20 23 12 6.3 5.5 6.8 13 10 10 10 Table 3. — Determination of ^^Fe content of Hemin isolated from Human Erythrocytes. Combustion AS Stated in Table 2, iron Deter- mined Colorimetrically With Sulfosalicylic Acid Activity units per microgram iron Sample Combustion 1 Combustion 2 1 0.37 0.35 2 0.24 0.24 3 0.16 0.16 4 0.031 0.029 5 0.049 0.051 6 0.27 0.23 7 0.24 0.23 8 0.27 0.29 THE DETERMI.NATIOX OF RADIOIROX 653 ing the aluminum dish under the Geiger counter. Since the 500 micro- gram of iron are about equally distributed over a surface of 1.2 cm- we are measuring the radioactivity of an almost infinitely thin iron layer. The intensity of the /3-radiation emitted by ^^Fe is reduced to half its value when passing a layer of 10.0 mgm of aluminum per cm^. A few examples of the method described are given in Table I which contains data on added and recovered amounts of ^^Fe obtained when preparing "standard" samples. Since the procedure is rapid and easy to carry out and requires no special equipment but a Geiger counter, it can be used in most clinical laboratories. The accuracy obtained in clinical application of the method is seen from Tables 2 and 3. Duplicate determinations involving both combustion and counting technique are stated. Summary A simple and rapid method for the determination of ^"Fe is described. The radioiron is precipitated as FeS and after filtration of the solution counted on the filter paper. References P. F. Hahn, W. F. Bale, E. O. Lawrence and G. H. Whipple (1939) Radio- active iron and its metabolism in anaenia. Its absorption, transportation and utihzation. J. Exptl. Med. 69, 739. P. F. Hahn and W. M. Balfour (1942) Radioactive iron used to study red blood cells over long periods. The constancy of the total blood volume- in the dog. Am. J. Physiol. 135, 600. 654 ADVENTURES IN RADIOISOTOPE RESEARCH Comment on papers 61 — 65 The method of in vitro labelling of plasma by adding minute amounts of iron of high specific activity worked out in the Donner Laboratory by Huff et al. (1950) proved to be most useful and found a very extended apphcation. In paper 61 by making use of this device, the effect of adrenahn on the rate of extrusion of 5^Fe from the plasma was investigated. The marked increase of extrusion rate observed can hardly be due to the effect of adrenahn on the permeabihty of the capillary wall, since extrusion of ^^Na from the plasma was not found to be accele- rated after injection of adrenalin. It is due to an increased rate of uptake of s^Fe by the tissue cells. Even when exposing a rabbit to a heavy dose of 1500 r, 1 hr after exposure the rate of extrusion of ^^Fe from the plasma, which is mainly due to a transport to the bone marrow, is not much reduced. If we wait for 48 hr after exposure, the effect of 800 r is found to be more effective in reducing the exodus of ^^Fe from the plasma than are 1500 r after the lapse of 1 hr. Hennessy and Huff (1950) have already previously shown that the optimal depression of ^^Fe incor- poration into erythrocytes is obtained 1 to 2 days after exposure. This result, and the observations stated in paper 61 suggested the explanation that it is not the haemoglobin synthesis which is radiosensitive, but the blocking of haemoglo- bin formation under the effect of irradiation is due to interference with cell divi- sion and to cell destruction. The correctness of this view was brought out by the autographic investigations of Lajtha et al. (1955). In following up cellular pro- cesses the autographic technique proved to be a most powerful line of approach. As described in paper 62 the nuclear fraction of the rat and rabbit liver contains a small amount of haemoglobin non-extractable in aqueous medium. The incorpo- ration of ^*Fe into this fraction was also found to be radiosensitive, not however its incorporation into ferritin or cytochrome b. Due to the blocking of incor- poration of^^Fe into haemoglobin under the effect of irradiation, the specific acti- vity of ferritin, ferrosiderin and other iron compounds of the hver increases. Quite recently Theorell and Akeson succeeded in preparing highly pure myo- globin. By availing ourselves of this method when isolating myoglobin from rat muscles we found the hemin moiety of myoglobin to be radiosensitive only. Since the availabihty of the scintillation counter the activity of blood and other labelled iron containing samples is mostly measured by making use of this apparatus. Prior to the availability of the latter we precipitated the iron after wet combustion of the sample as sulphide and measured the activity of the preci- pitate with the Geiger counter as described in paper 65. Minute activities are still preferably measured by making use of the last-mentioned procedure. References R. L. Huff, T. G. Hennessy, R. E. Austin, J. F. Garcia, B. M. Roberts and J. H. Lawrence (1950) J. Clin. Inv. 29, 1041. T. G. Hennessy and R. L. Huff (1950). Proc. Soc. Biol. Med. 73, 436. K. Agner, R. Bonnichsen and G. Hevesy (1954) J. Lab. Clin. Inv. 6, 261. L. G. Lajtha and H. D. Suit (1955) Brit. J . Haematolog. 1, 55. Originally published in Acta Physiol. Scand. 38, 184 (1956). 66. EMBRYONAL IRON TURNOVER. G. Ehrenstein and G. Hevesy From the Institute of Organic Chemistry and Biochemistry, University of Stockholm VoSBURGH and Flexner (1950) were the first to inject labelled iron as FeClg into the circulation of the guinea-pig and to study the passage of radioiron through the placenta. The amount of iron passed into the foetus of the guinea-pig varied between 16 and 119, with an average of 56 microgram of iron per gram of placenta per day. They found no correlation with gestation age. This finding induced them to suggest that the passage of iron across the placenta appears to involve a different mechanism than that which is concerned with other substances which they studied. The rate at which water, sodium and inorganic phosphate cross a unit weight of the guinea-pig's placenta increases about 10 times during the last half of pregnancy. This increase of rate is corre- lated with thinning of the barrier between maternal and foetal circu- lation and increased area of exchange, and is in the predicted direction if the process is essentially diffusion. Iron, however, crosses the placenta at a rate which shows no correlation with the duration of pregnancy, i. e. there is no evident difference between the rates in early and late stages. In addition, there may be a considerable difference in the amount of iron transferred to members of the same litter during the course of the experiment. They emphasize that these characteristics of iron trans- port across the placenta suggest the existence of a rather complex regulatory mechanism which may be analogous to that concerned with the absorption of iron from the gastrointestinal tract, since ferritin has Ijecn demonstrated in the placenta of the guinea-pig by Latham and VosBURGH (1950) and in the human placenta by Mazur et al. (1955). The above view recently received much support by the work of Wohler (1955), discussed by him and lay Heilmeyer (1956). Wohler injected 1.25mgmof labelled ferrosulfite into the circulation of pregnant rabbits, and found the ferritin extracted from the placenta to show a marked radioactivity already 40 minutes after injection. He demonstrated, furthermore, the marked dependency of the total iron content of the placenta and the foetal plasma iron on the plasma iron content of the mother, in contrast to the ferritin content of the placenta. The latter 656 ADVENTURES IN RADIOISOTOPE RESEARCH varied to a restricted degree only with a varying maternal plasma iron level. From these observations he inferred that much of the maternal plasma iron reaches the embryo after being previously incorporated into ferritin of the placenta. We wished to study iron metabolism at an early stage of pregnancy in which the liver of the embryo is to a large extent involved in hemo- poiesis and furthermore, the effect of exposure to radiation on iron tur- nover in the pregnant rabbit. Plasma samples of control and irradiated rabbits obtained 21 days after mating, were incubated at 37° C for 20 minutes with 0.5 microgram of iron as citrate labelled with ^^Fe of 5 microcurie of activity per one ml plasma, and then reinjected . The animals were killed 11.5 to 17 hours after injection. The activity and iron content of the plasma of the mother, total iron and activity of the livers of the mother, and the foetus and the specific activity of their liver-ferritin was then determined. We measured also the specific activity of the circulating hemoglobin and of the hemoglobin present in the nuclear fraction of the liver of mother and foetus. The amount of radioiron found in the embryos after removal of the liver was determined as well. The weight of the rabbits varied between 3.2 and 4.1 kgm the number of embryos between 4 and 11, their aggregate weight between 11 and 58 gm, and the aggregate Uver weight of the embryos between 1.4 and 5 gm. After wet ashing of the samples to be investigated, a knoA\Ti aliquot of the sample was used for colorimetric determination of its iron content. The iron deter- minations were made essentially according to the sulfosalicyhc acid method by LoBBEB (1927). Another known ahquot of the ashed sample, after bringing its iron content up to 500 microgram by adding FeSO^, was precipitated as sulphide and filtrated through perforated aluminium dishes covered with filter-paper, as described previously by Agner et al. (1954), prior to placing the dish under the Geiger counter. The livers of the animals were homogenized in 9 vol. 0.25 molar sucrose contain- ing 0.0018 m CaClg, centrifuged for 10 min at 2000 r. p. m. (Internat. refrigerated centrifuge horizontal head No. 269), and the ferritin was precipitated from the supernatant by addition of half a volume of ammoniumsulphate. The precipitate was taken up in water and repeatedly fractionated with anunoniumsulphate. The precipitate was then dissolved in water, heated to 70° C for a few minutes and the cold solution filtered. The filtrate, which contained the ferritin, was then asnalysed. The hemin of the purified red corpuscles was extracted with a mixture of acetone and HCl (10 ml 20 p. c. HCl in 1 liter of acetone). It was twice recrystallized from acetic acid and the crystals were washed with diluted HCl. The non-water soluble hemoglobin of the liver fraction was obtained from thoroughly purified nuclei, prepared according to Hoge- boom-Schneider as described hj Bonnichsen, Ehrenstein and Hevesy (1956). % 50H 40- 30- 20- _ T3 O «> ••- O c a. o >c u Fig. 9. Ratio of the specific activity of nuclear hemoglobin Fe and circulating hemoglobin Fe. EMBRYONAL IRON TURNOVER 66 1 in the embryo of the exposed rabbit is seen in Fig. 8. The specific acti- vity of the iron of this fraction is much smaller than that of the iron of circulationg hemoglobin in the maternal organism, but appreciably larger in the embryo (cf. Fig. 9). Presumably hemoglobin synthesis lakes place l)esides in the cyto- plasm of the hemopoietic liver cells in their nuclei as well. It is of interest in this connection to recall the finding of Stern, Allfrey, Mirsky and Saetren (1952), according to which almost ^g of the hemoglobin of the avian nucleated red corpuscles is present in the nuclei. In our experiments during the purification process of the nuclei, the water soluble hemoglobin, present possibly in appreciable amounts, may have been removed, in contrast to the small amounts of water insoluble hemoglobin which amounted to not more than 4.1 mgm in our maternal and 17.5 mgm in our foetal samples. It was found in a previous investigation [BoNNiCHSEN, (1956)] that the radiation sensitivity of the nuclear hemoglobin becomes very marked after the lapse of about 1 day, as does that of the circulating hemoglobin. Summary Seventeen hours after labelling the circulating iron-/Sjglobuhn with ^'Fe, 14_ 34 per cent of the labelled iron is found in the 21 days old foetus of the rabbit. Exposure of the rabbit to 500 r of X-rays increases the amount of ^*Fe passing the placenta to almost twice the value in non-exposed animals. Seventy-three— eighty-three per cent of the ^*Fe content of the embryo is found in the Uver. The specific activity of the hemoglobin iron present in the nuclear fraction of the hver amounts to one fourth of that of the circulating hemoglobin in the maternal and to about twice in the embryonal organism. Exposure to radiation one day prior to labelling the plasma depresses the frac- tion of embryonal ^^Fe incorporated into the hemoglobin of the nuclear liver fraction to about half of the value observed in non-irradiated rabbits. References K. Agner, R. Bonnichsen and G. Hevesy (1954) Scand. J. din. Lab. Inv. 6, 261. E. H. Belcher, J. G. Gilbert and Ll F. Lamerton (1954). Brit. J. Radiol. 27, 387. R. Bonnichsen and G. Hevesy (1955 a) Acta Chem. Scand. 9, 509. R. Bonnichsen (1956) Acta Chem. Scand. 9, 1045. R. Bonnichsen, G. Ehrenstein and G. Hevesy (1956) To be published. L. Heilmeyer (1956) Vortrdge aus dem Gebiet der klinischen Chemie und Cardiologie. Thieme Verlag, Stuttgart. T. G. Hennessy and R. L. Huff (1950) Proc. Soc. exp. Biol. 73, 436. R. L. Huff, W. P. Bethard, J. F. Garcia, B. M. Roberts, L. O. Jacobson and J. H. Lawrence (1950) /. Lab. din. Med. 36, 40. 662 ADVElf.TURES IN RADIOISOTOPE RESEARCH E. F. Latham and G. J. Vosburgh, cf. G. J. Vosburgh and L. B. Flexneb (1950) Amer. J. Physiol. 161, 202. F. LoBBER (1927) Biochem. Z. 181, 391. A. Mazur, S. Baez and E. Shorb (1955) J. Biol. Chem. 213, 417. H. Stebn, V. Allfrey, A. E. Mirsky and H. Saetren (1952) J. gen. Physiol. 35, 559. G. J. Vosburgh and L. B. Flexner (1950) Amer. J. Physiol. 161, 202. F. WoHLER (1955) Dtsch. med. Wschr. 30, Comment on paper 66 As already shown by Vosburgh and Flexner (1950) the ^^Fe introduced into the maternal plasma of pregnant guinea-pigs is soon detectable in the embryos. In paper 66 it is shown that about % day after labelling of the maternal plasma iron 1 gm of foetal hver contains 143 times more ^'Fe than does the same weight of the maternal hver. In the former about 80 per cent of the ^^Fe which passed the placenta accumulates, demonstrating conspicuously the fact that in the rabbit embryo the Hver is the main haemopoietic organ. Exposure of the rabbit to 500 r increases the amount of ^^Fe passing the placenta to twice the value in non-exposed animal. This is a result of the higher plasma ^^Ye concentration of the maternal plasma in the exposed animal. (1959) It was recently found by us that the maternal ^^Fe which reaches the mouse foetus is much better conserved during life than ^^Fe injected into the mouse intraperitoneally. ^/g ^j^ per day is lost daily from the former, i/g ^j^ from the latter Reference G. J. Vosburgh and L. B. Flexner (1950) Amer. J. Phijsiol. 161, 202. G. Ehrenstein and G. Hevesy (1959), Acta Haematol. 22, 311. Originally published in Acta Physiol. Scand. 5, 237 (1943). 67. R4TE OF FORMATION OF NUCLEIC ACID IN THE ORGANS OF THE RAT G. Hevesy and J. Ottesen From the Institute of theoretical Physics, University of Copenliagen The rate of formation of nucleic acid of the thymus nucleic acid type was investigated in the organs of the rat by administering labelled phosphate to rats and by determining the labelled P content of the desoxyribose nucleic acid extracted from the organs after the lapse of some days. The percentage of labelled nucleic acid present indicates the percentage of the total nucleic acid of the organs which is built up in the course of the experiment, as described in this note. Preliminary figures on the rate of formation of nucleic acid in some of the organs of the rabbit were communicated at an earlier date (Hahn and Hevesy, 1940). Data are furthermore available on the rate of for- mation of labelled "nucleoprotein" in some organs of the mouse (Tuttle, Erf and Lawrence, 1941). In our previous work, we extracted the nucleic acid with sodium chloride solution. Tuttle and his colleagues removed the acid soluble and the phosphatide P fractions from the organs investigated and considered the residual P to be phosphorus of the "nucleoprotein" fraction. Extended studies carried out in this laboratory lead to the result that it is hardly possible to obtain nucleo- protein sufficiently purified from non-nucleoprotein phosphorus by the last mentioned procedure. Muscles and other organs of the frog con- taining labelled P were treated for weeks daily alternately with tri- chloroacetic acid solution and with ether alcohol mixtures. The specific activity of the remaining "nucleoprotein" P was determined subse- quently. It was found much higher than the specific activity (activity per mgm P) of phosphorus obtained from properly purified nucleic acid. As shown in this note, the rate of formation of nucleic acid in most organs is very slow and, correspondingly, the specific activity of the nucleic acid P few hours and even some days after the administration of labelled P is low also. After the lapse of 2 hours, 1 mgm nucleic acid Pof the liver of the rat, for example, contains but 2 • 10^^ per cent of the labelled phosphorus administered, while the corresponding figure for 1 mgm acid solul)le P of the liver is about 1 per cent. If only 10"^ part of the isolated nucleic acid P 664 ADVENTURES IN RADIOISOTOPE RESEARCH is composed of acid soluble P present as an impurity, a grossly erroneous value will be found for the specific activity of the nucleic acid P, viz. 12 • 10~* instead of 2 • 10"*. This example illustrates the necessity of an exceedingly careful purification of the nucleic acid fraction from all non-nucleic acid phosphorus. In our work, we are not faced with the great difficulties which were surmounted by Hammahsten" in his experiments which lead to the preparation of non-depolymerized nucleic acid. On the other hand, w^e have to avoid the presence of even minimal amounts of non-nucleoprotein P, the presence of which in any other but the radioactive investigations would certainly not be found disturbing. EXPERIMENTAL PROCEDURE We applied the method of extraction and purification described by Klein and Beck (1935) adapted to work with tissue containing radioactive phosphorus, as previously used by H. von Euler and one of the present writers (1942) in their work on the rate of formation of nucleic acid in the Jensen sarcoma of the rat. The washed tissue is stirred with an equal weight of 5 per cent sodium chloride solution brought to boihng. Acetic acid is added until the major part of the pro- teins present is precipitated. Sodium acetate and sodium hydroxyde are then added and the alkaline solution is heated until the tissue is dissolved. The next operation is carried out in a slightly acid solution. This is obtained by adding acetic acid. From this solution, the protein present is removed by adding a dialysed colloidal iron hydroxide solution containing 5 per cent FgOg. An excess of acetic acid is added and the hot solution is filtered. By adding an equal volume of methylalcohol to the filtrate, the crude nucleic acid precipitates. The crude nucleic acid is dissolved in sodium hydroxide and is precipitated with hydrochloric acid and methylalcohol. Before re-precipitating the nucleic acid, we added about 10 mgm (NH4)2HP04 for each mgm nucleic acid. By doing so, we diluted the free radioactive phosphate possibly present in the nucleic acid. If the crude nucleic acid carried before and after the precipitation 1 mgm free phosphate, the free phosphate will be but 1/100 as active after precipitation as previously. This procedure is repeated several times, and each time inactive (NH^)2HP0^ is added to the alkaline solution. The purification process entails a substantial loss of nucleic acid. However, it is not the total desoxyribose nucleic acid content of the organs in which we are interested, but the percentage of the desoxyribose nucleic acid content which is built up during the experiment, i. e. the rate of renewal of the nucleic acid molecules. We are interested in the activity of 1 mgm nucleic acid P and not in the activity of the total nucleic acid present in the organs. The purified nucleic acid is brought into solution by wet ashing, i/g is reserved for colorimetric P determination, while ^/^ are precipitated as ammonium magne- sium phosphate; the activity of the precipitate is determined. The interpretation of the activities of which are to be compared, have the same weight. To obtain this, a suitable amount (about 80 mgm) of NagHPO^ is added to the solution before precipitating the ammonium magnesium compound. An aliquot of the solution administered by subcutaneous injection is treated in the same way. If this "standard preparation" has, for example, 1/1000 of the activity administered and the nucleic acid fraction containing 1 mgm P has 1/100 NUCLEIC ACID IN THE ORGANS OF THE RAT G65 of the activity of the standard preparation, we find U.OUl pei' cent of the labelled P administered to be present in 1 mgm nucleic acid P. The weight of the male adult albino rats used varied between 250 and 320 gm. They were kept in a normal diet. The labelled phosphate administered by subcu- taneous injection had an activity corresponding to '.i //Curie. CONTROL OF THE EFFECTIVITY OF THE PURIFICATION PROCESS To a crude nucleic acid fraction containing 200 mgm nucleic a(;id we added 75,000 relative units of radioactive phosphorus (^^p). Each time, decreasing amounts of inactive ammonium phosphate were added to the filtrate containing the nucleic acid. The amount of (NH^)2HP0^ added varied between 100 and 30 mgm. After successive purifications, the following activity figiires were obtained for the nucleic acid. Xumber of Activity of purifications fractions 75,000 1 — 2 34 3 9 The nucleic acid purified 3 times thus contained but 1/8000 part of the labelled phosphate added. The successive purification of nucleic acid P from other than free P can be controlled in the following way. The specific activity of an ahquot of the nucleic acid P of the purified sample is determined. Another aliquot of the sample is dissol- ved subsequently and is re-precipitated as described above, but, without adding phosphate. The specific activity of the P of the precipitate obtained is then again determined. If no other phosphorus than nucleic acid P is present in the sample the specific activities determined should be identical. When controUing the purity- of nucleic acid extracted from the liver and purified twice in the manner described above, a further purification reduced the specific activity of the phosphorus obtained from the nucleic acid by 5.5 per cent. Furthermore, we investigated whether the nucleic acid obtained is exclusively of desoxyribose type or contains also some nucleic acid oi' the ribose nucleic acid type. To 7.6 mgm active thymus nucleic acid dissolved in 1.0 cc. 7io N. NaOH about 60 mgm yeast nucleic acid, dissolved in 2 cc. 7io N- NaOH were added. After precipitating but once, the nucleic acid, as described on p. 664, we redetermined the specific activity of the nucleic acid P. This was found to be 76 per cent of the specific activity of the value measured at the start of the experiment. A single precipitation sufficed thus to remove 96 per cent of the yeast nucleic acid added. 666 ADVENTURES IN RADIOISOTOPE RESEARCH SPECIFIC ACTIVITY OF THE FREE P OF DIFFERENT ORGANS The fact that greatly divergent figures are obtained for the specific activity of the nucleic acid fractions is due mainly to the highly dif- ferent rate at which nucleic acid is formed in the different organs, molecules are built up. To calculate the rate of formation of nucleic acid, it is thus necessary to know the specific activity of the "free" phosphate present in the tissue cells. It does not suffice to know the specific activity of the free P at the end of the experiment. We have to determine this magnitude at different times in order to arrive at a value of the average specific activity of the free P during the experiment. The results obtained are seen in Table 1 which contains data on the specific activity of the "free" P extracted from the organs. They are obtained on rats killed at different times. In the case of the muscles, Table 1. — Specific Activity of the Free Phosphate Extracted from the Organs(i) Organ Duration of the experiment in hours Average value of the specific 2 5 8.5 13 25 50 72 94 activity during the experiment Plasma Liver Kidney Spleen Mucosa of the small intestine Muscle Testes Brain 0.91 0.67 0.214 0.286 0.204 0.114 0.099 0.069 1.61 0.95 0.42 0.59 0.17 01.5 0.10 0.10 1.10 0.78 0.43 0.57 0.18 0.16 0.096 0.10 0.77 0.67 0.31 0.43 0.19 0.16 0.11 0.10 0.72 0.51 0.35 0.51 0.15 0.13 0.11 0.11 0.27 0.13 0.12 0.38 0.062 0.084 0.090 0.055 0.13 0.12 0.086 0.14 0.062 0.079 0.072 0.06 0.044 0.052 0.045 0.083 0.047 0.057 0.053 0.051 0.18 0.25 0.24 0.21 0.19 0.11 0.080 0.054 ''' Specific activity usually denotes the activity of 1 mgm P in arbitrary units (often, the specific activity of the plasma P is taken = 100). The above specific activities denote the •percentage of the ^-P administered present in 1 mgm P. the free P was extracted from tissue samples taken from the narco- tized rat. The tissue was placed in liquid air and extracted at once by grinding with 10 per cent trichloroacetic acid. This precaution has to be taken in order to avoid the decomposition of creatine phosphoric acid present in large amounts in the muscle. While, in experiments of long duration, as discussed below, the specific activity of the creatine phospho- ric acid P does not differ from the specific activity of the free P, in ex- periments of shorter duration, however, large differences were found, and in these experiments a decomposition of creatine phosphoric acid prior to the removal of the free P would result in a lowering of the specific activity of the free P. NUCLEIC ACID IX THE ORGANS OF THE RAT 667 The specific activity of the creatine phosphoric acid was determined in the following way. After the removal of the free P as ammonium magnesium salt, the filtrate was slightly acidified and heated for a very short time. The free P obtained by the decomposition of creatine phos- phoric acid was then again precipitated as ammonium magnesium salt. While, after the lapse of one day or more, the specific activity of the creatine phosphoric acid phosphorus was found to be just as high as the specific activity of the "free" P present in the muscle tissue, after the lapse of 2 hours the ratio of the specific activities was found to be only 0.6- SPECIFIC ACTIVITY OF THE NUCLEIC ACID EXTRACTED FROM DIFFERENT ORGANS The results of the determination of the specific activity of the nucleic acid phosphorus extracted from the different organs is seen in Table 2. I and II denote the values obtained in the first and second experiments respectively. In each experiment the organs of 8 rats were pooled. The values shown in the last column indicate the percentage ratio of the specific activity of the nucleic acid P and the specific activity of the "free" phosphate P of the different organs. Table 2. — Specific Activity(i) of the Nucleic Acid Phosphorus Extracted from Different Organs of 8 Rats 4 Days after the Administration of Labelled Phosphate X 1000 Organ Specific activity x 1000 11. Percentage ratio of the specific activity of nucleic acid P and free P (Percentage renewal)'-' Small intestinal mucosa Spleen Muscle Liver Testes Kidney Brain 12.7 15.4 5.93 6.24 1.05 1.42 1.01 1.66 0.83 0.97 0.60 0.67 0.09 0.22 59 23 8.8 4.2 10 2.1 2.3 (1) Percentage of the '"P administered present in 1 mgm P. ''> When calculating the above ratio, we must take into account that the nucleic acid has been extracted from the organs of 8 rats. When calculating the figures of the last column, we have not taken the figures of the specific activity of the free phosphate P as stated in Table 1, but corrected these for the presence of labelled P in the extracellular space of the organs. The extracellular P is not utilized to build up nucleic acid, and we have to consider the ratio of the specific activity of the nucleic 668 ADVEXTURES IN EADIOISOTOPE RESEARCH acid P the specific activity of the cellular free P. The figures for the size of the extracellular space of the organs were taken from a paper by Manery and Hastings (1939) and it was assumed that the labelled phosphate concentration of the extracellular fluid is identical with the labelled '6 -1 8- ■ Doily percentage renewal of desoxyribose nucleic acid 0> a S •o c o Fig. 1. phosphate concentration of the plasma water. The correction for the presence of labelled phosphate in the extracellular space was largest for the testes, but even in this case only 12 per cent of the value stated in Table 1. We did not correct the specific activity found for the free P of the brain in view of the uncertainty prevailing as to size and compo- sition of the extracellular fluid of the brain. Therefore, it is possible that the rate of renewal of the brain nucleic acid is not slightly larger, but smaller than the corresponding value found for the kidneys (cf. Table 2). The percentage ratio of the specific activity of nucleic acid P and free P (the percentage renewal of the nucleic acid) in different organs is seen in Table 2 and Fig. 1. The highest percentage of new nucleic acid is found in the small intestine, while the lowest figure is shown by the brain. Remarkably low figures are found for the liver. In the NUCLEIC ACID IX THE ORGANS OF THE RAT 669 course of 1 hour, a very large part of the acid-soluble P compounds and a few per cent of the phosphatides present in the liver are renewed. Compared with these figures, the rate of renewal of nucleic acid in the liver is negligible. The percentage ratio of the specific activity of the nucleic acid P and the free P indicates the percentage of new nucleic acid present, i.e. nucleic acid formed in the course of the experiment. We cannot state with certainty whether this new nucleic acid is formed in the organ in which it is found or transported from another organ in which it was built up. It would be conceivable that the nucleic acid molecules built up in the intestinal mucosa, for example, where we find far the greatest rate of renewal of nucleic acid, reach the circulation and are deposited in the muscles. Information on this point can be obtained on the same line or on similar lines on which the origin of the phosphatides present in the yolk was investigated (Hevesy and Hahn, 1938). The nucleo- proteins are probably built up in the nuclei of the cells and not carried from organ to organ. The low new (labelled) nucleic acid content of the liver can be interpreted as an argument against the last mentioned interpretation. The liver takes up easily constituents present in the cir- culation and, if any organ takes up from the circulation nucleoproteins and thus nucleic acids, we would expect the liver to do so. The active nucleic acid content of the liver nucleic acid is, however, very low and this fact supports the view that the active nucleic acid molecules present in the liver are synthesized in this organ. The rate of renewal of the nucleic acid molecules in the liver may be identical with the rate of new formation of liver cells^^^ The figures for the rate of formation of nucleic acid in the organs of the rat found in this investigation are very much lower than those for the renewal of nucleic acid or of "nucleoproteins" by different experiments both in the organs of the rabbit and in the organs of the mouse. In the liver of the rabbit (Hahn and Hevesy, 1940), for example, 6 per cent of the nucleic acid present were found to be renewed in the course of 11.5 hours. In the liver of the mouse (Tuttle, Erf and Lawrence, 1941), in the course of 6 hours, about 40 per cent of the "nucleoproteins" present w^ere found to be labelled. In these experiments, the nucleic acid P and the "nucleoprotein P", respectively, contained presumably some strongly active acid-soluble or phosphatide phospho- rus, the presence of which was presumably responsible for the high values obtained for the rate of renewal of the nucleic acid and the "nucleo- proteins". ^1^ The rate at which hver cells are renewed is not known. While this rate may- be smaller than the rate of formation of nucleic acid in the liver cells, it can hardly he larger. 670 ADVENTURES IX RADIOISOTOPE RESEARCH AMOUNT OF NUCLEIC ACID FORMED DAILY IN THE DIFFERENT ORGANS OF THE RAT If we assume that the labelled desoxyribose nucleic acid found in an organ is synthesized in the organ in question, we can estimate from the data of Table 2 and the desoxyribose nucleic acid content of the organs the total amount of desoxyribose nucleic acid which is built up daily in the different organs. Data are available on the total nucleic acid content of the organs of the rat. These data are given in Table 3. With the exception of the figure stated for the nucleic acid content Table 3. — Upper Limit of the Desoxyribose Nucleic Acid Content of Different Organs of the Rat Desoxyribose nucleic acid Organ \ t. ^ , s content (mgm per gm) Muscle Heart Brain Kidney Testes(i) Mucosa of the small intestine Liver Spleen Thymus(2) 1.4 1.4 2.5 3.3 5.7 5.1 6.5 10 30 •'> Horse testes. (Javiilier and Allaire, 1926.) '■^1 Horse thymus. For calf thymus, 36 was found. (Javillier and Allaire, 1926.) of the intestinal mucosa, they are taken from a paper by Javillier et al. (1928). These workers state the nucleic acid P content of the tissue investigated ; we multiplied their figures by 12 to arrive at the nucleic acid content. As no data were available for the nucleic acid content of the mucosa of the intestine, we determined the desoxyribose nucleic content of the mucosa small intestine by using Dische's method (1930) in a slightly modified form, as applied by Vowles (1940). This method is based on the fact that, when heating a solution of desoxyri- bose nucleic acid in the presence of diphenylamin, acetic acid and sul- phuric acid, a violet colouring is obtained, the intensity of the colour being proportional to the concentration of the nucleic acid. As standard preparation we used a thymus nucleic acid preparation kindly presented us by Professor Hammaesten. As the reaction used is not strictly spe- cific for desoxyribose, the figure obtained has also to be considered an upper limit of the desoxyribose nucleic acid content of the intestinal mucosa. NUCLEIC ACID IN THE ORGANS OF THE RAT 671 If the bulk of the proteins present were not previously precipitated, the colorimetric determination gave a higher value (6.9 mgm). The same observation was made by Vowles (1940). The upper limit of the amount of desoxyribose nucleic acid built up daily in the different organs of the rat is given in Table 4. Table 4. — Upper Limit of the Amount of Desoxybibose Nucleic Acid Built UP Daily in the Different Organs of Rats Weighing on the Avkrage 275 gm Organ Weight in gm Nucleic acid present in the organ in mgm Upper limit of desoxy- ribose nucleic acid built up in the course of a day in mgm Brain Kidney Testes Spleen Liver Mucosa of the small intestine Muscle 1.43— L53 1.72— L76 1.93—2.57 0.86—0.81 9.24—9.06 4 111 — 109 3.7 5.8 12.8 8.3 57.5 21 154 0.02 0.03 0.34 0.48 0.61 3.0 3.4 As already mentioned previously, when calculating the above figures we assume that the nucleic acid found in an organ is built up in the organ. This assumption may not hold strictly, as the blood contains some nucleic acid which may have been carried from the organs into the circulation. However, this amount is small. Assuming the rat blood to contain the same nucleic acid concentration as human blood which, according to Javillier and Allaire (1931) amounts to 0.3 mgm per gm blood,, the total amount of desoxyribose + ribose nucleic acid present in the circulation of a rat weighing 275 gm amounts to about 6 mgm, thus about ^/jQ of the total nucleic acid content of the rat. Summary Labelled pliospliate is administered to adult rats by subcutaneous injection.. After the lapse of 4 days, the rats are killed and the desoxyribose nucleic acid present in different organs is extracted. By comparing the activity of X mgm desoxy- ribose nucleic acid P with the activity of 1 mgm cellular inorganic P of the same organ, data on the percentage renewal of the desoxyribose nucleic acid present in the organs are obtained. In the course of 4 days, only a minor part of the desoxyribose nucleic acid pre- sent in all the organs investigated is found to be labelled, i. e. formed in the course of the experiment. The greatest daily formation of nucleic acid (15 per cent) takes place in the mucosa of the small intestine. This is followed by the spleen (5.8 per cent), the testes (2.6 per cent), the muscles (1.9 per cent), and the liver (1.0 per cent). The lowest figures aie shown by the kidneys and the brain (0.6 per- cent) . 67 2 NUCLEIC ACID IN THE ORGANS OF THE RAT References Z. DiscHE (1930) Hoppe-Seyl Z., 192, 56. H. EuLER and G. Hevesy (1942) Kgl. Danske Vidensk. Selsk. Biol. Medd. 17,8. L. Hahn and G. Hevesy (1940) Nature, 145, 549. G. Hevesy and L. Hahn (1938) Kgl. Danske Vidensk. Selsk. Biol. Medd. 14, 2. M. Javillieb and H. Allaire (1926) Bull. Soc. Chim. Biol., Paris 8, 924. M. Javillier, a. Cremieu and H. Hinglais (1928) 10, 327. M. Javillier and M. Fabrykant (1931) Ihid. 13, 685. G. Klein and J. Beck (1935) Z. Krebsforsch. 42, 163. J. F. Manery and B. Hastings (1939) J. Biol. Chem. 127, 657. L. W. TuTTLE, L. A. Erf and J. H. Lawrence (1941) J. Clin. Invest. 20, 57. R. B. VowLES (1940) Ark. Kemi, 14, No 10. Originally published in Acta Physiol. Scand. 11, 335 (194G). 68. RATE OF RENEWAL OF RIBO- AND DESOXYRIBO NUCLEIC ACIDS E. Hammaesten and G. Hevesy From the Institute for Research in Organic Chemistry and the Chemistry Laboratories of the Karolinska Institute, Stockhokn Enzymic processes coupled with phosphorylation often take place at a remarkedly rapid rate. A large percentage of the molecules of many of the acid-soluble phosphorus compounds and, to a minor extent, also those of phosphatides present in the liver and some other organs are renewed within a short time. This is demonstrated by the observation that shortly after the administration of ^^P these molecules are found to contain labelled phosphate. That the presence of ^^P in the molecules of organic phosphorus compounds indicates an enzymic synthesis of such molecules is most strikingly demonstrated by a recent experiment of Chaikoff and his associates (1942). These authors have shown that labelled phosphatides are formed when surviving liver slices are shaken with bicarbonate Ringer solution containing labelled phosphate, that this formation is, however, impaired in the absence of oxygen, and homogenized liver tissue completely loses its ability to incorporate ^^P into the phosphatide molecule. A non-enzymic process could hardly be dependent on the intactness of the tissue cells. In contradistinction to the above mentioned compounds, as found in previous (Hevesy and Ottesen, 1943 ; Ahlstrom, Euler and Hevesy, 1944 ; Brues, Tracy and CoHisr, 1944) and in the present investi- gations, desoxyribo nucleic acid molecules present in the liver are at a very slow rate only. This result falls in line with the view that deso- xyribo nucleic acid is present in the nuclei of the cells and is involved in the process of cell division. As the mitotic process in the liver of a fully grown rat takes place at a very slow rate only, the low rate of formation of desoxyribo nucleic acid in the fully grown liver is in no way surprising, neither is the much higher rate of formation observed in the liver of the only few days old rat. The rate of forma- tion on new desoxyribo nucleic acid molecules present in the liver of 3 to 4 days old rats was found to be about 20 times that of the corresponding figure in outgrown rats (Ahlstrom, Euler and Hevesy, 1944). 43 Hevesy 674 ADVENTURES IN RADIOISOTOPE RESEARCH In contradistinction to the desoxyribo nucleic acid, a large part of ribo nucleic acid is located in the cytoplasm and, according to the view developed by Caspersson (1940), is involved in protein synthesis. As such a synthesis takes place at a marked rate in the liver, the rate of renewal of the ribo nucleic acid can be expected to be larger than the rate renewal of desoxyribo nucleic acid in this organ. The present note contains the results of experiments in which the rate of renewal of both types of nucleic acid was determined, viz. in the liver, the spleen, and the intestinal mucosa of the rat and also in the total rat body. The methods applied in the isolation of the nucleic acids will shortly be published by one of the authors. RESULTS If we assume the free phosphate to be built into the nucleic acid molecules without the formation of an intermediary phosphorus com- pound of comparatively long life, the ratio of the specific activity of the nucleic acid P and the free P is a measure of the rate of formation of new nucleic acid molecules and thus of the extent of renewal of such molecules. If, previous to the formation of labelled nucleic acid mole- cules, labelled precursors would be built up at a rate slower than the rate of formation of new nucleic acid molecules, the ratio of the specific activity of the nucleic acid P and that of the free P would no longer be a proper measure of the rate of renewal of the nucleic acid. In the latter case, namely, during part of or possibly throughout the whole experiment, new nucleic acid molecules would be built up without participation of =^2P. The participation of such labelled intermediary compounds of a very long life in the formation of desoxyribo nucleic acid in the liver is, however, highly improbable in view of the comparatively short hfetime of most of the acid-soluble phosphorus compounds and the very long lifetime of desoxyribo nucleic acid molecules present in the liver. In our experiments the specific activity of the nucleic acid P is com- pared with the specific activity of the free P at the end of the experiment. As the specific activity of the free P changes throughout the experiment, the specific activity of the nucleic acid P at the end of the experiment should, however, be compared with the mean specific activity of the free P during the experiment. As the liver takes up ^ap at a very rapid rate, and its free ^^p content culminates within the first 2 hours, the end value and the average value of the specific activity of the free liver P do not differ essentially, the average value being about 5 per cent lower than the end value (Ahlstrom, Euler and Hevesy, 1944). In the case of the spleen the corresponding figure is about 25, and even larger differences are found in the case of the intestinal mucosa. The figures RENEWAL OF DESOXYRIBO NUCLEIC ACIDS 675 of columns 2 and 3 of Table 1 should therefore be multiplied by 1.05 in the case of the liver, for example, to obtain a correct value for the amount of the rate of renewal of the desoxyribo nucleic acid present in the liver. In the figures of Table 1 we have not introduced this cor- rection, as we are mainly interested in the relative rate of renewal of the desoxyribo and ribo nucleic acids. Table 1 contains the results of an experiment in which three rats in nitrogen-equilibrium weighing 252, 182 and 215 gm were injected sub- <'utancously with respectively 7.5, 6.6 and 5.6 microcuries of ^^p per 100 gm body weight. After 2 hours the animals were killed and the organs prepared according to a method which will soon be published by one of the authors. Table 1. — Ratio of the Rate of formation of the Ribo Nucleic Acid and Desoxyribo Nucleic Acid in the organs OF the Rat in the Course of 2 Hours Percentage ratio of the specific activity of the nucleic acid P and that of the free P Ratio of the rate of format ion Organ Eibo Desoxyribo of ribo and desoxyribo nucleic acid Liver 3.3; 3.6 3.1; 10.2 7.1 4.1 0.12; 0.09 2.2; 2.2 3.4; 2.3 33 Snleen 3 Intestine 2 As recorded in Table 1, the rate of renewal of ribo nucleic acid in the liver is as much as 33 times larger than the rate of renewal of deso- xyribo nucleic acid. In spite of the finding that ribo nucleic acid is renewed at an even larger rate in the spleen and the intestinal mucosa than in the liver, the ratio of the rate of renewal of ribo- and desoxyribo- nucleic acids in these organs is only 3 and 2, respectively. This low ratio is due to the comparatively high rate of formation of desoxyribo nucleic acid in these organs. From the above figures it follows that the rate of renewal of both types of nucleic acid is highest in the intestinal mucosa and in the spleen. The specific activity of both the desoxyribo and the ribo nucleic acid P extracted from the rat liver was determined by Brues, Tracy and CoHN (1944) in experiments lasting 3 to 8 days. In these experiments the ribo nucleic acid P was found to be only 5 to 6 times as active as the desoxyribose nucleic acid P. The discrepancy between these figures and those obtained by us may be due, at least mainly, to the much longer duration of the last mentioned experiments. 43^ 676 ADVENTURES IX RADIOISOTOPE RESEARCH Specific Activity of the Nucleic Acid Phosphorus Extracted from the Total Rat In another experiment the specific activity of both the total clesoxy- ribo nucleic acid P and the total ribo nucleic acid P extracted from a rat weighing 194 gm was determined. The activity of labelled sodium phosphate amounted to 8.1 microcuries per 100 gm animal weight. The time of the experiment was 2 hours. The results of this experiment are seen in Table 2. Table 2. — Specific Activity of the Nucleic Acid P Extrac- ted FROM THE Total Rat Compared with the Corresponding Values for Liver, Spleen, and Inte.stinal Mucosa. The Value for the Specific Activity of the Total Rat Ribo P IS Assumed to be = 100 Specific activity Sample Ribose Desoxyribose Free P nucleic acid Total rat Liver Snleen 100 164 292 112 60 4.4 63 63 5100 2850 Intestine 2770 As shown in Table 2, the specific activity of the average nucleic acid P of the rat is almost identical with the corresponding value of the ribo and desoxyribo nucleic acid, respectively extracted from the intestine. The interpretation of the significance of the specific activity figures obtained for the total rat encounters some difficulties, as the specific activity of the free P utilized in the formation of the labelled nucleic acid molecules is unknown. If the specific activity of the free P utilized in building up the average body nucleic acid would correspond to the specific activity of the free liver P, the percentage "rate of renewal" of the body ribo and desoxyribo nucleic acids would be 2.0 and 1.2, respectively. If the specific activity of the free P utilized in building up the average nucleic acid of the organism would correspond to the specific activity of the intestinal P, larger figures, i.e. 3.6 and 2.2, respectively, would be obtained. When calculating the last mentioned figures, we compared the specific activity of the nucleic acid P at the end of the experiment with the spe- cific activity of the free intestinal P at the end of the experiment. Cor- rectly we should have considered the mean value of the specific activity of the tliree intestinal P prevailing during the experiment. The mean RENEWAL OF DESOXYRIBO NUCLEIC ACIDS 677 value of this magnitude is about almost half of its end value, we have therefore to multiply the figures mentioned above (3.6 and 2.2 respec- tively) by about 2 to obtain an approximate value of the percentage renewal of the ribo- resp. desoxyribo nucleic acid in the course of 2 hours. It is improbable that a so highly active free phosphate is utilized in the building up of the nucleic acid molecules as found in a 2 hours experiment in the liver. Liver and kidneys have a privileged position concerning the rate of intrusion of phosphate. The amount of nucleic acid present in the liver and the kidneys makes out, furthermore, only a small percentage of the total nucleic acid content of the organism. It is much more probable that free P of similar specific activity as found in the intestine is applied in the building up of the labelled nucleic acid molecules. In fact, the amount of nucleic acid present in the mucosa of the digestive tract makes out a large percentage of the body nucleic acid. While the body nucleic acid contains also slightly radioactive fractions, viz. those originating from the liver, the kidneys, and the brain and fractions of restricted radioactivity originating from the muscles (Hevesy and Ottesen, 1943), it contains also fractions of higher activity than found in the intestinal mucosa, viz. those originating from the bone marrow, the thymus and lymph nodes (Andreasen and Ottesen, 1944, 1945). The lymphocytes secreted into the organism can also be expected to contain pronouncedly active nucleic acid. This makes it understandable that the rate of renewal of the average body nucleic acid corresponds to about the rate of renewal of the intestinal nucleic acid and is thus quite pronounced for both types of nucleic acid in contradistinction to the rate of renewal found in the liver, which is very low in the case of desoxyribo nucleic acid and appreciably higher in the case of ribose nucleic acid. DISCUSSION In view of the high desoxyribo nucleic acid content of the lympho- cytes and because they are partly produced in the spleen, the compa- ratively high rate of turnover of desoxyribo nucleic acid in the spleen is in agreement with our expectance. As to the high figures found for the rate of renewal of desoxyribo nucleic acid in the intestinal mucosa, those are presumably due to the rapid new-formation of cells mechanically destroyed in the course of the digesture success. If we accept the view put forward by Casperssox, the high rate of renewal of ribo nucleic acid is in no way surprising. The high figures found for the rate of renewal of ribo nucleic acid in the intestine, the .spleen and the liver is just what we would expect in view of the impor- 678 ADVENTURES IN RADIOISOTOPE RESEARCH tance of these organs in protein metabolism. The incorporation of labehed sulfur into protein sulfur is found to be higher in the intestine than in any other organ (Tarver and Schmidt, 1942) and the i^N content of the proteins isolated from the intestinal wall of the rat after adminis- tration of isotopic 1( — )-leucine is larger than the corresponding value for any other organ investigated. Somewhat smaller values for the i^N content of the proteins isolated from the spleen were found, and still smaller values for the ^^N of the proteins isolated from the Hver (Schoen- HEiMER, Ratner and Rittenberg, 1939). The rate of formation of ribose nucleic acid in these three organs diminishes in the same sequence. If we want to state, not as above the percentage, but the amount of nucleic acid formed during the experiment, we must know the nucleic acid content of the organs of the rat and of the total rat. Some preliminary figures for the total nucleotide P of the liver, spleen, intestine and total rat and also some preliminary figures for the share of polydesose and polyribo nucleotides in the total nucleotides is seen in Table 3. The method applied in obtaining these figures and more accurate data will be shortly published by one of the authors. Table 3. — Polydesose Nucleotide Phosphorus and Polyeibo Nucleotide Phosphorus Content of Some Organs and of the Total Rat Approximate share of poly- desose nucleotides in the total nucleotides % gm nucleotide P per 100 gm dry weight Total rat Liver . . , Spleen . . Intestine 45—50 35 75 57 gm polydesose nucleotide P per 100 gm dry weight 0.232 0.350 0.643 0.669 0.11 0.12 0.48 0.38 gm polyribo- nucleotide P per 100 gm dry weight 0.12 0.23 0.16 0.29 Assuming the percentage formation of the polydesose nucleic acid of the total rat in the course of 2 hours to be 4 (cf. p. 676) and the fresh weight of the rat to amount to five times its dry weight, in a 200 gm rat in the course of 2 hours about 2 mgm polydesose nucletoide P will be incorporated. The corresponding figure for the polyribo nucletoide P works out to be 3. In the total rat the rate of formation of the 2 types of polynucleotides does thus not differ very appreciably. A very different result is obtained when comparing the amount of polydesose and polyribo nucleotide phosphorus incorporates in the liver. The figures work out, assuming the liver to weigh 6 gm,to be 0.0017 mgm and 0.094 mgm respectively. Fifty-five times more polydesose nucleotide than ribonucleotide is thus formed in the liver during the same time. RENEWAL OF DESOXYRIBO NUCLEIC ACIDS 679 Assuming the spleen to weigh 0.8 gm, both the amount of polydesose nucleotide P and that of polydesose nucleotide P formed and still present in the spleen works out to be about 20 microgram. Sumiuary Labelled sodium phosphate is administered to rats and after the lapse of 2 hours the specific activity of the ribo-nucleic acid phosphorus and that of the desoxyribo-nucleic acid phosphoinis determined. In the hver the specific activity of the ribo-nucleic acid P is found to be 33 times larger than the specific activity of the desoxyribo-nucleic acid P. In the course of 2 hours about 0.1 and 3.3 per cent respectively of these compounds were formed. In the intestine and in the spleen in which the specific activity of the desoxy- ribo-nucleic acid is found to be about 20 times larger than the corresponding value in the liver, the specific activity of the ribo-nucleic acid phosphorus is only 2 to 3 times larger than the corresponding value of the desoxyribo-nucleic acid phos- phorus. The ribo- and the desoxyribo-nucleic acid phosphorus extracted from the total rat have a very similar specific activity to the corresponding phosphorus extracted from the intestine. In the total rat the difference in the rate of formation of the two types of nucleic acid is not very pronounced. In a rat weighing 200 gm approxima- tely about 2 mgm desoxyribo-phosphorus and 3 mgm ribonucleic acid phosphorus are incorporated in the couree of 2 hours. References L. Ahlstrom, H. v. Euler and G. Hevesy (1944). Sv. Vet. Akad. Ark. Kemi 19, No. 9. E. Andreasen and J. Ottesen (1944) Acta Path. Microbiol. Scand. Suppl. 54. E. Andreasen and J. Ottesen (1945) Acta Physiol. Scand. 10, 258. A. M. Brtjes, M. Tracy and W. E. Cohn (1944) J. hiol. Chem. 155, 619. T. Caspersson (1940) Chromosoma 1, 562. I. L. Chaikoff (1942) Physiol. Rev. 22, 291. G. Hevesy and J. Ottesen (1943) Acta Physiol. Scand. 5, 237. R. Schoenheimer, S. Ratner and D. Rittenberg (1939) J. biol. Chem. 130, 703. H. Tarver and C. L. A. Schmidt (1942) Ihid. 146, 69. Originally published in ArMv for Kemi 26 A, 4 (1948). 69. TURNOVER OF RIBOSENUCLEIC ACID IN THE JENSEN-SARCOMA OF THE RAT H. EuLER, G. Hevesy and W. Solodkowska From the Institute for Research in Organic Chemistry, Stockholm In previous papers the rate of formation of labelled desoxyribosenucleic acid in the Jensen-sarcoma in rats upon the administration of labelled phosphate was investigated. In the present paper the results of investigations into the rate of renewal of the ribosenucleic acid in the Jensen-sarcoma of the rat will be reported. EXPERIMENTAL A few microcuries of labelled phosphate of negligible weight dissolved in 0.1 ml physiological sodium chloride solution were subcutaneously injected into rats (weighing 125— 165 gm) inoculated with Jensen-sarcoma. Two hours after the injection the rats were sacrificed. The blood plasma and the sarcoma were secured and the inorganic phosphate was extrac- ted with a 7 percent CCI3COOH solution from both the plasma and an aliquot of the sarcoma tissue. The specific activity of the nucleic acid was determined on the greatest part of the sarcoma tissue, a minor part being applied in the determination of the specific activity of the in- organic P. The first mentioned determinations were made by the method described by Schmidt and Thanhauser^i). This method is based on the assumption that after thorough extraction with trichloracetic acid and ether, alcohol, chloroform and methanol the nucleic acids are the only phosphorus compounds present in the tissue. If the tissue purified in this way is dissolved in IN NaOH solution at 37°, the desoxyribose- nucleic acid remains unchanged, while the ribosenucleic acid is split into mononucleotides<2). From the alcaline solution, CCI3OOH precipitates desoxyribosenucleic acid and the ribosenucleotides are present in the filtrate. Then the desoxyribose and the ribose fractions were ashed and ^i^G. Schmidt and S. J. Thanhauser, J. Biol. Chem. 161, 83 (1945). ^2^ G. Schmidt, R. Arbiles, B. H. Swartz and S. J. Thanhauser, J. Biol. Chem. 170, 760 (1947). RIBOSEXUCLEIC ACID IN THE JENSEN SARCOMA OF THE KAT 681 an aliquot of the solutions obtained was used for the colorimetric deter- minations and another aliquot for the determinations of the radio- activity. 40 mgm Na2HP04 were added to the aliquot, which was used for the determinations of the radioacti\'ity, and then the phosphorus was precipitated as magnesium-ammonium phosphate. As already observed by Schmidt and Thanhauser and as shown below, the ribonucleic fractions always contain some inorganic P which possibly originates from some protein phosphorus present in the tissue and decomposed in the alcaline solution. The presence of inorganic P might also be explained by assuming that despite thorough extraction either some inorganic P or organic P compound was left in the tissue, which are identified as inorganic P in the alcaline solution of the tissue. It is also possible that a minute amount of nucleic acid P is split off in the alcaline solution. The inorganic P present in an aliquot of the solution of ribosenucleotides was determined by the method of Delory^^). Owing to the fact that large amounts of proteins are present in the solution containing the ribosenucle- otids it was not possible to use the conventional methods for determining P. When calculating the specific activity of the ribosenucleic acid P from the inorganic P content and the activity of the ashed aliquot of the final solution, it must be taken into account that the whole amount of in- organic P present does not originate through ashing of the ribosenucleic acid P, because part of it is a remnant of the inorganic P, which has been present before ashing (see above). We have to deduct the values corre- sponding to the contaminating P both from the activity measured and from the inorganic P colorimetrically determined in the ashed ribose- nucleic P fraction. Since the total activity of the last mentioned component is mostly much lower than the activity of the- ribose P fraction, the corrected values do not differ much from the non-corrected ones. The following example illustrates the method used in determining the specific activity of nucleic acid P. Three sarcomata of an aggregate weight of 17.6 g weie pooled. The minced tissue was extracted with ice-cooled 7 per cent trichloracetic acid. The solution was filtered off through a Biichner fuel covered by a thick layer of hyflo. The tissue was repeatedly washed with 1 per cent CCI3COOH. The last wash water did not show any colouring of amidol. The total value of 1 per cent CCI3COOH added amounted to 800 ml. Trichloracetic acid present in Ihe tissue was quantitatively removed by washing with water. The tissue was then refluxed with 40 volumes of absolute alcohol for 14 hours. The ether — drycd tissue was again extracted with 20 volumes of 7 per cent CCI3COOH and the pro- cedure described above repeated. After having been dried in vacuum, the tissue was refluxed with 3 : 1 alcohol-ether mixtuie for 11 hours. After removing the alcohol by washing with ether and drying in vacuum, the tissue was a third time (i^Delgry, Biochem. J. 32, llGl (1938). 682 ADVENTURES IIST RADIOISOTOPE RESEARCH extracted with CCI3COOH. The washed and dried tissue was then refluxed with a chloroform methanol mixture (3 : 1) for 12 hours. After another extraction with CCI3COOH, the extract had a negative reaction, which indicated the absence of inorganic P. After one more extraction with CCI3COOH and chloroform-ether the tissue was dissolved in 180 ml. KOH by keeping it at 37° for one night. Only the hyflofilter was not dissolved. It was removed by centrifugation and the centri- fugate was brought up to 200 ml. In order to be able to study the effect of repeated precipitation of desoxyribosenucleic acid two halves of this volume were treated separately. 5 per cent CCI3COOH and 20 ml 0.2 N HCl were added to each solution. From the 2 desoxyribosenucleic acid precipitates obtamed, one (A) was dis- solved in 1 N KOH solution and repricipitated and ashed, while the other (B) was ashed after the first precipitations. The filtrate containmg the ribosenucleoti- des, obtained after precipitation of the desoxyribosenucleic acid, was neutrahzed and brought to 300 ml. After neutraUzation a minor precipitate was thrown down. This precipitate was found to be only shghtly radioactive. The total P of the neutrahzed solution was ashed and its P content (total P) determined. A larger aliquote, i.e., 125 ml, was used to determine the inorganic P content of the solu- tion. The method of Delory was used and 2 ml cone. NH3 -f 10 ml 2.5 per cent CaClg + 10 ml 0.5 per cent MgC03 were added to the solution. The precipitate obtained was centrifuged, washed with 2 per cent NH3 and dissolved in a 10 per cent CCI3COOH solution. An aUquote was used for the colorimetric determination and from another ahquote, to which 80 mgm inactive Na^HPO^ had been added. P was precipitated as magnesiumammonium phosphate. RESULTS Table 1. — Values obtained for the Phosphorus Content of Fractions Sample Plasma (3 ml) Sarcoma tissue (0.593 gm) , A. (reprecipitated) Desoxyribosenucleic acid Total P in filtrate obtained after removal of desoxyribose- nucleic acid Inorganic P in filtrate Ribosenucleic acid Etheric phase B. (not reprecipitated) Desoxyribosenucleic acid Total P in filtrate, obtained after removal of desoxj^ribose- nucleic acid Inorganic P in filtrate Ribosenucleic acid P Etheric phase P content in mgm 0.165 (inorganic) 0.265 4.425 0.3087 7.1163 0.0026 5.375 7.80 0.327 7.473 0.0071 P content per gm tissue in mgm 0.055 (inorganic P per ml plasma) 0.447 50.2 80.6 61.1 84.7 Ribosenucleic acid P = total P in filtrate — inorganic P in filtrate. RIBOSENUCLEIC ACID IN THE JENSEN-SARCOMA OF THE RAT 683 To ascertain if the filtrates containing the ribosenucleotides are free of phosphatides, we were shaking the two filtrates (A and B) with 2 volumes of ether and 0.2 volume of N 0.1 HCl to extract phosphatides present. As seen above, the etheric phases contained a negligible amount of P only. Table 2. — Results of Activity Measurements (32P Content of Fractions) Sample Counts/niin. of aliquote precipitated Counts/min. of total volume Oounts/min. per mgm P Plasma inorg. P Sarcoma inorg. P A. Desoxyribosenucleic acid P ] Total P in filtrate after removal of desoxy j ribosenucleio acid I Inorganic P in filtrate Ribosenucleic acid P Residual fraction B. Desoxyribosenucleic acid P Total P in filtrate obtained after removal of desoxyribosenucleic acid Inorganic P in filtrate Ribosenucleic acid P Residual fraction 461.5 597.5 260.5 96.3 126.9 11.7 290.8 121.2 101.5 57.1 1792.5 651.3 2890 846 204.4 14.6 727 3030 951 2079 71.4 8758 6766 147 389 2745 288 135 398 2910 279 Specific Activity Ratios ("Percentage New-formation Figures") Activity of 1 mgm desoxyribonucleic acid in percentage of activity of 1 mg : A. Sarcoma inorganic P 2.18 B. Sarcoma inorganic P 2.0 A. Plasma inorganic P 1.68 B. Plasma inorganic P 1.55 Activity of 1 mgm ribosenucleic acid in percentage of activity of 1 mgm : A. Sarcoma inorganic P 4.27 B. Sarcoma inorganic P 4.13 A. Plasma inorganic P 3.29 B. Plasma inorganic P 3.19 684 ADVENTURES IN RADIOISOTOPE RESEARCH Ratio of the specific activities of ribosenucleic acid P desoxyribosenucleic acid P A B 1.96 2.06 Table 3. — "Pekcentage Formation" of Nucleic Acids in the Jensen-Sabcoma OF THE Rat(1) (Time = 2 hours) Activity of 1 mgm nucleic acid P in Activity of 1 mgm nucleic acid P in No. of Experiment per cent of the activity of 1 mgm sarcoma inorganic P per cent of the activity of 1 mgm plasma inorganic P Ratio of the specific activities of ribosa P and desoxyribose P ribose desoxyribose ribose desoxyribose 232 5.38 1.79 4.23 1.33 3.0 233 A 4.27 2.18 3.29 1.68 2.0 233 B 4.13 2.0 3.19 1.54 2.1 234 5.62 1.53 5.78 1.57 3.7 235 3.88 1.90 4.04 1.98 2.0 236(2) 3.72 1.74 3.24 1.51 2.1 (fasting 237 3.55 1.60 3.75 1.09 2.3 rats) 238 2.74 1.76 5.0 1.77 1.6 239 7.71 2.73 5.0 3.2 2.8 Average value 4.6 1.9 4.2 1.7 2.4 Ratio of the "P content of ribosenucleic acid and desoxyribosenucleic acid in the Jensen-sarcoma = 2.4 O We came to tlie "Percentage Renewal" figures, contained in the above Table by comparing the end values of the specific activities of the nucleic acid P and inorganic, P. Correctly, we have to consider the mean value of the specific activity of the inorganic P during the experiment. The correction to be applied to arrive at correct figures is discussed on p. 685. <') In this experiment the specific activity of the pyrophosphate P was determined as well. Tlie ratios of the specific activities of pyrophosphate P : plasma inorganic P : sarcoma inorganic P was 1 : 1.10 : 0.97. Similar values for the rate of formation of ribosenucleic acid and of desoxyribosenucleic acid were obtained in a number of other experiments, the result of which are summarized in Table 3. The average value, obtained for the percentage new -formation of desoxyribosenucleic acid, 1.9, compares fairly well with the average value, obtained in our previous investigations (2.05, 2.17), which involved a very large number of determinations. In all our previous work^^) the desoxyribosenucleic acid P was purified from contaminating phosphorus compounds by repeated alternative boiling with cone. NaOH and reprecipitation of the dissolved desoxy- ribosenucleic acid P with methanol, containing HCl. This method has the disadvantage that a poor yield is obtained. The method of Schmidt ^i^ H. EuLEB and G. Hevesy, Kgl. Danske 17, No. 8 (1942). Videnskab. Selskab. Biol. Medd. RIBOSENUCLEIC ACID IN THE JENSEN-SARCOiL\ OF THE RAT 685 and Thanhauser has the advantage that it permits a quantitative or almost quantitative isolation of desoxyribosenucleic acid and can thus be carried out even if only small tissue samples are available. The Average Value of the Specific Activity of the Inorganic P in the Sarcoma during the Experiment The specific activity of the cellular inorganic P of the sarcoma de- pends on the rate (a) at which the labelled phosphate reaches the surface of the cells, (b) at which it penetrates into the cells of the tissue, (c) at which it is incorporated into organic P compounds in the cells (the incorporation of the inorganic ^'^P into organic compounds goes hand in hand w ith the corresponding formation of inactive phosphate by the degradation of the inactive organic compounds present in the cells of the tissue). The magnitudes of these rates varies between the sarcomata. When comparing the specific activities of the inorganic P fractions extracted from fresh and necrotic sarcoma 2 hours after the adminis- tration of ^^P tissue, we found previously^^) that the values determined in the fraction of necrotic sarcoma were roughly only half the magnitude of those computed in the fraction extracted from fresh sarcoma. Also, the values obtained for the specific activity of inorganic P extracted from different parts of the fresh sarcoma tissue varied widely^^\ In the experiments here reported, each rat was first inoculated with Jensen- sarcoma and 15 days later 0.1 ml physiological sodium chloride solution, containing a negligible amount of phosphorus of about ^4 microcurie activity was administered to each rat. The 12 rats were sacrifced Yo, ^, iVo and 2 hours respectively after the administration of ^'^P. The ratio between the end values and the average value of the specific activities of the tissue inorganic P was found to vary between 1.17 and 1.57. In view of these variations it would be advisable to determine in each case the average specific activity of the sarcoma inorganic P. This presents great difficulty as such determinations would necessitate to secure samples from the same sarcoma at different dates. We determine therefore usually only the end value of the specific activity of the sar- coma inorganic P and assume this to be 1.3 times the average value. We arrive at the last mentioned figure by taking the average of the results obtained in experiments as described above. Correspondingly, we have to multiply the values of the "percentage new-formation", obtained l)y comparing the end values of the specific activity of the nucleic acid P with the specific activity of the end- value of the inorganic P, by 1.3 to arrive at the correct value of the percentage renewal of the sarcoma nucleic acids in experiments lasting 2 hours (cf. Table 3 in which the ^2>n. EuLER and and G. Hevesy, Ark. Kemi A 17, No. 30 (1944). 686 ADVENTURES IN RADIOISOTOPE RESEARCH uncorrected values of the "percentage renewal" are set out). The ratio between the average and end- value of the specific activity of the sar- coma inorganic P can vary appreciably from sarcoma to sarcoma. These variations influence unfavourably the accuracy of the determination of the percentage new-formation of the nucleic acids in the sarcoma. It may be assumed that the increase in the ribosenucleic acid content of the growing sarcoma during the course of the experiment (2 hours) amounts to about the same value as the increase of the desoxyribose- nucleic acid, i.e., 0.9 per cent of the amount present. Out of the 6 per cent newly formed ribosenucleic acid during the experiment, i/ is thus due to additional formation of ribosenucleic acid, while ^/g are due to renewal of old molecules. The value of the rate of renewal of ribosenucleic acid in the Jensen- sarcoma is about twice of the corresponding value (3.5) determined in the liver. The Nucleic Acid P Content of the Jensen- Sarcoma From the colorimetric determination of the P of the desoxyribose- nucleic acid and the ribosenucleotides we computed the values set out in Table 4. The ratio between the desoxyribose and ribosenucleic acid of the Jensen-sarcoma is appreciably smaller than that between those in liver and most other organs. This is mainly due to the large desoxyribose- nucleic acid content of the sarcoma tissue. Table 4. — Nucleic Acid P Content of Jensen-Sarcoma mgm% in fresh tissue Desoxyribose P 42 54 Ratio Ribose P Ribose P 64 87 65 91 46 77 36 52 57 63 50 68 52 78 41 58 52 67 57 67 Mean value 50 86 Desoxyribose P = 1.7 RIBOSENUCLEIC ACID liST THE JENSEN-SARCOMA OF THE RAT 687 Summary Radiopbosphorus is injected into full-grown rats inoculated with Jensen-sarcoma. The specific activity of 1 mgm ribosenucleic acid extracted from the Jensen-sarcoma at the end of 2 hours from the begimiuig of the experiment was found to be 6 per cent of the average specific activity of the inorganic P of the sarcoma through- out the experiment. On the basis of the assumption that the precursor of the ribosenucleic acid P is either the cellular inorganic P of the sarcoma or organic P which is rapidly brought in exchange equilibrium with the ceUular inorganic P, the above figure represents the percentage of the new-formation of the ribose- nucleic acid present in the Jensen-sarcoma in the course of 2 hours. Out of 6 labelled ribosenucleic acid molecules 1 represents additional formation due to growth of the sarcoma during the experiment, while the formation of roughly 5 new molecules is compensated by the simultaneous disappearance of 5 "old" ribosenucleic acid molecules. The ratio between the specific activity of the ribosenucleic acid P and the specific activity of the desoxyribosenucleic acid P of the Jensen-sarcoma is 2.4. Originally published in Nature 156, 534 (1945). 70. LIFE-CYCLE OF THE RED CORPUSCLES OF THE HEN G. Hevesy and J. Ottesen From the Institute for Theoretical Physics, Univeisity of Copenhagen The life-cycle of the mammalian red corpuscles is not known with cer- tainty. Values varying between 30 and 200 days are recorded. One would expect the problem to be easily solved by making use of an isotopic indicator, that is, by labelling the corpuscles. In trying to find a suitable indicator, great difficulties are encountered due to the fact that almost every compound present in the corpuscles is renewed at a comparatively rapid rate. Only such labelled molecules which have a longer life-time than the red corpuscles in which they are located can be used as indi- cators. Iron atoms incorporated with haemoglobin molecules remain unchanged duriug the life- time of the red corpuscles^^\ Hahn and his colleagues^^\ however, found that the iron atoms contained in the debris of the haemoglobin of decayed corpuscles are preferentially used in the formation of new corpuscles. This fact makes radioactive iron unsuitable for the determination of the life-cycle of the red corpuscles. We found desoxyribose nucleic acid phosphorus to be a suitable indi- cator for the determination of the life-cycle of nucleated corpuscles. In contradistinction to desoxyribose nucleic acid molecules present in some organs, those found in the red corpuscles of the hen are not renewed at an appreciable rate. In experiments in vitro, in which hen blood was shaken in an oxygen atmosphere in the presence of labelled sodium phosphate, no active desoxyribose nucleic acid was found to be formed, in contradistinction to other active phosphorus compounds. Furthermore, activity was absent in the desoxyribose nucleic acid present in the circulating red corpuscles of the hen up to four days after administration of radioactive phosphate. Hen corpuscles, labelled by their active desoxyribose nucleic acid content, can be used in two different ways. We can administer, for (i^Hahn, p. F., Bale, W, F., Ross, J. F., Hettig, R. A., and Whipple, G. H., Science, 92, 131 (1940). ^2) Hahn, P. F., Bale W., F., and Balfour, W. M., Amer. J. Physiol., 135, 800 (1941- -42). RED CORPUSCLES OF THE HEN" 689 example, labelled phosphate to the hen, and after the lapse of a week replace part of the corpuscles of a second hen by labelled corpuscles of the first one. When taking blood samples at intervals, we can deter- mine what percentage of the transfused corpuscles is still present in the circulation of the hen. In a note to be published later, we shall commu- nicate the results obtained in such experiments. In this note we shall describe another method in which, by avoidance of blood transfusion, the uncertainty about the equality of the life-time of the transfused corpuscles and the endogenous corpuscles can be eliminated. 100 80 60 40 20 / 9 / 10 20 30 40 Life-cycle of the red coipuscles of two hens. Abscissae : days after start of experiment ; ordinates : specific activity of desoxyribose- nucleic acid phosphorus extracted from the corpuscles secured at different dates. In the latter method, labelled phosphate is administered twice a day to the hen in such quantities that the plasma phosphate is kept at a constant or almost constant level of activity. The active phosphate penetrates into the marrow and participates in the formation of the nucleic acid of the corpuscles, which thus become labelled. The percentage of labelled corpuscles will increase with time, and finally the circulation will contain labelled corpuscles only; thus the activity of 1 mgm cor- puscle desoxyribose nucleic acid phosphorus will be equal to the activity of 1 mgm marrow phosphorus and I mgm plasma phosphorus re- spectively. The results of such experiments are shown in the accompanying graph, which makes it clear that in the first four days the nucleic acid present in the corpuscles is inactive. This may be interpreted by assuming that. 44 Hevesy 690 ADVENTURES IN RADIOISOTOPE RESEARCH in the first phase of the experiment, corpuscles containing inactive nucleic acid reach the circulation, and that it is about four days before corpuscles containing labelled nucleic acid are given off by the sinusoids to the circulation. The maturing of the corpuscles in the marrow thus takes about four days. The graph also shows that, after the lapse of about thirty-three days, the maximum value of the activity of the desoxyribose nucleic acid is reached. Taking into account that in the first four days no labelled corpuscles intrude into the circulation, the life-time of the red corpuscles will be 29 days. It is of interest finally to note that the results obtained indicate that all or almost all corpuscles present in the circulation have a similar life-time. 691 COMMKNT ON PAPERS 67 — 70. We started our investigations on the application of radio-phosphorus as a tracer with the study of the incorporation of ^ap into the minoial constituents of the skeleton, then took up the study of the formation of labelled phosphatides and acid-soluble compounds. In 1939 an investigation of incorporation of ^^p into desoxyribonucleic acid was started. A note on the results of these studies was published by Hahn and Hevesy (1940) and a more detailed account is given in paper 67. Outgrown organs which do not secrete DNA containing leucocytes as the liver and kidney were found to incorporate minimal amounts of ^^P into DNA only. The spleen which secretes leucocytes incorporates appreciable amounts of ^^P into its DNA since the secreted molecules have to be replaced and the new formation of DNA molecules takes place under the incorporation of ^^p. The production of labelled DNA molecules is very pronounced in the intestinal mucosa in which the cells destroyed in the course of the digestive processes have to be replaced. In paper 67 it is stated that " The rate of renewal of the nucleic acid in the liver may be identical with the rate of formation of liver cells". A statement, supported by the results of recent investigations. In later studies (paper 68) the rate of formation of both labelled DNA and ENA was investigated. In the meantime Brues et al. (1944) found, in experiments tak- ing over a week, the latter to be larger than the former. The very great difference between the incorporation of ^ap into DNA and the UNA of the rat liver was brought out in the investigation described in paper 68 in which incorporation was determined after 2 hr only. The specific activity of RNA phosphorus was found to be thirty-three times as large as that of the DNA phosphorus. In the spleen and intestine in which DNA is built up at a rapid rate the corresponding ratio makes out 3 and 2 only. In the rapidly growing Jensen-sarcoma of the rat in which formation of DNA takes place at a very appreciable rate as it does in the spleen and in the intestinal mucosa, the corresponding ratio was found to be 2.4 (pa- per 69). The great stabihty of the DNA present in nucleated erythrocytes mad. it possible to determine the previously unknown Ufe cycle of avian red corpuscles. Doubts were expressed on the correctness of the results obtained when these were communicated at the Solvay Congress in Brussels.Their correctness was, however, brought out by later investigations (ShemIn, 1948; Ottesen, 1955). Since those early days when ^ap was first incorporated into DNA, labelled desoxyribonucleic acid found a very extended apphcation in numerous studies. References A. M. Brijes, M. M. Tracy and W. E. Cohn (1944) J. Biol. Chem. 155, 619. L. Hahn and G. Hevesy (1940) Nature 145, 549. J. Ottesen (1955) The Life-Cycle Of Hen Erythrocytes. Ejnar Munksgaard, Copen- hagen. D. Shemin (1948) Cold Spring Harbour Symphosia On Quantitative Biology 13, 185. 44* Originally published in Kgl. Danske Vidensk. Selskab Biol. Medd. 17, No. 8 (1942). 71. THE EFFECT OF X-RAYS ON THE RATE OF NUCLEIC ACID FORMATION IN JENSEN- SARCOMA H. EuLER and G. Hevesy The Vitamin Institute of the University of Stockholm and the University of Theoretical Physics, University of Copenhagen The detection of the effect of X-rays on sarcomas is usually studied in the following way. Fragments of the irradiated sarcoma are transferred by inoculating normal animals and tests are made to find out whether or not the sarcoma has grown after a few days. We have tried to replace this procedure by a chemical test. There are numerous experiences which support the statement that doses which are effective against sarcoma do not substantially affect the metabolic processes taking place in the tissue cells, and that are the processes taking place in the cell nucleus which primarily succumb to the action of the radiation. In the search for a chemical test for the effect of X-rays on the sarcoma it was there- fore evident that the processes occurring in the cell nucleus should be studied in more detail. The nucleic acids are among the most important constituents of the cell nucleus. Nucleic acid plays an essential part in cell division. Kossel, for example, has shown that the changes occurring in the course of sperma- togenesis consist in degradation and synthesis of proteins and in the synthesis of the histonamine or protamine nucleate, which is compara- tively poor in protein and Caspersson's^^^ studies of the absorption of ultra-violet radiation by dormant and dividing cell nuclei have made it apparent that Kossel's scheme of protein synthesis is valid also for the ordinary mitotic cell, division. The mitotic division of cells is retarded by comparatively small doses of X-rays and, on the basis of the above discussion, it might be expected that the irradiation of tissue with X-rays would cause a diminution in the of rate of formation of desoxyribo nucleic acid in the cell nucleus. This view has induced us to study the formation of desoxyribo nucleic acid in the Jensen-sarcoma of the rat, before and after irradiation with X-rays. ^T. Caspersson, Chromosoma 1, 562 (1940). EFFECT OF X-KAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 693 The investigation was performed by using the radioactive indicator method. This method makes it possible to distinguish between tlie mole- cules of nucleic acid which have been formed before and after the beginn- ing of the experiment. The latter, which have been formed in an active medium, viz. in cells containing active phosphate, will be active (i. e. will contain radioactive phosphorus) as opposed to the inactive mole- cules already present before commencement of the experiment. DESCRIPTION OF THE METHOD When a very small amount of sodium phosphate, labelled with an admixture of radioactive phosphorus (i?P), is introduced by, for example, injection into the experimental animal, the labelled phosphate ions soon enter the sarcoma cells and take part in the synthetic processes occurring in these cells with the same probability as the other phosphate ions already present in the sarcoma cells. If molecules of nucleic acid are synthesized in the sarcoma cells they will be radioactively labelled. If all the nucleic acid molecules present in the sarcoma at the end of the experiment have been formed in the course of the experiment, then 1 mgm of nucleic acid phosphorus will show the same content of ^^p^ i. e. the same radioactivity, as 1 mgm of phosphate phosphorus. If, on the other hand, at the conclusion of the experiment 1 mgm of nucleic acid phosphorus shows an activity amounting to, say, only 1 per cent of the activity of 1 mg of phosphate phosphorus, then the amount of nucleic acid formed during the experiment will be 1 per cent of the total amount of nucleic acid present in the sarcoma. By making this state- ment we assume that the measured activity of 1 mgm of phosphate after completion of the experiment is equal to the activity present at any other point in time during the course of the experiment (cf. the discus- sions on p. 705). Preparation and Measurement of the Activity of Radioactive Phosphorus The radioactive phosphorus used in the experiments which will be described below has usually been obtained by the action of neutrons on carbon disulphide^-^^ A mixture of radium-beryllium containing 600 mgm of elementary radium served as the source of neutrons. We are very greatly indebted to Professor Niels Bohr for the loan of this source and also for many other pieces of equipment. In addition to this, radio- active phosphorus was available which had also been produced by the 1 O. Chievitz and G. Hevesy, Kgl. Danske Vidensk. Selskab. Biol. Medd. 13, 9 (1937). 694 ADYEA^TURES IX RADIOISOTOPE RESEARCH t action of fast neutrons on carbon disulphide but in this case the neutrons had been obtained by means of a high voltage apparatus, usuahy at the Institute of Theoretical Physics of the University of Copenhagen but occasionally at the Research Laboratory of Philips Gloeilampfen- fabrik in Eindhoven. The samples produced with the help of the radium sources tiid not contain any chemically detectable amount of phosphorus. They were obtained by shaking the 10 1. of carbon disulphide used for irradiation with dilute nitric acid. The active phosphate remained in the residue after evaporating the nitric acid solution; it was taken up in water and the solution was filtered through a glass filter. This process was repeated a few times. The activity was finally dissolved in physiological saline solution and then injected into the experimental animals; 0.1 — 1.0 cm^ was injected into each rat. The activity of the injected solution amounted to about 0.1 ^c. Measurement of the Radioactivity of the Nucleic Acid Phosphorus The rat was killed 2 hr after injection and the nucleic acid of the sarcoma was isolated. The Klein and Beck^ method of isolation was used for this purpose. Besides the weakly active nucleic acid, very strongly active acid-soluble compounds and strongly active phosphatides are also formed in the sarcoma in the course of the experiment; contamina- tion of the nucleic acid with the slightest amount of acid-soluble phos- phorus or with phosphatide phosphorus can therefore interfere with the results of measurement of the nucleic acid activity. For this reason, the samples of nucleic acid were purified still more thoroughly than in the above-mentioned experiments of Klein and Beck. The purified nucleic acid was wet-ashed with sulphuric acid and 30% HgOa. One-fifth of the solution obtained was used for the colorimetric deter- mination of phosphorus; four-fifths of the solution were treated with 80 mgm of sodium phosphate and the phosphorus content was isolated as magnesium ammonium phosphate. By this means the active phos- phorus content of the sample was present in a mixture with about 70 mgm of inactive magnesium ammonium phosphate. All our preparations were treated in this way and we were thus able, in determining the activity, always to compare samples of nearly the same weights and volumes. Any contingent correction for different absorption of ^^-radiation in the samples being compared, was thus avoided. The activity of the magnesium ammonium phosphate or of a known fraction of the sample was measured with a Geiger— Miiller counter. The activity of 1 mgm of phosphorus was calculated from the measured 2 O. Klein and Beck, Z. Krebsforsch. 42, 172 (1935). EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 695 activity, the weight of the magnesium ammonium phosphate sample and the chemically determined phosphorus content of the nucleic acid as shown in the following example: The activity of 1 mgm of nucleic acid phosphorus is equal to 8.60 X X80x5/2.44x71x4 = 4.98 counting-tube pulses/min. The figure 8.60 denotes the activity of the magnesium ammonium phosphate containing the nucleic acid phosphorus, 2.44 is the phosphorus content of the total nucleic acid in the sarcoma (mgm); 80 is the weight of the whole of the precipitated magnesium ammonium phosphate sample (mgm), and 71 is the weight of sample placed under the counting tube; 5/4 is the correction to be applied because only four-fifths of the solution obtained by ashing the nucleic acid was used for the radioactive investigation. After this it is necessary to compare the radioactivity of 1 mgm of nucleic acid phosphorus with the radioactivity of 1 mgm of free-phosphate phosphorus in the sarcoma. The postulate mentioned on p. 690, that the phosphate phosphorus isolated from the sarcoma has the same activity which would have been found at any time during the experiment, does not hold good. The absorption of the injected phosphate requires several minutes and although the membrane of sarcoma cells is, as discussed elsewhere^^\ highly permeable to the penetration of phosphate, the infiltration of phosphate from the plasma (or from the extracellular fluid) into the sarcoma cells likewise requires time. Furthermore, the activity of the plasma phosphate changes during the experiment. At first it increases and then decreases, as a result of the inflow of labelled phosphate into the cells of the organ and vice versa. Strictly speaking, therefore, we should measure the activity of 1 mgm of sarcoma phosphate at different times and in this way calculate the mean value of the phos- phate activity during the experiment. This average activity of 1 mgm of phosphate should then be compared with the activity of 1 mgm of nucleic acid phosphorus determined at the conclusion of the experiment. We are, however, not so much interested in the accurate values of nucleic acid metabolism of this acid in the sarcoma as in the effect of X-rays on the nucleic acid metabolism. We have, therefore, replaced the above somewhat tedious procedure by the following one. The activity of 1 mgm of nucleic acid phosphorus was compared with the activity of 1 mgm of free plasma phosphorus determined at the end of the experiment. The activity of the plasma phosphorus at first increases during the experiment and later decreases; the activity value measured two hours after injection is not very different from the mean value in the course of the experiment. We have also compared the activity of 1 mgm of nucleic acid of the sarcoma with the activily of 1 mgm of free phophorus in the liver, and have thus attained a second scale for com- iQ. Hevesy and H. Euler, Ark. Kern ISA, 15 (1940). 693 ADVENTURES IN RADIOISOTOPE RESEARCH parison. In our later experiments we have also compared the activity of the nucleic acicl with that of the free phosphorus in the sarcoma. In the latter procedure a portion of the sarcoma must be sacrificed in order to isolate the free phosphate; in spite of this the procedure is definitely preferred to the one first described and at first applied in our experi- ments . Plasma and liver samples were treated with 10 and 25% trichloro- acetic acid, respectively; 80 mgm of sodium phosphate was added to four- fifths of the solution, the free phosphate in the solution was precipitated as magnesium ammonium phosphate and the radioactivity of the sample was measured, as described above, with the Geiger— Miiller counter. One-fifth of the solution w^as used for colorimetric determination of the free phosphorus. The magnesium ammonium phosphate samples were placed in aluminium trays, 1.2 cm in diameter and 2 mm deep, before the activity was measured; the trays were pushed beneath the window of the counter. Whereas the liver fractions measured yielded more than 1000 pulses/min in the counting tube, the activity clue to the nucleic acid of the sarcoma usually amounted to only a few pulses per minute; in isolated cases, indeed, it was necessary to measure activities amount- ing to only a few-tenths of a pulse per minute. Such measurements were performed by alternating measurements of the activity of the sample and of inactive magnesium ammonium phosphate for periods of 24 hr each. The background amounted to about 4 pulses/min and could be reproduced with an accuracy of 2 per cent. Temperature variations of the counting apparatus in excess of about 1° C must be avoided if such accuracy is to be attained. Determination of the Percentage of Injected ^^P Found in the Nucleic Acid of the Sarcoma In determining the percentage of the injected radioactively labelled phosphorus incorporated in the nucleic acid of the sarcoma, the procedure is as follows: 80 mgm of sodium phosphate are added to a known small fraction, e. g. one-threehundredth of the solution used for injection; the P content of the solution is then precipitated as magnesium ammo- nium phosphate. The activity of this sample is compared with the acti- vity of the nucleic acid phosphorus, isolated from the sarcoma, which is also in the form of magnesium ammonium phosphate and of the same weight as the above sample. If the activity of one-threehundredth of the injected solution is equal to 100 pulses/min, then 30,000 pulses were injected into the rat. If the activity of 1 mgm of nucleic acid phos- phorus is found to be 10, then 1 mgm of nucleic acicl phosphorus contains 0.033 per cent of the injected ^^p. The values recorded in Tables 1 to EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 697 6 are obtained by dividing by the colorimetrically determined weight of the phosphorus in the nucleic acid. The Sarcomas Used Jensen sarcomas of rats, which had been cultivated by transphmting a sarcoma obtained from Professor Domagk were used for all the experiments which we have described up to this point. The sarcomas were always transplanted subcutancously by installing a tissue section about 1 mm thick. The sarcomas developed in our strain of rats up to a size of about 20 gm in about 3 weeks. Rats with a sarcoma weight of about 20 — 30 gm were ordinarily used in the irradiation experi- ments. Isolation of the Nucleic Acid The sarcoma tissue was finely minced and worked up for desoxyribo nucleic acid by the method described by Klein and Beck^^\ The crude precipitate was dissolved in 1 N caustic soda, treated with about 80 mgm of disoclium hydrogen phosphate and precipitated with 5% ferric hydro- xide solution. This purification of the nucleic acid was repeated two more times to remove any radioactive phosphate. The product obtained in this way was then twice dissolved and reprecipitated, with methyl alcohol containing hydrochloric acid, in accordance with the method of Klein and Beck. The most careful purification of the nucleic acid from foreign phosphates is of the utmost importance because the acid- soluble phosphate is much more active than the phosphate from the nucleic acid. The phosphate in the finally purified precipitate was determined colorimetrically by the method of Fiske and Subbarow^^^ (Theorell's modification^^^) . Another portion was precipitated as magnesium ammo- nium phosphate which was then used for determining the activity. With regard to the quantities employed in this procedure, the reader is referred to the description supplied as an example (p. 692) and also to Tables 1 — 6 (pp. 699 — 702). The samples of blood and liver were worked up by the methods describ- ed below: The blood, which had been collected in a vessel containing a few milligrams of sodium citrate, was centrifuged; the plasma (usually 2—3 cm^) was treated with 3 cm-"^ of 10% trichloroacetic acid, the 10. Klein and Beck, Z. Krehsforsch. 42, 163 (1935). 2 Fiske and Subbabow, J. Biol. Chem. 66, 375 (1925). ^Theorell, Biochem. Z. 230, 1 (1931). 698 ADVENTURES I^^ RADIOISOTOPE RESEARCH solution centrifugecl and filtered, and the centrifuged residue was again extracted with 2 cm^ of trichloroacetic acid and filtered. The whole of the filtrate was diluted with water to 25 cm^; 2 cm^ of this was used for colorimetric determination of the phosphorus by the Fiske— Subbarow— Theorell method; 20 cm^ of the filtrate was treated with 80 mgm of disodium hydrogen phosphate, and the whole of the phosphate was precipitated as magnesium ammonium phosphate. After drying at 110° C this preci- pitate was used for determination of the activity. The liver (usually 5 to 8 gm) was first treated for 15 min with 20 cm^ of 25% trichloroacetic acid and for a second period of 10 min. The trichloroacetic acid extract was filtered and the filtrate diluted with water to 100 cm^. Phosphorus was determined colorimetrically in 1 cm^. After addition of 80 mgm of sodium phosphate, 80 cm^ of the solution was used for precipitation of the magnesium ammonium phosphate required for the determination of the radioactivity. Irradiation of the Sarcoma The sarcomas were irradiated for periods of from 26 — 67 min with X-rays emitted from a tube operated at 165 kV and 7 mA. A 0.5 mm copper foil and 1 mm aluminium foil were used as radiation filters. The irradiation was made at distances between 25 and 42 cm. Only the sarcoma was irradiated; the other parts of the body of the rat were protected against the action of the radiation by covering them with lead plates. The NaC'l solution containing the radioactively labelled sodium phosphate was injected 20 min after completion of the irradiation. The performance of experiments lasting for such a short time has the advantage, among others, that the effect of the X-rays can be studied soon after the ending of the irradiation. It is well known that in the course of time considerable changes take place in irradiated tissue and the effect of these changes on the nucleic acid metabolism can be studied by injecting the radioactive one or more days and not immediately after interruption of the irradiation. The results of such experiments w^ill be discussed below. EXPERIMENTAL RESULTS The results of the experimental work can be seen in Tables 1—6. Tables 1 — 3 contain data on the rate of formation of desoxyribo nucleic acid in unirradiated and in slightly and more strongly irradiated sarcomas. Tables 4—6 contain data on the fraction of injected ^^P which is to be found after the passing of 2 hr in 1 mgm of free plasma phosphate phos- phorus and in 1 mgm of free liver phosphate phosphorus. EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 699 Table 1. — Nttcleic Acid Formatiox ix Unirradiated Jensen-Sarcoma (Time of experiment, 2 hr.) Rat ao. Weight of iniri- fied sarconiu fem) Xucleic acid phosphorus con- tent in 1 gm sarcoma (mgm) '*P content of 1 iiil'iu rmriric acid phosphorus, as a piK mtiL/c of the '*P content of 1 mgm of inorg. P of the liver 1 mgm of inorg. P of the plasma I + II III + IV V + VI VII +vni IX + X XI + XII XIII XIV XV XVI Mean value . 36.2 34.5 19.3 31.9 23.2 2.9 16.7 22.5 17.9 17.7 22.3 3.8 12.3 7.5 6.6 8.3 0.9 1.3 6.3 2.4 5.7 5.5 2.60 2.23 3.10 1.76 1.30 1.35 2.33 1.14 1.13 1.25 1.82 2.98 2.15 1.76 1.06 1.21 1.52 1.78 Table 2. — Nucleic Acid Formation in Slightly Irradiated Jensen-Sarcoma (Dosage of 77 to 310 r [international Roentgen units]. Time of experiment, 2 hr.) Marking of rat Dosage (r) Weight of purified sarcoma (gm) Nucleic acid P content of sarcoma (gm) '^P content of 1 mgm nucleic acid P as a percentage of the '^P content of 1 mgm of inorg. P of the liver 1 mgm of inorg. P of the plasma A B C D E F G H J K L M N 77 77 77 155 155 155 310 92 92 92 186 186 186 6.2 11.2 5.7 13.0 9.4 15.0 13.8 12.3 10.0 3.1 6.3 6.8 3.8 10.1 16.0 11.0 6.4 13.7 35.0 1.3 2.3 1.0 2.5 5.0 1.2 0.47 1.74 0.26 1.33 1.39 1.80 1.92 1.20 0.95 1.04 1.65 2.14 1.93 0.54 1.53 0.22 1.74 1.03 3.41 2.54 1.47 0.80 1.53 1.51 3.32 3.71 0.80 Mean value — 9.0 8.1 1.38 1.82 700 ADVENTURES 11^ RADIOISOTOPE RESEARCH Table 3. — Nucleic Acid Formation in Irradiated Jensen-Sarcoma (Dosage of 460 to 7000 r [international Roentgen units]). Time of experiment 2 hr) Rat no. Dosage (r) Weight of purified sarcoma (gm) Xucleic acid P content of sarcoma (gm) ^=P content of 1 mgm nucleic acid P as a percentage of the '"P content of 1 mgm of inorg. P of the liver 1 mgm of inorg. P of the plasma 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Mean value 2080 2080 2080 2080 2080 2080 1000 1000 1000 2080 1240 620 1025 460 460 1025 1025 1025 1025 465 465 620 900 1180 1395 1025 1025 1025 1180 1400 1730 2550 7000 7000 11.1 18.2 18.5 5.3 5.5 6.0 14.9 13.0 6.8 18.0 13.4 17.0 13.9 12.0 8.6 5.9 2.2 11.8 3.4 9.7 10.0 6.3 7.7 4.8 4.6 7.3 17.5 6.7 12.3 6.0 9.3 8.2 9.1 14.2 9.9 1.33 2.33 8.30 1.36 2.30 1.86 4.05 2.05 1.70 0.36 1.33 2.11 6.20 5.30 5.31 21.0 6.71 52.0 6.70 16.2 27.2 8.80 25.0 2.31 2.00 7.12 10.9 11.0 4.72 3.70 2.27 1.80 0.28 0.65 7.5 0.20 0.41 0.37 0.17 0.20 0.21 0.22 0.35 0.39 0.10 0.25 0.13 0.36 0.02 0.56 0.28 1.04 0.31 0.51 0.84 2.12 1.31 0.78 0.60 0.42 0.80 0.076 0.65 0.34 0.47 1.40 0.75 0.40 0.11 0.50 0.32 0.27 0.44 0.19 0.48 0.24 0.21 0.01 0.29 0.35 1.50 0.54 0.66 1.04 1.29 3.41 0.99 0.82 0.56 0.91 0.18 0.70 0.34 0.64 1.51 0.95 0.50 0.080 0.65 EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SAKCOMA 701 Table 4. — '^P Content of the Free Phosphate in the Blood Plasma and Liver (Unirradiated) Rat no. Weight of liver (gm) P content of liver phosphate (mgm%) P content of plasma phosphate (mgm%) % of injected ^=P present in 1 mgm liver P 1 mgm plasma P I + n in + IV V + VI VII +VIII IX + X XI + XII XIII XIV XV XVI 16.9 17.0 15.2 13.6 14.5 12.2 6.0 5.0 6.3 6.4 39 50 52 57 58 45 53 58 47 46 5.7 6.4 8.2 8.5 7.7 9.6 0.48 0.44 0.48 0.85 0.89 1.37 1.35 1.52 1.42 1.63 0.59 0.61 1.79 1.61 1.32 1.33 Mean value 7.1 51 7.7 1.04 1.21 Table 5. — ^^P Content of the Free Phosphate in the Blood Plasma and Liver (Slightly irradiated, 77—310 r.) Marking of rat Weight of liver (gm) P content of liver phosphate (mgm%) P content of plasma phosphate (mgm%) % of injected '^P present in 1 mgm liver P 1 mgm plasma P A c::;;:;;;::;;::: D E F G H J K L M N 6.6 6.8 6.5 6.2 7.0 5.9 6.6 8.3 6.6 5.9 5.7 6.0 5.2 49 54 53 54 41 54 46 50 48 42 43 40 53 4.0 4.3 4.4 4.0 7.8 5.6 9.2 6.4 6.5 5.6 7.4 7.9 6.0 2.27 2.31 2.84 2.19 2.31 2.23 1.47 1.55 2.23 1.88 1.89 1.92 1.59 2.60 2.74 2.75 2.97 1.08 1.64 1.21 1.84 1.52 2.05 1.22 1.00 1.81 Mean value 6.3 47 6.1 2.05 1.83 702 ADVENTURES IN RADIOISOTOPE RESEARCH Table 6. — ^^p Content of the Free Phosphate in the Blood Plasma and Liver (Ii-radiated, dosage 460—7000 r.) Eat no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Mean value Weight of liver (gm) P content of liver P content of plasma phosphate phosphate (mgm%) (nigm%) 4.8 79 _ 5.2 69 — 8.0 52 5.9 50 7.4 7.1 55 6.8 4.0 48 6.8 7.0 47 — 6.6 36 — 5.2 41 — 4.4 48 10.0 5.0 45 6.9 4.6 48 8.3 5.5 79 5.7 5.1 89 4.5 4.1 98 6.0 5.9 49 9.2 7.2 45 5.6 4.0 53 4.4 5.8 52 4.9 6.2 60 7.9 6.1 64 7.7 6.6 46 9.2 4.8 69 7.6 5.5 60 .5.9 4.9 67 4.0 7.3 50 6.6 7.5 59 6.7 6.7 57 5.0 5.1 27 5.4 5.4 46 5.0 6.5 54 4.5 6.2 52 5.8 6.3 58 5.8 5.5 62 9.6 5.7 59 6.0 % of injected '^p present in 1 mgm liver P 1 1 mgm plasma P 0.81 0.88 0.79 1.76 1.25 3.15 0.67 0.43 0.37 4.55 4.45 3.90 1.94 1.70 1.77 3.0 2.5 4.7 3.6 1.5 1.1 1.7 1.8 1.7 1.7 1.2 1.8 1.3 3.6 1.9 0.9 1.3 1.7 1.5 2.0 0.94 0.91 1.51 2.31 2.33 2.24 3.50 3.21 3.23 2.5 2.1 2.7 2.7 1.0 1.8 0.6 1.3 1.1 1.7 1.1 0.9 0.7 1.5 1.4 0.9 1.0 1.4 2.1 1.7 DISCUSSION OF RESULTS The data in Tables 1 to 3 and the summarizing presentation in Table 7 show that the formation of new (radioactive) molecules of desoxyribo nucleic acid is in most cases markedly reduced by the action of an X-ray dosage exceeding 450 r. In sarcomas irradiated with less than 450 r EFFECT OF X-RAVS OX XUCLEIC ACID IN JENSEN-SARCOMA 703 the effect of irradition on Ihe formation of radioactivelylabelled nuc- leic acid is ]ess pronounced. In five of the thirty-four sarcomas irradiated with more than 450 r we were unable to detect any effect of irradiation on the formation of Table 7. — Nucleic Acid Metabolism in Unirradiated, Slightly Irradiated (with less than 450 r) and More Strongly Irradiated (more than 450 r) Jensen Sarcoma Sarcoma ""P content of 1 mpm nucleic ac-id P as a percentage of the P'^ content of 1 mgm inorg. liver P 1 mgm inorg. plasma P Unirradiated Slightly irradiated Irradiated Unirradiated Slightly irradiated Irradiated Unirradiated Slightly irradiated Irradiated Mean 1.82 1.38 1.50 Maximu 3.10 2.14 2.12 Minimu 1.13 0.26 0.076 value 1.78 1.81 0.65 m value 2.98 3.11 3.41 m value 1.06 0.22 0.080 nucleic acid. One of these sarcomas was irradiated with 1730 r, a second with 1025 r, a third with 620 r and the remaining two with 465. Presum- ably these sarcomas were more resistant to the action of radiation. In the investigation of the growth of irradiated sarcomas after transplanta- tion, it was found that the individual sarcomas vary in sensitivity toward the action of radiation. Russ and Scott^^\ for example, state that of one hundred Jensen rat sarcomas irradiated with 1000 r, seventy-five recovered, and Suguria^ states, with regard to the sensitivity of the closely related "sarcoma 180", that only half of the sarcomas withered after irradiation with 1000 r. In most cases the difference in the formation of radioactively labelled (and thus newly formed) nucleic acid molecules in unirradiated and irradiated sarcomas is so striking that an unirradiated sarcoma can be distinguished from an irradiated one by just placing the sample under the counting tube. Complete suppression of the formation of active nucleic acid molecules cannot of course be achieved even by using very powerful doses. For example, after the most effective irradiation with IS. Russ and G. M. Scott, Brit. J. Radiol. 13, 267 (1940). 2K. SuGURiA, Radiology 29, 352 (1937). 704 ADVENTURES IN RADIOISOTOPE RESEARCH 7000 r the activity of 1 mgm of nucleic acid phosphorus was found to be equal to 0.1 per cent of the activity of 1 mgm of free liver phosphorus and to 0.08 per cent of the activity of 1 mgm of free plasma phosphorus. The experiments performed up to the present time with Ehrlich carcinomas on mice have to some extent produced results differing from those obtained with Jensen sarcomas, as will be shown in a future communi- cation. By means of the possibility of measuring the effect exerted by X-rays on the formation of nucleic acid molecules in a sarcoma with the aid of radioactive indicators, their action on the sarcomas can be followed chemically. It should be noted that the experiments described are easily performed, preferably by comparing the activities of the nucleic acid phosphorus in the sarcoma and of the free phosphorus in the sarcoma, and also that it would be important to establish the effective dose of X-rays causing a detectable reduction on the formation of radioactively labelled nucleic acid. In the experiments described, the formation of radioactively labelled nucleic acid was determined during the 2 hr following irradiation (the irradiation itself lasted for at most 42 min). There is nothing to hinder determination of the formation of radioactively labelled nucleic acid in the first hour, the first half-hour or even shorter times after the irradia- tion. It is thus possible to determine the chemical effect of X-rays on the sarcoma immediately after irradiation. Phosphorus Content of Liver and Plasma The data in Tables 4—6 show that there is no essential difference between the phosphorus contents of the plasma and liver in unirradiated, slightly irradiated and more strongly irradiated animals (in all cases only the sarcoma was irradiated). The average free phosphorus content of the liver amounts to 51, 47 and 59 mgm %; the corresponding values for the free phosphorus content in the plasma are 7.7,6.1 and 6.0 mgm %. Two hours after injection there is 1.04^^^ 2.05 and 2.00 per cent of the injected ^^Pin 1 mgm of free liver phosphorus in unirradiated, slightly irradiated and more strongly irradiated rats, respectively. The liver therefore contains at this time (mean value of liver weight equals 7.1, 6.3 and 5.7 gm) about 7 per cent of the injected ^^P as free P. In 1 mgm of free plasma phosphorus 2 hr after injection there is 1.21, 1.83 and 1.72 per cent of the injected ^^P. Therefore 1 mgm of free liver 1 In later experiments (see p. 706) 2.13 and 2.16 per cent of the injected ^^P was found, respectively, in 1 mgm of free liver pliosphorus in unirradiated rats and in animals irradiated with 2000 r. The corresponding values for the plasma phosphorus were 1.21 and 1.15. EFFECT OF X-RAYS OX XUCLEIC ACID IX JEXSEX-SAECOMA 705 phosphorus contains nearly the same amount of ^ap as 1 mgm of free plasma phosphorus 2 hr after injection. Comparison of the Activity of Nucleic Acid Phosphorus with the Activity of the Free Phosphorus in the Sarcoma It is evident from the preceding sc^ction that there is less active nucleic acid formed in irradiated than in unirradiated sarcoma. It cannot be concluded unconditionally from this statement, however, that the irradi- ation decreases the rate of formation of nucleic acid in the sarcoma. It might be thought that irradiation would make it difficult for the radio- active tracer to penetrate into the sarcoma cells and that the observed would be due to a decreased permeability of the cell wall to phosphate under the action of the irradiation. The experiences which have been accumulated from various biological systems concerning the action of irradiation on permeability, and which will be reported shortly, are opposed to the latter interpretation of the observed decreased forma- tion of active nucleic acid in the irradiated sarcoma; we have, however, set up experiments, in which the activity of the nucleic acid phosphorus was compared with that of the free phosphorus in the sarcoma, in order to eliminate the explanation by the effect of irradiation on the phosphate permeability. The major portion of the free phosphorus in the sarcoma consists of phosphorus situated within the cells; if a comparison of the ratio of the activities of the nuclei acid P and the free P of the sarcoma indicates a considerably smaller value in the case of the irradiated sarco- ma, then this finding proves unequivocally that irradiation inhibits the metabolism of nucleic acid^. The results of these experiments are seen in Tables 8 and 9. These tables show that the radioactively labelled nucleic acid, i.e. the nucleic acid formed during the 2 hr experimental period after irradiation with 2000 r, was about one-third the amount formed in the unirradiated sarcoma. This result is supported by data obtained by a comparison of the activities of nucleic acid phosphorus and free sarcoma phosphorus in the fresh tissue material. (The behaviour of the necrotic tissue will be discussed in the next section.) As has already been mentioned, the free phosphorus of the sarcoma consists partly of phosphorus originating in the extracellular fluid of the tissue, and this phosphorus has a specific activity (activity per mgm P) which differs from that of the intracellular P. The specific activity of ^ In our earlier experiments we have omitted a study of the activity of free phosphorus in the sarcoma to enable us to use all the sarcoma tissue in obtaining nucleic acid; later, however, we found it decidedly advisable to determine also the specific activity of the free phosphorus in every sarcoma under investigation. 45 Hevesy 706 ADVEXTURES IX RADIOISOTOPE RESEARCH the extracellular P corresponds closely to the activity of the free plasma phosphorus. The extracellular share of the free sarcoma phosphorus, however, is small and, since the activity of the sarcoma phosphorus does not differ considerably from that of the plasma phosphorus, the error committed in disregarding the complex nature of the free sarcoma phosphorus is not important. The rat 47 : 1, for example, contains 1.45 mgm % of extracehular P and 44.8 mgm % of intracellular P. These figures are obtained by assuming that one-quarter of the sarcoma con- sists of extracellular fluid in conjunction with the P content of the plasma (5.8 mgm %) and of the sarcoma (46.3 mgm %). The specific activity of the 1.45 mgin % of extracellular P is 5 per cent higher than that of the 44.8 mgm % of intracellular P (see Table 8) ; by neglecting the extracellular quota, therefore, the specific activity of the intracellular P is overestim- ated by 0.07/44.8 = 0.14 per cent. With regard to the sarcoma cells there is possibly a hmited effect of irradiation on their permeability. Whereas the ratio of the activity of 1 mgm of sarcomaP to that of 1 mgm of plasma P in unirradiated sarco- mas is on the average 1.07 (see Tables 8 and 9), the corresponding ratio in irradiated sarcomas is 0.94; this difference, however, cannot explain the major part of the observed effect. Nucleic Acid Metabolism and Phosphate Permeability of the Necrotic Sarcoma Tissue It is shown in Table 8 that the nucleic acid formation in necrotic sarcoma tissue is indeed considerably less than in fresh tissue but that Table S. — Nucleic Acid Formation in Unirradiated Sarcomas Activity of 1 mgm of nucleic acid phosphoru s as Rat Volume of sarcoma (cm') a percentage of the activity in 1 mgm of liver P plasma P sarcoma P 47 : 1 (200 gm) 38 J fresh 1 necrotic 0.77 0.41 1.13 0.55 1.18 1.08 47 : 2 (145 gm) 13 ( fresh ( necrotic 1.58 3.31 2.12 47 : 3 (1.58 gm) 14 j fresh \ necrotic 1.15 0.71 1.67 1.03 1.23 1.20 47 : 4 (168 gm) 18 j fresh 1 necrotic 0.81 0.29 2.77 0.97 1.93 1.15 51 : 1 (160 gm)| +51 : 2 (132 gm)} 19 + 15 j fresh 1 necrotic 0.97 0.78 1.23 0.96 4.64 1.06 48 : 4b (188 gm) 23 1.13 1.47 1.20 Mean value 1 fresh 1 necrotic 1.07 0.55 1.93 0.88 2.05 1.12 EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 707 the former is not negligible. The same is true of the speed of replacement of intracellular phosphate by plasma (lymphatic) phosphate. The exchange equilibrium in necrotic tissue has not proceeded as far as in the fresh tissue; in the course of 2 hr, however, a considerable portion of the free sarcoma phosphate is replaced by plasma phosphate. Hence it follows that a good circulation of blood (lymph) must exist in part of the necrotic tissue. The intrinsically decreased rate of formation of nucleic acid in necrotic tissue is reduced still further by the action of the X-rays. Pcrcenlagr of injected ^^P present in 1 mgm Liver P Plasma P Sarcoma P Nucleic acid P fresh necrotic fresh necrotic 47 : 1 47 : 2 1.73 1.99 1.83 3.73 1.98 1.51 1.18 0.95 1.26 1.12 1..56 1.21 1.12 1.49 1.72 1.61 0.46 1.43 0.635 1.29 1.08 0.95 1.45 0.0133 0.0313 0.0212 0.0311 0.019 0.017 0.0065 47 : 3 47 : 4 51 : +2 48 : 4b 0.0130 0.0109 0.015 Mean value 2.13 1.21 1.30 1.08 0.022 0.011 Table 9. — Nucleic Acid Synthesis in Sarcomas Irradiated at 80 r/min FOR 25 min Rat Tolume of sarcoma (cm3) Activity of 1 mgm nucleic acid phosphorus as a percentage of the activity in 1 mgm of liver P plasma P sarcoma P 49 : 1 49 : 2 49 : 3 49 : 4 50 : 1 50 : 2 50 : 3 Mean vahie 18 ( fresh 0.68 0.90 0.72 ' necrotic 0.40 0.60 0.88 23 1 fresh \ necrotic 1.00 0.42 0.85 0.33 0.79 0.46 13 1 fresh \ necrotic 0.70 0.025 1.01 0.036 1.00 0.036 19 1 fresh I necrotic 0.12 0.50 0.54 7 1 fresh 0.45 1.14 0.80 1 necrotic — — — 8 J fresh 0.21 0.37 0.31 (necrotic — — — S'i / fresh 0.14 0.13 0.33 1 necrotic 0.073 0.063 0.23 f fresh I necrotic 0.47 0.63 0.65 0.23 0.26 0.16 45^ 708 ADVENTURES IN RADIOISOTOPE RESEARCH Percentage of injected P^^ present in 1 mgm Sarcoma P ISTucleic acid P Liver P Plasma P fresh necrotic fresh necrotic 49 : 1 1.53 0.92 1.15 0.67 0.0083 0.0059 49 : 2 0.64 1.00 0.82 0.58 0.012 0.0026 49 : 3 1.71 1.18 1.19 1.17 0.012 0.00043 49 : 4 6.23(?) 1.60 1.48 1.27 0.0077 , 50 : 1 1.69 0.74 1.05 0.25 0.0084 _ 50 : 2 2.04 1.15 1.38 — 0.0043 _ 50 : 3 1.29 1.50 0.55 0.42 0.0018 0.00095 Mean value 2.16 1.16 1.09 0.73 0.0078 0.0025 The intrinsically decreased metabolism of nucleic acid in necrotic tissue is re- duced Still further by the action of the X-rays. Table 10. — Comparison of the Mean Values of Nucleic Acid Formation and OF Phosphate Permeability in Fresh and Necrotic Tissue Activity of 1 mgm of nucleic acid phosphorus as a percentage of the activity of the free sarcoma P Katio of the activity of 1 mgm of free P of | the necrotic and fresh sarcoma tissues / Unirradiated Irradiated with 2000 r Unirradiated Irradiated Fresh tissue 2.05 0.65 Necrotic tissue 1.12 0.16 0.66 0.76 Amount of Nucleic Acid Newly Formed in the Sarcoma Tissue in the Course of 2 hr We found (Table 8) that two hours after subcutaneous injection of the radioactive phosphate, the activity of the nucleic acid phosphorus amounts to 2 per cent of the activity of the free sarcoma phosphorus. If the activity of the free sarcoma phosphorus were the same during the experimental period as at the end of the experim.ent, it would be possible to conclude from the above data that nucleic acid molecules constituting 2 per cent of the total nucleic acid present in the sarcoma, i. e. on the average 0.18 mgm/g of tissue for sarcomas weighing less than 40 gm (see Table 12), had been formed during the 2 hr experimental period. The activity of the sarcoma P, however, changes in the course of the experiment. If we assume, for example, that it increases linearly with EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 70^ time, then the percentage of newly formed nucleic acid molecules is not 2 per cent but 4 per cent. The change in the specific activity of the free phosphorus in the sarcoma cells does not follow a simple propor- tionality; initially it is practically equal to zero for a few minutes, since the absorption of the injected P and its penetration into the extra- cellular and intracellular volumes require time. On the other hand, the specific activity rises only slightly with time in the last phase of the experiment; when equilibrium between the activity of the plasma (extracellular) P and the cellular P is almost attained, then the specific activity of the sarcoma P changes only slightly with time. In a number of cases has there been not only equalization of the specific activities of the sarcoma and liver phosphorus after two hours, but the former has indeed exceeded the latter. The cause of this process is that the activity of the plasma P reaches a maximum within the first ^2 1^^ after subcutanous injection and then decreases gradually. Very active P penetrates into the cells from the highly active plasma; this P is, of course, again replaced by newly arriving less active plasma P, but the equalization of activity between the sarcoma P and plasma P takes place more slowly than the changes in the plasma activity, and thus is explained the fact that sarcoma P more active than plasma P can be encountered after 2 hr. In the above discussions it must be borne in mind that the free phosphorus of the sarcoma cells has not only the ability to enter and escape through the cell wall, but also has various chances of incorporation into the organic phosphorus-containing mole- cules of the sarcoma cells. After the entry of the very active plasma P there is a correspondingly rapid entry of P into adenosine triphosphate, hexose monophosphate and similar molecules, which then serve as a storage space of the highly active P. If this phosphorus flows back again into the plasma, which has meanwhile become impoverished, then the loss in activity of the free sarcoma P will be compensated by an escape of strongly active P from the storage space and, therefore, this process contributes to maintaining the higher level of activity of the free sarcoma P. In certain conditions the activity of the sarcoma P can indeed undergo a decline, instead of a nonlinear increase with time, in the later phases of the experiment. By multiplying the final value of the specific activity of the sarcoma P by about 1.5, we should obtain the average value for the free sarcoma P during the course of the experi- ment and of the raw material serving for the formation of the active nucleic acid. We then have to multiply by 1.5 the value obtained for t he ratio of the activities of 1 mgm of nucleic acid P and 1 mgm of sarcoma P in order to obtain the value of the percentage increase of nucleic acid in the sarcoma in the course of 2 hr. This value therefore amounts to about 3 per cent of the total quantity of nucleic acid, or on the average 0.26 mgni'/gm of sarcoma. 710 ADVENTURES IN RADIOISOTOPE RESEARCH Growth and Renewal The nucleic acid formed during the course of the experiment, and for that reason radioactively labelled (containing ^^P)^ is either to be found in the newly formed tissue or to be attributed to the renewal of nucleic acid already present. In the case of adenosine triphosphoric acid and some other acid-soluble phosphorus compounds, renewal of the mole- cules in the sarcoma and in other organs takes place at a very high rate. These compounds exhibit radioactivity after the passage of only a very short time when radioactively labelled phosphate is present. The nucleic acid molecules, on the other hand, are only very slowly renewed in the normal organs. Data on the speed of renewal of nucleic acid in the organs of adult rats will be communicated shortly. We assume that the nucleic acid content of the sarcoma is proportional to its weight or volume. This assumption is supported by the observations which are discussed below. The percentage growth of the nucleic acid content is then equal to the percentage volume increase of the sarcoma. By means of the radioactive experiments, we determine the percentage of nucleic acid molecules which have been formed in the period of 2 hours before the rat is killed and we ascertain the growth by studying the volume increase of the sarcoma in the same period. The size, how- ever, is too small to be ascertained by measuring the dimensions of the sarcoma. The growth which has taken place in the last 24 hr, on the other hand, can be measured and hence the growth occurring in the last 2 hr can be calculated. It seems to be more correct, however, to base the calculation of the volume increase in the 2 hr period not on a single measurement, which is affected by uncertainties (see Table 11), but to use a series of measurements which have been made over the last 6 days of investigation. The results of these and other measurements are to be seen in Tables 11 and 12.^^^ The latter table contains a summary of the results obtained for sarcomas which weigh less and more than 40gm.The sarcomas studied by the radioactive methods were mostly lighter than 40 gm and our interest is therefore more particularly in the volume increase of this group of sarcomas. The dry weight of the sarcomas studied varied between 18.5 and 20.7% (average 19.2%) of the weight of the sarcoma. It is readily appreciated that in sarcomas which have nearly attained the limit of their possible growth, the daily percentage volume increase is considerably less than in sarcomas which are able to grow to an almost unlimited extent. The average content of nucleic acid per gm of sarcoma in both cases, however, is found to be essentially the same; it amounts 1 Values of 26.5 and 14.3, respectively, are obtained by calculating the percen- tage volume increase of the sarcoma from measurements taken at the beginning and end of the last day. EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 711 Table 11. — Increask in Volume of the Sarcoma Rat Weight and specific weight of the sarcoma; total nuc- leic acid content per gm Date Volume of sareoma(i) (cm») Daily per cent volume increase Average of daily percen- tage volume increase in the course of the last 6 days 16/5 2.77 48 : la 15 gm; (2.5) 3.6 mgni 18/5 19/5 4.88 5.63 38.1 15.3 25.02 20/5 6.10 8.4 18/5 0.80 — 19/5 1.13 41.2 48 : 2a 8 gm; (2.3) 20/5 21/5 1.62 2.16 43.2 33.4 34.4 22/5 2.49 15.3 23/5 18/5 3.46 38.8 1.09 — 19/5 1.59 46.0 48 : 3a 8 gm; (2.4) 4.9 mgm 20/5 21/5 2.64 2.64 66.6 27.93 22/5 3.35 26.8 23/5 26/5 3.35 1.42 — 27/5 2.01 41.5 48 : lb 19 gm; (2.5) 9.5 mgm 29/5 3/05 3.81 4.23 44.8 11.0 30.5 1/6 6.03 21.5 2/6 7.75 28.5 26/5 3.23 — 27/5 5.24 62.3 48 : 2b 28 gm; (2.0) 7.1 mgm 29/5 30/5 7.37 8.77 20.4 19.0 20.3 1/6 14.25 31.1 2/6 14.25 29/5 0.84 30/5 1.42 69 1/6 1.80 13.4 48 : 5b 11 gm; (1.8) 12.6 mgm 2/6 3/6 2.47 3.52 37 42.6 28.0 4/6 4.53 28.7 5/6 6.03 33.1 ' The volume of the sarcoma was calculated from the length, breadth and depth, assuming an elliptica from of the sarcoma. * Increase observed for 4 days only. ' Increase observed for 5 days only. 712 ADVENTURES IN RADIOISOTOPE RESEARCH Table 11. — (contd.) Eat Weight and specific weight of the garcoma; total nuc- leic acid content per gm Date Volume of sarcoma(') (cm') Daily per cent volume increase Average of daily percen- tage volume increase in the course of the last 6 days 26/5 0.34 — 27/5 1.01 197 • 29/5 1.17 7.9 30/5 1.34 14.5 1/6 1.34 48 : 6b 15 gm; (1.7) 12.3 mgm 2/6 3/6 1.84 2.89 37.3 57 22.7 4/6 2.80 5/6 3.81 36.1 6/6 4.53 18.7 8/6 6.54 22.2 9/6 8.97 34.2 9/6 1.8 48 : 12b 26 gm; (1.9) 13/6 15/6 6.12 11.28 60 42 36.3 16/6 9/6 14.08 1.93 25. — 48 : 13b 20 gm; (1.9) ft 13/6 15/6 5.7 7.84 49 18.8 24.0 16/6 18/5 10.56 34.5 1.93 — 19/5 2.81 45.7 20/5 3.69 31.4 21/5 5.36 45.3 49 : 1 36 gm; (2.0) 22/5 6.34 18.8 23.6 9.1 mgm 23/5 8.13 27.6 26/5 13.49 22.0 27/5 13.49 28/5 18/5 18.44 36.9 1.47 • — 19/5 1.84 25.0 20/5 2.60 .52.2 21/5 3.18 22.5 49 : 3 20 gm; (2.1) 9.3 mgm 22/5 23/5 3.77 5.24 18.5 38.8 25.0 26/5 7.04 11.4 27/5 11.0 56.4 28/5 13.28 20.7 EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SAR(^OMA 713 Eat Weight and specif ic weight of the sarcoma; total nuc- leic acid content per gm Date Volume of sarcoma(') (cm») Dailj- per cent volume increase 1 Average of daily percen- tage volume increase in the course of the last 6 days 18/5 1.51 19/5 2.43 61.0 20/5 3.27 34.5 49 : 4 30 gm; (1.6) 13.7 mgm 21/5 22/5 23/5 4.69 6.37 9.05 43.4 35.9 42.0 21.5 26/5 11.56 9.2 27/5 16.13 39.9 28/5 19.32 19.6 •jO : 1 18 gm; (2.5) 9.9 mgm 4/6 9/3 4/6 9/6 26/5 2.6 7.25 35.6 35.6 50 : 2 18 gm; (2.1) 4.6 mgm 3.35 8.40 30.2 30.2 2.93 27/5 4.32 47.8 29/5 8.46 47.7 48 : 3b 51 gm; (1.7) 13.0 mgm 30/5 1/6 12.57 14.54 21.7 7.8 21.3 2/6 15.50 6.6 3/6 18.81 18.2 4/6 20.53 9.1 5/6 29.30 42.7 26/5 2.43 27/5 4.48 84.4 29/5 5.49 11.3 42 gm; (1.9) 30/5 8.46 .53.3 48 : 4b 14.5 mgm 1/6 11.10 15.0 19.4 14 mgm 2/6 13.20 18.9 3/6 15..50 1 17.4 4/6 21.37 37.9 5/6 22.63 5.9 26/5 2.51 — 27/5 4.06 6.16 48 : 7b 75 gm; (1.9) 14.5 mgm 29/5 30/5 8.42 9.23 ' 53.5 9.6 16.0 1/6 14.71 30 2/6 16.97 15.4 9.6 17.18 1.2 714 ADVENTURES IX RADIOISOTOPE RESEARCH Table 11. — (contd.) Rat Weight and specific weight of the sarcoma, total nuc- leic and content per gm Date Volume of sarcoma(') (cm') Daily per cent volume increase Average of daily percen- tage volume increase in the course of the last 6 days 4/6 22.46 — 48 : 7b (contd.) 75 gm; (1.9) 14.5 mgm 5/6 6/6 8/6 26.4 29.84 36.84 17.5 13.1 11.6 12.8 9/6 40.56 10.0 26/5 4.69 — 27/5 6.37 35.9 29/5 7.75 10.2 30/5 10.89 40.5 1/6 15.71 31.5 2/6 20.66 15.2 81 gm; (1.6) 3/6 21.34 3.4 48 : 8b 10.8 mgm 4/6 27.15 27.0 7.1 5/6 35.95 32.0 6/6 34.22 8/6 38.17 5.6 9/6 42.54 11.5 10/6 49.15 15.4 11/6 51.29 4.3 12/6 51.29 26/5 3.52 27/5 4.99 42 29/5 7.0 21 30/5 10.39 49 1/6 13.67 15.5 2/6 19.19 41.1 79 gm; (1.5) 3/6 23.51 22.5 48 : 9b — 4/6 29.41 25.3 11.9 5/6 29.86 1.5 6/6 31.68 6.0 8/6 41.36 15.1 9/6 37.75 10/6 47.85 29.2 11/6 45.77 12/6 51.33 12.1 48 : 10b 83 gm; (1.7) 26/5 27/5 2.35 3.69 57.2 35.4 29/5 5.53 25 EFFECT OF X-RAYS OX XUCLEIC ACID IX JEXSEX-SAKCOMA 715 Weight and specific weight Volume oC Daih' per Average of daily percen- Eat of the sarcoma; total nuc- leic acid content per gm Date sarcoma' (cm») cent volume increase tage volume increase in the course of the last 6 days 30/5 6.83 23.5 1/6 8.84 14.7 2/6 9.51 7.6 3/6 12.07 26.8 4/6 16.89 40.0 5/6 19.90 17.7 48 : 10b (contd.) 83 gm; (1.7) 6/6 8/6 9/6 20.82 26.15 26.15 4.6 12.8 8.7 10/6 29.87 2.8 11/6 34.91 16.8 12/6 39.13 12.1 13/6 39.13 15/6 41.94 3.6 16/6 48.27 14.8 26/5 1.13 — 27/5 1.76 56.0 29/5 2.81 30.0 30/5 7.04 93.0 1/6 9.22 15.5 2/6 11.31 22.6 78 gm; (1.8) 3/6 15.25 34.7 48 : lib 4/6 16.89 11.4 8.4 5/6 18.77 11.1 6/6 23.59 25.6 8/6 31.68 17.1 9/6 34.32 8.2 10/6 35.2 2.6 11/6 36.29 3.1 12/6 41.4 14.4 13/6 16/5 43.45 3.02 5.0 — 18/5 4.99 33 19/5 6.29 26.1 49 : 2 44gm; (1.9) 20/5 7.25 15.3 12.7 10.1 mgm 21/5 9.30 27.7 22/5 11.44 24.1 23/5 11.44 716 ADVENTURES IN RADIOISOTOPE RESEARCH Table 11. — (contd.) Kat Weight and specific weight of the sarcoma; total nuc- leic acid content per gm Date Volume of sarcoma(') (cm3) Daily per cent volume increase Average of daily percent- age volume increase iu the course of the last 6 days 49 : 2 fi4 gm; (1.9) 26/5 17.30 17 (contd.) 10.1 mgm 24/5 20.91 20.8 13.4 28/5 23.09 10.2 26/5 3.44 — 27/5 6.45 79 29/5 7.42 15.0 30/5 9.13 23.9 1/6 12.19 16.8 50 : 3 64 gm; (1.9) 2/6 15.08 24.0 12 2 10.2 mgm 3/6 17.56 14.5 4/6 20.11 16.4 5/6 21.24 5.6 6/6 27.36 28.8 8/6 33.44 11.1 9/6 33.44 to 8.8 and 12.0 mgm/gm of sarcoma, respectively. This finding, which rela- tes to two groups of sarcoma with very different sizes, supports the correctness of our assumption that the percentage increase in nucleic acid runs approximately in parallel with the percentage volume (weight) increase. Table 12 Average volume increase of 14 and 10 sarcomas wit- hin day Average nucleic acid content per gm of sarcoma Sarcomas weighing less tlian 40 gm 27.4% I 8.8 mgm Sarcomas weighing more than 40 gm 12.8% I 12.0 mgm The average specific weight of the heavy sarcomas varies between 1.5 and 1.9 gm with a mean of 1.8; the corresponding limits for the sarco- mas weighing less than 40 gm are 1.6 and 2.5; the mean value for these amounts to 2.1. For the sarcomas in which we are interested the increase in volume and the corresponding increase in nucleic acid amount to 27 per cent daily, or about 2 per cent in the course of 2 hr.^^^ The newly formed quota 1 The above result must be interpreted with care since the volume increase of the sarcoma is possibly rhythmical during the course of the day. EFFECT OF X-RAYS ON NUCLEIC ACJJJ IX JENSEN-SARCOMA 71 of nucleic acid was obtained from the radioactive measurements within about 3 per cent. A very substantial fraction of the radioactively labelled nucleic acid must therefore be ascribed to the growth process. Effect of X-rays on the Formation of Acid-Soluble Phosphorus Compounds in the Sarcoma In contrast to tlie synthesis of nucleic acid molecules, the formation of acid-soluble phosphorus compounds in the sarcoma is not noticeably affected when exposed to a dosage of a few thousand roentgens, as is shown in Table 13. Table 13. — Formation of Radioactively Labelled Acid-Soluble Phosphorus Compounds IN THE Irradiated Sarcoma Rat Dosage (r) Fraction dc'iioted by its h3'firolysis time of (min) Percentage of injec- ted '^P per mgm P I 1395 7 180 100 95 85 II 1670 7 180 100 88 84 III 2040 7 180 100 100 84 IV 2000 7 100 180 100 100 98 93 V 2000 7 100 180 100 85 85 100 VI 2100 7 100 180 100 92 98 98 It is evident from Table 13 that the 7-min fraction has nearly- the same specific activity as the 0-min fraction, and that the bulk of the remaining fractions is also radioactively labelled, i. e. it has been newly 718 EFFECT OF X-EAYS ON NUCLEIC ACID IN JENSEN-SAECOMA formed in the course of the experiment. X-ray dosages of 2000 r units do not, therefore, prevent the almost complete renewal of the molecules of hydrolysable acid-soluble phosphorus compounds in the sarcoma. The metabolism of the acid-soluble phosphorus compounds is closely related to the oxidation and reduction processes taking place in the cells, and it is well known that these latter processes are quite insensitive towards the action of X-rays. ^^^ Enzyme Activity in Irradiated Jensen- Sarcomas An effect of X-rays on the enzymes taking part in the processes of degradation and synthesis of the acid-soluble phosphorus compounds was not to be expected, having regard to the insensitivity of these pro- cesses to X-rays, as mentioned in the last section. Experiments in which the effectiveness of the catalase isolated from the sarcoma 1 hr after irradiation with 3000 r was studied, showed that this effectiveness had not suffered as a result of irradiation of the sarcoma. While the catalase activity of the muscle of normal rats has been found to be 0.0252, the muscle catalase of the irradiated rats showed a value of 0.0385.^2) The effect of nuclease, obtained from sarcoma irradiated with 3000 r, on desoxyribonucleic acid used as substrate did not in any way fall short of the effectiveness of the nuclease obtained from unirradiated sarcoma. Whereas the former decomposed 66 per cent of the desoxyribo- nucleic acid in the course of 4 hr, the corresponding value for the unirra- diated sarcoma amounted to 46 per cent. From the chemical point of view^ the cell division is a consequence of the very intensive synthetic process which takes place in the nuclei of the cells. Interference with these synthetic processes can stop the cell division. Interference by means of the action of irradiation can take place either by more rapid destruction of the molecules indispensable in the synthesis than the rate at which they are formed, or by the form- ation of noxious products, as a result of the effect of radiation, which enter into the chemical processes occurring in the cell nuclei or in certain parts of these nuclei and thus inhibit the normal course of these processes and their sequel, cell division. Among the effects of X-rays there is, for example, splitting of high polymer molecules, as is known from the 1 Whereas four-fifths of the sarcomas have been prevented from growing after transplantation, by irradiation with 1800 r, the oxygen consumption of these sarcomas was not different from that of the control sample (W. Keil, Arch, exp. Pathol, 167, 338 (1932). 2 These experiments are described in more detail elsewhere. EFFECT OF X-RAYS ON NUCLEIC ACID IN JENSEN-SARCOMA 7 1 9 work of SvEDBERG and Brohult^^\- or degradation of plasma proteins into particles of lower molecular weight. In addition to the cleavage fragments from high polymers, a series of other constituents are found in irradiated tissue such as, for example, nascent oxygen which permits Ihe formation of hydrogen peroxide and other oxidation products. The X-rays have no selective affinity for one or other iyipe of molecule in the tissue; any atom in the irradiated tissue has approximately the same probability of being ionized as any other atom; it can be assessed at 4xl0~" for irradiation with a dose of 1 r.^^^ It is possible that the X-rays and the ions liberated by the radiation act directly upon a con- stituent required for the synthesis of nucleic acid, e. g. by cleaving such a molecule; it is not less probable, however, that the entry of noxious products into the synthetic phase leading to cell division is the determin- ing step. Suniniary Rats with Jensen -sarcoma are injected subcutaneously with a radioactivelj^ labelled phosphate solution; after the passage of 2 hr desoxjribonucleic acid of the sarcoma is isolated. If the nucleic acid is found to bo radioactive it follows that nucleic acid mole- cules have been synthesized in the sarcoma during the course of the experiment.^ A comparison of the activity of 1 mgm of nucleic acid P with the activity from 1 mgm of free sarcoma P makes it possible to determine the amount of newly formed nucleic acid. The nucleic acid molecules formed in the course of 2 hr amount to 2 — 3 per cent of the total nucleic acid content, which on the average constitutes 9 mgm/gm of sarcoma. The irradiation of the sarcoma with 1000 international roentgen units and even with a dose of 450 r or less, causes a decrease of the foimation of nucleic acid, in the great majority of cases, to an average of half to one-third of the value found in an unirradiated sarcoma. Individual sarcomas (five of forty five), which presumably are particularly resistant to radiation, exhibit normal formation of nucleic acid even after irradiation. The method described permits detection of the effect of radiation on the growth of the sarcoma by a chemical method directly after irradiation. The nucleic acid formation in necrotic sarcoma tissue amounts to about half to one-quarter of that found in fresh tissue. The free phosphate of the sarcoma cells is replaced more slowly by plasma phosphate in necrotic tissue than in fresh tissue, but in the necrotic tissue the bulk of the free phosphate originallj^ present in the sarcoma cells is also replaced in a period of 2 hr. iTh. Svedberg and S. Brohult, Nature 143, 938 (1938). 1 P. Jordan, Radiologica 2, 25 (1938). 720 ADVENTURES IX RADIOISOTOPE RESEARCH One milligram of the acid-soluble organic P of the sarcoma, which is decomposed in the course of 180 min hydrolysis, shows nearly the same content of radio - actively labelled phosphorus, 2 hr. after injection, as 1 mgm of free P of the sarcoma. Almost all molecules of the above mentioned acid-soluble phosphorus compounds, of the sarcoma are thus renewed in a period of 2 hr. Irradiation with a dose of 2000 r does not produce any detectable effect on the rate of renewal of the acid-soluble P compounds of the sarcoma. Originally published in Ark. Kern. 19A, No. 9 (1944). 72. THE EFFECT OF X-RAYS ON NUCLEIC ACID FORMATION IN THE ORGANS OF THE RAT LrciE jAhisteom, Hans Euler and George Hevesy From the Institute for Research in Organic Chemistry, Stockholm On an earlier occasion^^^ we found that the synthesis of desoxyribonucleic acid molecules in the Jensen-sarcoma of the rat is inhibited by the action of X-rays. We have extended our studies to the effect of radiation on the metabolism of nucleic acid in the normal organs of the rat and we describe the outcome of these experiments in the following pages. EXPERIMENTAL Albino rats with ages between 3 days and 12 months were available for this work. In order to obtain sufficient amounts of nucleic acid it w^as necessary simultaneously to work with organs of several animals, because the requisite detailed purification of nucleic acid from other phosphorus-containing compounds is accompanied by considerable losses of material. As we described previously^i^ the nucleic acid was purified with hydrochloric acid and methanol, three times with the addition of phosphate and then twice more without. One-fifth of the nucleic acid obtained was used for measuring the radioactivity and another fifth for colorimetric determination of phosphorus. The radioactive phosphate was administered to the rats by sub- cutaneous injection at the beginning of the experiment, using 0.1 cm^ of a physiological saline solution which contained some active sodium phosphate. The dose administered to one rat had an activity of from 3 to 6 //c. The animals were killed two hours after injection. The irradiated animals were given the injection immediately after irradiation. The irradiation dose varied between 1300 and 3000 r. The X-ray tube was operated at a voltage of 165 kV. A 0.5 mm copper foil and 1 mm aluminium foil were used as radiation filters. The field size was 10X10 cm2. 1. H. Euler and G. IIevesy, Kgl. Danske Vidensk. Selskab. Biol. Medd. 17, No. 8 (1942); Ark. Kern. 17A, No. 30 (1944). 46 Hevesy 722 ADVENTURES IN RADIOISOTOPE RESEARCH An average sample of the tissue from the organ was used for isolating the free P in the organ. In some instances the total P content of the organs also was isolated. In these fractions also, one-fifth was used for determining the radioactivity and one-fifth for colorimetric determination of the P content. The radioactivity was measured by means of a Geiger — Miiller counter. EXPERIMENTAL RESULTS The ratio of the ^^P content of 1 mgm of nucleic acid P at the end of the experiment to the ^^p content present on the average in 1 mgm of free P in the organ during the experiment was used as a measure of the rate of formation of nucleic acid. The ^^P content of the free P in the organ was determined at the end of the experiment. The values quoted in the tables are, therefore, not an exact measure of the nucleic acid formation. The rate of formation of nucleic acid in the organs of the rat w as determined in collaboration with J. Ottesen^^\ taking into account the mean activity of free P prevailing during the experiment. In the present study it was desired to investigate the effect of X-rays on the rate of formation of nucleic acid, and for this purpose it is sufficient to compare the activity of the nucleic acid Pat the end of the experiment with the activity of the free P in the organs at the same time. As we shall discuss later (p. 725), the difference between the final and average activities of the free P in the organs involved is not very considerable and in the case of the liver is almost negligible. Table 1 exhibits the rate of formation of nucleic acid in the organs of the unirradiated rat. Table 2 show the metabolism of nucleic acid determined in the organs of the irradiated rat. We have not included any of the results obtained on 5-w^eek old rats in Table 1 since no rats of this age have been irradiated; the figures in Table 1 should be compared with those in Table 2, which contains data on irradiated rats. The results which have been obtained with 5-week old rats are pre- sented in Table la. Finally, a study was made of the rate of formation of nucleic acid in the organs of adult rats which had been starved for 5 days. Table lb shows that the rate of formation of nucleic acid was not much affected by the fasting. It has already been demonstrated by one of us in collaboration with J. OTTESEN^^Hhat the rate of formation of nucleic acid, in contrast to that 1 G. Hevesy and J. Ottesen, Acta Physiol. Scani. 5, 237 (1943). EFFECT OF X-RAYS ON NUCLEIC ACID IN THE RAT 723 Table 1. — Formation of Nucleic Acid in the Ohgans of Unirradiated Adult Rats in a Period of 2 lir Expt. no. 80 82 83 84 85 88 93 97 97 99 103 151 I 151 II Xo. of rats Age aud sex Organ 15 20 1 8 1 year F 8 3 months F 8 1 year F 10 months F 15 months F 6 months F 10 7.5 months F 15 11 months F 11 months F 5 months F 5 months F 6 months F 3 months F Average valnes Activity of 1 mgrn of nucleic acid P as a percentage of the activity of 1 mgm of Free P in the organ liver spleen kidneys liver liver kidneys liver kidneys liver spleen kidneys liver spleen liver kidneys liver spleen kidneys intestinal mucosa liver spleen kidneys intestinal mucosa spleen intestinal mucosa liver liver liver spleen kidneys intestinal mucosa 0.052 1.13 0.119 0.20 0.034 0.070 0.031 0.14 0.0.54 1.41 0.19 0.32 3.57 0.26 0.15 070 3.19 0-18 5.05 0.21 0.25 6.8 2.9 2.4 0.13 0.11 Plasma P 0.188 0.218 0.12 0.069 0.077 0.049 0.13 0.064 1.00 0.18 0.12 2.2 0.30 0.14 0.103 2.51 0-26 2.40 0.18 1.29 0.21 2.6 1.97 0.87 0.31 0.13 0.136 0.203 2.50 1.79 0.158 0.15 1.8 2.1 of most of the acid-soluble phosphorus compounds and phosphatides, in the liver is very small and also that the spleen and especially the intestinal mucosa are characterized by a high formation of nucleic acid; Andreasen and Ottesen^^^ have shown recently that the meta- bolism of nucleic acid in the thymus exceeds that in all other organs and is twice as large in this organ as in the small intestinal mucosa. ^ E. Andreasen and J. Ottesen, Acta Path. Microbiol. Scand. Suppl. 54. (1944). 46^ 724 ADVENTURES I?f RADIOISOTOPE RESEARCH Table la. — Foemation of Nucleic Acid in the Organs OF 5 ^\'EEK Old Rats Activity of 1 mgm of nucleic acid P as a percentage of the activity No. of Age and sex Organ of 1 mgm of Free P in the organ Plasma P 22 5 weeks F liver 0.059 0.02 spleen 4.8 1.1 kidneys 0.159 0.18 20 5 weeks F liver 0.24 0.32 spleen 2.9 2.2 Table 16. — Formation of Nucleic Acid in the Organs of Starved Adult Rats No. of Age and sex Organ Activity of 1 mgm of nucleic acid P as a percentage of the activity of 1 mgm of Free P in the organ Plasma P 6 6 months M 2 — 4 months M liver spleen 0.23 1.08 0.29 8.5 0.22 0.93 0.20 0.62 kidnev.s 0.20 7 intestinal mvicosa liver snlepn 3.9 As we have already stated, the ratio of the specific activity of the nucleic acid P at the end of the experiment to the specific activity of the free P at this same time is not a correct measure of the nucleic acid molecules newly formed during the experiment. An accurate calculation of this quantity requires a knowledge of the average specific activity of the free P in the course of the experiment. With regard to the liver, of course, these last mentioned quantities are very little different from the values which have been obtained for unirradiated rats, as is shown by the following data which we obtained on a previous occasion^-^\ The above figures prove that the average specific activity of the free P in the 2 hr experiment is only about 5% less than the final activity. The number 0.13 in the last horizontal line of Table 1 should, therefore, be multiplied by 1.05 in order to arrive at the percentage rate of nucleic acid formation in the liver in a period of 2 hr. Similar conditions are involved in the kidneys, where the added phosphate ions likewise pene- 1 H. EuLER and G. Hevesy Ark. Kern. 17A, No. 30 (1944). EFFECT OF X-RAYS ON NUCLEIC ACID IN THE RAT 725 trate rapidly into the cells and then gradually leave again. The rate of penetration of the labelled phosphate into the spleen cells is slower and should not differ very considerably from the rate of penetration into the cells of the Jensen-sarcoma, where the final value of the specific activity of the free P was found to be about 25 per cent greater than Time after injection of "n^ (ill-) Percentage of the injected ^^P present in 1 nigm of free liver P ¥2 1 2 2.3 2.8 2.1 the mean value. If the final value 2.5, in the last horizontal row of Table 1, is multiplied by about 1.25 we approach the percentage renewal of nucleic acid in the spleen taking place in a period of 2 hr. We also determined the variation in the specific activity of the free phosphorus in the liver and spleen of 31/2 day rats with time. The follow- ing percentages of the ^^p injected into each rat (0.05 cm^ of radio- actively labelled physiological sodium phosphate solution), were found in 1 mgm of free P as shown : Time (hr) % of injected '''P present in 1 mgm of free liver P % of injected "P present in 1 mgm of free spleen P 'A 1 2 2.12 2.39 2.11 2.13 2.19 2.17 In these experiments also, the final value of the specific activity of 1 mgm of free liver P differs only a little from the mean value of the activity during the experiment. A comparison of the values obtained for the spleens of 3I/2 day rats with those for adult rats (cf. Table 3) yields the result that the phosphate ions penetrate into the spleen cells of 3I/0 day old rats at about the same rate as into the liver cells, whereas the liver cell walls of the adult animals are appreciably more permeable to phosphate than are the spleen cell walls. Effect of Irradiation on the Rate of Formation of Nucleic Acid Table 2 contains data for the rate of formation of nucleic acid in the or- gans of irradiated rats. The animals were exposed to total body irradiation, except that the head was covered with 5 mm thick lead sheet. The time of irradiation amounted to 20 — 40 min. Irradiation was performed with 726 ADVENTURES IN RADIOISOTOPE RESEARCH 165 kV X-rays. A 0.5 mm copper foil and a 1 mm aluminium foil were used as radiation filters. The rats were irradiated at a distance of 28 em. The ^^P was injected immediately after the end of the irradiation. Table 2. — Formation of Nucleic Acid Molecl-xes in the Organ.s of Irradiated Adult Rats Expt. no. No. of rats Age and sex Dose (r) Organ Activity of 1 mgm of nucleic acid P as a percentage of the activity of 1 mgm of Free organ P Plasma P 100 10 5 6 3 3 3 5 months 5 months 9 months 9 months 5^ months 10 months 1480 1480 1480 2000 3000 3000 liver spleen 0.034 0.55 1.7 1.2 2.6 0.036 ■ ■ 1.5 0.038 0.37 0.096 1.90 0.049 0.738 0.061 49 103 intestinal mucosa . . . liver spleen 1.2 0.18 0.89 104 intestinal mucosa . . . liver soleen 0.78 0.044 129 intestinal mucosa . . . liver snleen 0.8 0.056 20 138 liver spleen 0.133 1.45 137 liver spleen liver snleen 0.069 0.51 Mean values 0.0506 0.952 1.9 0.090 708 intestinal mucosa . . . 0.9 Expt. no. No. of rats Age and sex Dose (r) Organ Activity of 1 mgm of nucleic acid P as a percentage of the activity in 1 mgm of Free organ P Plasma P 1151 2 3 months 1300 liver snleen 0.337 2.08 0.353 1.38 ' Two rats having sarcoma were used in this experiment, the whole body being protected, except the ■sarcoma with an area amounting to 5 x 3.5 cm^ with a 5 mm thicl; lead sheet. A comparison of the activity of 1 mgm of nucleic acid P in unirradiated rats, as a percentage of the activity of 1 mgm of free phosphorus in the organ or plasma, with the corresponding figures for unirradiated animals (cf . Table 3) shows that the formation of nucleic acid in the organs which have been studied is inhibited by irradiation. Consequently, the effect of X-rays which has been observed on the sarcoma and which inhibits the formation of nucleic acid, extends to the non-growing normal organs •of adult animals. EFFECT OF X-RAYS ON NUCLEIC ACID I.V THE RAT 727 Table 3. — Comparison of the Activity of 1 mgm of Nucleic Acid P in Unirradiated Rats, Expressed AS A PERCENTAGE OF THE ACTIVITY OF 1 mgm OF FREE Phosphorus in the Organ or Plasma, with the Cor- responding Vali'e in Irradiated Rats Organ Ratio: unirradiated — irradiated Compared witti free P in the organ Compared witli free P in the plasma Liver Spleen Intestinal mucosa 2.3 2.5 2.3 Formation of Nucleic Acid in the Liver of 3V2 — 4V2-day old Unirra- diated and Irradiated Rats We have extended our investigations to the determination of the nucleic acid formation rate in the liver and spleen of strongly growing 3 — 4 day old rats. Twenty-seven rats aged S^v, days were each injected with 0.05 cm-^ of a solution containing ^^p (activity 2 //c). i\fter 2 hr the rats were killed and the nucleic acid P was isolated, as also the free P, from the liver and spleen. The formation of nucleic acid, determined by means of the activities of these fractions, can be seen in Table 4. Table 4. — Formation of Nucleic Acid in the Organs of Twenty-seven Si/o- day Old Rats in a Period of 2 hr Activit}' of 1 mgm of Crude nucleic Separated pure P content of the nucleic acid P as a per- r K a n acid nucleic acid nucleic acid centage of the activity (mgm) (mgm) % of 1 mgm of free P in the organ Liver 76.0 9.3 7.21 1.96 SnlftftTi 20.1 2.1 7.95 9.76 In order to determine the effect of X-rays on the formation of nucleic acid in the liver of strongly growing rats, we irradiated seven 3— 4-day old rats with 2000 r immediately before injecting with radio- active phosphate. The rate of formation of nucleic acid during a period of 2 hr was found to be 0.81 per cent and therefore less than half the value (1.96) determined in the unirradiated animals. This magnitude of nucleic acid formation in the liver of unirradiated or irradiated strongly growing (4-day old) rats is an order of magnitude greater than the formation found in the liver of adult rats. The per- 728 ADVENTURES IN RADIOISOTOPE RESEARCH centage inhibition of the nucleic acid formation due to the action of X-rays is, on the contrary, about the same in the liver of strongly growing and adult rats and, furthermore, it is not greatly different from the percentage inhibition observed in the case of the Jensen-sarcoma . Summary The metabolism of desoxyribonucleic acid has been determined in the organs of 250 rats, aged between 3 days and 1 year, by using radioactivelly labelled phosphorus as an indicator, and a study has been made of the effect of X-rays on the rate of nucleic acid formation. Immediately after irradiation of adult rats with from 1500 to 3000 r, the for- mation of new molecules of desoxyribonucleic acid in the liver, spleen and intesti- nal mucosa is found to be reduced in the course of 2 hr to one-third to one-half, i.e. to an extent similar to that found in the Jensen-sarcoma. The percentage of newly formed desoxyribonucleic acid, in a period of 2 hr, in the hver and spleen of 31/^ -day old rats is forty and ten times, respectively, the amount found in the corresponding organs of adult animals. The percentage reduction in the formation of nucleic acid in the organs of 3 — 4 -day old rats due to the action of X-rays is not greatly different from that observed in the organs of adult rats. 729 Comment on papers 71 and 72 That DNA is built up prior 1o cell division was inferred at that date (1939) when the study, the result of which is communicated in paper 71, was initiated. A sup- pression of DNA formation should thus lead to a mitoitic arrest, and vice ve^rsa and a suppression of DNA formation under the effect of exposure to irradiation should reflect itself in a depressed ^^p incorporation into the DNA molecules formed in the tissue of rats studied (papers 71 and 72 and Euler and Hevesy, 1944). To determine a change of 1 per cent in the DNA content of a tissue with cytochcmical methods, difficulties are encountered even today (in the meantime great progress was made in this field). The tracer method is very suitable for demonstrating small differences in the new-formation of DNA molecules. If during the experiment in the non-exposed Jensen sarcoma 1 per cent of the DNA mole- cules get labelled (thus newly formed) a formation of 0.96 per cent only in tlic irradiated sarcoma (thus a difference of 0.04 per cent in the total DNA content) will be easily detectable. That DNA is built up prior to cell division was first proved by Howard and Pelc (1951). In Vicia faha which they investigated, the synthesis was already found to be terminated 6 hr prior to the beginning of visible prophase. They made use of the powerful autoradiographic method in their investigations. After inadiation with a Roentgen lay dose of 300 r or more, incorporation of ^-P into the DNA of the Jensen-sarcoma of most of the few hundred investigated rats (paper 71 and Euler and Hevesy, 1944) was found to be strongly diminished, as well as incorporation into the normal organs of 250 partly growing and paitly adult rats (72). The cells of the organs investigated were in different stages of the mitotic cycle, and, correspondingh^ the results obtained indicate a resultant of the effect of exposure to radiation on cells which were in different stages of the division process. Holmes (1947, 1948) investigated the effect of irradiation with Rontgen rays both on the incorporation of P^^ into DNA and RNA. These investigations brought out as well, as did very numerous later studies, the blocking effect on ^^p incorpora- tion into DNA, while incorporation of ^^P into RNA was found by Holmes to be only slightly affected. The result arrived at in papers 71 and 72 and by Euler and Hevesy (1944) that exposure to Roentgen rays diminished ^^p incorporation to about half the value observed in controls, was substantiated by a great number of investigations (as for example, by those of Pelc and Howard, 1955) publis- hed in the course of the last fifteen years, not, however, a possible interpretation of this effect. According to the latter, incorporation of '^P into DNA may be partly due to additional formation of DNA molecules and partly to renewal of DNA molecules already present. It was suggested that irradiation with Roentgen-rays possibly interferes with the formation of additional DNA molecules but not with their turnover. We know today that in the growing tissue the formation of labelled DNA molecules takes place at least in most cases in connection with mitotic processes only. This view was already put forward in the first investi- gation in this field (paper 67) where it is stated: "The rate of renewal of the nucleic acid molecules in the liver may be identical with the rate of new-for- mation of livor cells" Irradiation with ionizing radiation can interfere with DNA formation in diffe- rent ways. Cell destruction produced by irradiation will stop DNA synthesis. 730 ADVENTURES IN RADIOISOTOPE RESEARCH As first shown by Howard and Pelc (1953) irradiation can stop cell division even in that part of the interphase in which the DNA synthesis, preceding mitosis, is already terminated. Cell division being blocked, DNA synthesis is bound to stop after the lapse of hours the length of which depends on the system investi- gated. We meet here a second indirect way of interference with DNA formation. The third way is a direct interference with DNA synthesis. This is due, as made very probable by Orden and Stock and others, to a disturbance of the template, to a macromolecular lesion as denoted by Mitchell, and also to a change produced in the phosphorylating enzymes. According to te results obtained by Lajtha et al. (1958) investigating i*C incorporation into bone marrow cells, doses below 300 r produce in cells which are in the presynthctic period at the time of radiation a 40—50 per cent depression of the number of cells entering the subsequent synthe- tic period in a given time, without affecting the rate of subsequent DNA synthesis. As to the 50 per cent depression of DNA formation due to exposure to radiation Lajtha et al. put forward two alternative suggestions, (a) There are two cell populations in the presynthctic phase, one sensitive and one resistant to small doses of radiations. The sensitive cells can be prevented from entering the synthe- tic period; the resistant ones will enter into and proceed in it undisturbed, (h) All presynthctic cells are sensitive, and the maximum damage results in slowing down the rate of entry into the synthetic period about half. This occurs not in a form of accumulation of presynthetic stage cells just before the beginning of the synthe- tic period, but more likely by slowing down the "progress through the presynthetic period". Thus an unambiguous explanation of the 50 per cent depression of DNA synthesis due to exposure to irradiation is still outstanding. References H. EuLER and G. Hevesy (1944) Ark. Kemi. A 17, No. 30. B. E. Holmes (1947) Brit. J. Radiol. 20, 450. B. E. Holmes (1949) Brit. J. Radiol. 22, 260. A. Howard and S. R. Pelc (1951) J. Exp. Cell Res. 2, 178. S. R. Pelc and A. Howard (1955) Radiation Research 3, 135. A. Howard and S. R. Pelc (1953) Heredity Suppl. 6, 261. L. G. Lajtha, R. Oliver, T. Kumatori and F. Ellis (1958) Rad. Res. 8, 1. Originally published in Arkiv for Kemi 24 A, 12. (1!)47). 73. TURNOVER OF NUCLEIC ACID IN RETROGRADE SARCOMATA L. Ahlstrom, H. Euler, and G. Hevesy From the Institute for Research in Organic Chemistry, Stockholm Desoxyribosenucleic acid is wholly or mainly confined to the cell nuclei. That desoxyribosenucleic acid vanishes in certain phases of the mitotic cycle and accumulates in others, was conspiciously shown by Caspersson^ in making use of the technique of ultraviolet absorption. We have, therefore, to expect an appreciable formation of "new" desoxy- ribosenucleic acid molecules in mitotic cells. The ultraviolet absorption method has the great adventage that it makes it possible to carry oul micro-determinations //? situ. When applying the radioactive method, we have to isolate the desoxyribosenucleic acid P from all other phos- phorus fractions present. This requires the employment of substantial quantities of tissues. The radioactive method has, however, 2 advent- ages: (a) It indicates all new formation of the desoxyribosenucleic acid, including any such formation which takes place without a perceptable change in the total amount of desoxyribonucleic acid present. (b) The formation of small amounts of new desoxyribosenucleic acid as correspond to only 0.1 percent, or even less of the total amount of desoxyribonucleic acid present in the tissue sample under investigation, can be determined. The unique sensitiveness of the radioactive method is due to the fact, that the "old" molecules not being radioactive, are not registered at all; and, correspondingly, if the number of molecules increases during the experiment from 1000 to 1001, the radioactivity of the sample increases from to 1. The result arrived at is thus in contrast to all other methods, not based on a small difference between two large figures. Former investigations into the percentage of new formation of des- oxyribosenucleic acid in the growing Jensen sarcomata led to the result that in the course of 2 hours about 2 out of 100 desoxyribosenucleic acid molecules are newly formed. 'i)Cf. T. Caspersson and L. Santesson, Acta Rad. Suppl. 17 (1942). 732 ADVENTURES IN RADIOISOTOPE RESEARCH Among the numerous Jensen sarcomata obtained by grafting, there were found occasionally those, which showed a spontaneous regression. It seemed to be of interest to investigate if the enzyme cycle leading to the formation of new desoxyribosenucleic acid molecules is undisturbed in such regressive sarcomata, i. e. if the percentage renewal of the des- oxyribosenucleic acid molecules present in the regressive sarcoma deviates from the figure obtained in the growing sarcoma, or not. EXPERIMENTAL RESULTS In Table 1 the date of inoculation, the weight of the rat and the volume of the sarcoma at different dates is given, thus demonstrating the rate of regression of the sarcomata. Furthermore, the ratio of the activity of 1 mgm desoxyribosenucleic acid P to that of 1 mgm inorganic P from the sarcoma, the plasma and the liver respectively, is stated. Table 1 Experiraent 176: I to III Male rat born ^^9 1944, inoculated with Jensen sarcoma 22/^, Date Weight in pm Volume of sarcoma length of axes in cm I. ^7l2 98 Vi 140 1.3 X 2.2 X 1.5 ^7i 138 1.4 X 2.5 X 1.5 ^Vi 1.4 X 2.6 X 1.5 "A 1.3 X 2.3 X 1.5 II. Vi 164 1.1 X2.7 X 1.3 ^Vi 143 1.2 X 2.7 X 1.5 «/l 1.2 X 2.7 X 1.3 III. Vi 156 1.3 X 2.6 X 1.4 ^Vi 147 1.2 X 2.7 X 1.4 "A 163 1.1 X2.5 X 1.3 "A 1.1 X 2.35 X 1.3 Experiment 177: I to IX. Male rat born October 1944 inoculated 2-/12 Date Weight in gm Volume of sarcoma axes in cm I. ^Vl2 105 Vi 157 ^Vi 155 1.3 X 2.8 X 1.5 "A 153 1.3 X 3.1 X 1.7 NUCLEIC ACID IX RETROGRADE SARCOMATA 733 Date Weight in gm Volume of sarcoma axes in cm 30/^ 180 1.7 X 4.4 X 2.5 V2 200 1.8 X 3.5 X 2.7 '/a 1.6 X 3.5 X 2.6 II. "A 125 1.0 X 2.2 X 1.1 ''ll 0.8 X 2.0 X 1.3 30/^ 1.2 X 2.4 X 1.5 V2 1.0 X 2.0 X 1.3 III. . 30/^ 124 1.3 X 2.7 X 1.7 V3 147 1.0 X 2.8 X 1.7 IV. "A 1.0 X 2.3 X 1.3 -Vi 146 1.1 X 2.3 X 1.4 30/^ 156 1.0 X 2.2 X 1.3 V2 156 0.9 X 2.0 X 1.3 V. ^Vi 1.2 X 2.4 X 1.4 "A 145 1.4 X 3.1 X 1.7 '7i 148 1.4 X 2.7 X 1.55 V-a 170 1.0 X 1.9 X 1.2 VI. ^Vi 1.0 X 2.0 X 1.2 ^Vi 122 1.1 X 2.2 X 1.2 30/^ 124 1.15 X 2.4 X 1.3 V2 136 0.9 X 2.3 X 1.2 VII. ^Vx 1.1 X 2.1 X 1.2 "/l 145 1.2 X 2.2 X 1.2 30/^ 149 1.1 X 2.3 X 1.3 V2 160 1.0 X 2.2 X 1.2 VIII. 24/^ 161 1.3 X 2.7 X 1.7 30/ 165 1.5 X 2.7 X 1.9 V2 1.0 X 1.9 X 1.4 IX. 30/^ 124 1.2 X 2.7 X 1.4 V2 133 0.9 X 2.4 X 1.6 V2 0.9 X 2.2 X 1.3 Activity of 1 mgm desoxyribosenucleic acid P, expressed in percentage of the activity of 1 mgm inorganic P from the sarcoma plasma liver fresh tissue 1.98 1.82 1.53 necrotic tissue 2.08 2.04 1.72 734 ADVENTURES IN RADIOISOTOPE RESEARCH Experiment 180: I to IV Female rat bom "/lo 19^4, inoculated 2% 1945 I. "./2 ^72 II. ^72 ^72 III. -/2 ^72 IV. ^72 '-72 "Weight in gm Volume of sarcoma axes in cm 156 1.0 X 1.6 X 0.9 165 0.8 X 1.5 X 1.0 139 1.1 X 2.2 X 1.3 156 0.8 X 1.7 X 1.1 165 0.8 X 1.5 X 0.8 168 very small 150 149 very small Activity of 1 mgm desoxyribosenucleic acid P, expressed in percentage of the activity of 1 mgm inorganic P from the sarcoma P plasma P liver P 2.76 1.61 1.10 Experiment 186: I to X Male rat born January 1945, inoculated 174 Date "Weight in gm Volume of sarcoma axes in cm I. '72 215 1.3 X 2.4 X 2.0 24/2 1.3 X 2.3 X 2.0 II. '72 210 0.8 X 1.4 X 1.1 '72 0.7 X 1.2 X 1.1 III. 72 148 0.7 X 1.3 X 1.9 '72 165 0.7 X 1.2 X 0.9 IV. "A 215 0.6 X 1.5 X 0.8 '7i 0.6 X 1.4 X 0.9 V. "A 200 1.1 X 1.5 X 1.2 '7x 1.0 X 1.4 X 1.2 VI. "A 173 1.0 X 1.7 X 1.4 'Vi 1.0 X 1.5 X 1.4 VII. "A 175 1.4 X 2.4 X 2.0 '7i 1.3 X 2.3 X 1.9 VIII. "A 205 0.7 X 1.2 X 1.0 '7x 0.7 X 1.1 X 0.9 IX. "A 193 0.9 X 1.2 X 0.9 'Vi 0.8 X 1.2 X 0.9 X. '7i 194 0.9 X 1.3 X 1.0 24/^ 0.9 X 1.3 X 1.0 NUCLEIC ACID IN RETROGRADE SARCOMATA 735 Activity of 1 mgm desoxyribosenucleic acid P, expressed in percentage of the activity of 1 mgm inorganic P from the sarcoma plasma liver 1.12 1.05 1.28 Activity of 1 mgm desoxyribosenucleic acid P, expressed in percentage- of the activity of 1 mgm inorganic P from the sarcoma P plasma P liver P 2.57 1.18 0.94 Experiment 54: I to III. Volumina of the sarcomata are stated in K. Sv. Vet.-Akad. Arkiv f. Kemi A 17 No. 30, 0. 41. Activity of 1 mgm desoxyribosenucleic acid P, expressed in percentage of the activity of 1 mgm inorganic P from the sarcoma plasma liver 1.19 1.05 1.03 In Table 2, data on the mean renewal rate of desoxyribosenucleic acid in 26 sarcomata are given. (A) is the mean average for each separate experiment involving several animals. (B) is the mean figure, when all the sarcomata are considered col- lectively. Table 2 Activity of 1 mgm desoxyribonucleic acid P of retrograde Jensen sarcomata, expressed in percentage of the activity of 1 mgm inorganic P from the Sarcoma Plasma Liver (A) (B) 1.92 2.14 1.34 1.42 1.18 1.19 In Table 3 we state the ratio of the activity of 1 mgm desoxyribose- nucleic acid P, to that of 1 mgm inorganic P from the sarcoma, the plasma and the liver respectively, as found earlier. A comparison of the figures in Tables 2 and 3 leads to the conclusion that there is no pronounced difference between the percentages of newly formed desoxyribosenucleic acid in retrograde and in growing sarcomata; the difference amounting only to 12 per cent when consi- dering the "sarcoma scale", and to 25 per cent when considering the "plasma scale". The significance of these scales is discussed below. The autolysis of the tissue, followed by a regression of the sarcoma, leads to a desrease in the total desoxyribosenucleic acid content of 1 he- 73() ADVENTURES IN RADIOISOTOPE RESEARCH Table 3 Activity of 1 mgm desoxyribosenucleic acid P of growing Jensen sarcomata in percentage of the activity of 1 mgm inorganic P from the Sarcoma Plasma Liver r«j(^) (b)(') rc>i(^) 2.05 2.17 2.37 1.93 1.66 1.89 1.07 1.07 H. EULER, and G. Hevesy, Sv. Vet.-Akad. Arkiv /. Kemi, A 17, No. 30 (1944). •'' Average of 4 not previously published results. sarcoma, while the desoxyribosenucleic acid concentration in the sarcoma remains practically unchanged. We found the mean unpurified desoxy- ribosenucleic acid content of 1 gm growing sarcoma to be 9.2 mgm, this value representing the average of a very great number of investigated Jensen sarcomata. For the spontaneously retrograde sarcoma the corresponding figure was found to be 8.8 and for benzpyrene produced sarcoma (cf. p. 738) the value was 8.0 In the growing sarcoma an increase of the total desoxyribosenucleic acid content by 1 per cent during the experiment leads to a formation of labelled molecules up to at least 1 per cent of the desoxyribosenucleic acid content of the sarcoma. Such an increase is thus clearly indicated by the radioactive method. A decrease of the desoxyribosenucleic acid content by 1 per cent, however, will hardly be indicated by tracer experiments, since, to a very large extent, non-labelled molecules dis- appear. Should we find a decreased percentage new formation of des- oxyribosenucleic acid in the retrograde sarcoma, this result would thus indicate that the mechanism of desoxyribosenucleic formation does not function normally in the retrograde sarcoma. As seen above, this is not the case; or the case only to a very restricted extent. This result illustrates the fact that the tissue autolysis, followed by resorption, (the necrotic process) has very little in common with the more subtile enzyme "new formation" process; a result which is in no way surprising. Taurog and his associates(i) placed surviving liver slices for a few hours in a Ringer solution containing labelled phosphate. An appreciable part of the ^^p added was found to be present at the end of the experiment in the phosphatides isolated in the tissue slices. A quanti- tative determination of the extent of renewal of the tissue phosphatide was not carried out, but it can be estimated to have amounted to a few per cent. Simultaneously the decrease in the total phosphatide content of the tissue slices was determined. It was found to be as much as 20 ^1^ A. Taurog, J. L. Chaikoff and J. Perlman, J. Biol.Chem. 145, 281 (1942). NUCLEIC ACID IN RETIIOGKADE SARCOMATA 737 per cent. Thus, with a substantial autolytic decomposition of the tissue phosphatides, a less pronounced formation of labelled phosphatides takes place. The latter more subtle process can be almost quantitatively suppressed by 0.01 mgm KCN, while the extent of autolysis is not at all diminished by the presence of cyanide; a slight decrease of the rate of autolysis being even observed. In our experiments^^) on the formation of labelled desoxyribosenucleic acid in surviving slices of Jensen sarcoma, incubated at 37^ in a Ringer solution containing labelled phosphate, we found, in experiments taking 4 hours, about 0.1 per cent of the desoxyribosenucleic acid molecules present at the end of the experiment to be labelled. The desoxyribose- nucleic acid content of such slices was found to be diminished by 25%, due to autolysis, in the course of 24 hours at 20". In a retrograde sarcoma the tissue not involved in the necrotic process is thus showing a almost normal desoxyribosenucleic acid formation. Since in the retrograde sarcoma the percentage formation of labelled desoxyribosenucleic acid is not appreciably lower than in the growing sarcoma, we have to conclude that a growth of the intact parts of the retrograde sarcoma takes place as well; possibly at a somewhat lower rate as indicated by the figures of Table 2. In some of our earlier experiments we irradiated the rats all through the experiment. In these experiments the rats were still fixed to a table after the injection of the labelled phosphate had taken place. The ratio of the specific activity of the desoxyribosenucleic acid P, to that of the inorganic P of the sarcoma and the plasma respectively, was found in some of these experiments to be much lower than in our usual experi- ments, where the rats were fixed to a table during the irradiation, but not after the injection of the labelled phosphate had taken place. The low radioactivity found when the rats were fixed to a table, and irradia- ted all through the experiment, was discovered not to be due to a cor- respondingly diminished turnover of the desoxyribosenucleic acid under the action of radiation, but mainly to a disturbance in the circulation of the rat. We found, not in all, but in several of the control experiments in which rats were fixed to a table without being irradiated, very low activity figures for the desoxyribosenucleic acid. The disturbed circula- tion results in a low ratio of the specific activity of the inorganic P of the plasma and the sarcoma, which can become in such experiments with disturbed circulation as low as ^/^q. In such cases a very appreciable part of the inorganic P of the sarcoma is made up of extracellular inor- ganic P. Furthermore, the disturbed circulation also influences the resorption of the injected phosphate, and the difference between the ^-^ L. Ahlstrom, H. Euler and G. Hevesy, Sv. Vet-Akad. Arkiv f. Kemi, A 21, No. 6 (1945). 47 Hevesv 738 ADVENTURES IN RADIOISOTOPE RESEARCH end and the mean value of the specific activity of the tissue inorganic P during the experiment becomes very appreciable. That the fixing of the rat on to the table all through the experiment does not necessarily interfere with the normal circulation, is seen from the following activity ratios: Percentage ratio of the specific activity of the nucleic acid P to that of the inorganic sarcoma P Percentage ratio of the specific activity of the nucleic acid P to that of the inorganic plasma P Control Fixed to table 1.47 1.54 In this case the muscles of the rat presumably relaxed. That the effect of fixing can obstruct the circulation to a high degree, is seen from the following figures: Fixed to tab'.e 0.234 0.1 II In our later experiments, in which the animal was irradiated all through the experiment, we placed the rat in a beaker coated with black paper. The X-ray dose administered was controlled by placing a micro-ionising chamber on the sarcoma. In these experiments no appre- ciable difference was found between the results obtained either when the irradiation took place only before the injection or when it took place all through the experiment; as seen in Table 4. Table 4. Irradiation of Rats in Glass Beaker With a Total Dose of 1000 r Control Irradiated uefore injection only Irradiated A] through tlie experiment Specific activity of desoxyribosenucleic acid P, expressed in percentage of the specific activi- tv of sarcoma inoreranic P 2.95 0.983 1 1 0.980 1.17 1.08 Specific activity of desoxyribosenucleic acid P, expressed in percentage of the specific activi- ty of plasma inorganic P 2.29 0.775 1.02 1.15 Desoxyribosenucleic Acid Formation in Benzpyrene Sarcomata In a few cases we determined the rate of renewal of desoxyribosenucleic acid in sarcomata obtained by grafting tumour tissue produced by treatment of rats with benzpyrene. The results obtained are seen in Table 5. NUCLEIC ACID IX KETROGBADE SARCOMATA 739 Table 5. Percentage New Formation of Desoxyribosenucleio Acid IN Benzpyrene Sarcomata Aotivity of 1 nism nuc-leio acid P expressed Xo. of Weight of in perfentage of the activity of 1 mgm experiment sarcoma, in gm inorganic P from the sarcoma plasma liver 163: 1 4 1.71 1.91 1.20 163: 2 2 172: a 50 1.44 1.17 0.95 172: b 5 1.27 ].05 0.85 174: a 12 3.4 1.38 0.81 0.60 174: b 3 6 1.20 0.90 0.93 179 10 9 2.05 1.81 1.18 Mean value 1.51 1.24 0.97 Table 6. . — Percentage New Formation of Desoxyribo- .senucleic Acid in Retrograde Benzpyrene Sarcomata Xo. of experiment Date of inoculation 19M Weight of rating Sarcoma volume; axes in cm 163: 1 ■'Vs 125 "/9 153 2.0 X 3.9 X 1.5 VlO 193 1.4 X 3.1 X 1.5 7l0 1.3 X 2.4 X 1-3 163: 2 V9 180 1.2 X 2.4 X 1.3 79 1.0 X 1.8 X 1.3 Activity of 1 mgm nucleic acid P expressed in percentage of the activity oi' 1 mgm inorganic P from the sarcoma 1.71 plasma 1.91 liver 1.20 Activity of 1 mgm nucleic acid P expressed in percentage of the activity of 1 mgm inorganic P from the sarcoma 0.99 plasma 0.81 liver 0.66 The mean new formation figure obtained for the benzpyrene sarcomata is lower than the corresponding figure obtained for Jensen sarcomata. While we investigated several hundred Jensen sarcomata, the number 47* 740 ADVENTURES IN RADIOISOTOPE RESEARCH Table 6. (Cont) No. of experiment Date of inoculation 1944^1945 Weight of rating Sarcoma volume; axes in cm 178 : 1 ^Vl2 147 "A 166 1.75 X 3.2 X 2.0 ^Vi 175 1.4 X 3.1 X 1.7 ^7i 175 1.3 X 2.8 X 1.4 V2 210 1.2 X 2.4 X 1.0 V2 1.1 X 2.4 X 1.0 178 : 2 ^Vi 200 1.3 X 2.2 X 1.3 V2 230 1.3 X 1.9 X 1.4 V2 . 1.3 X 1.8 X 1.3 178 : 3 30/^ 212 1.7 X 4.9X2.6 V2 240 1.6 X 4.6 X 1.7 V2 1.5 X 4.0X1.7 of benzpyrene sarcomata investigated amounts to ten only; the difference obtained in the renewal rate of the tvi^o types of sarcoma is therefore to be interpreted cautiously. We investigated furthermore 5 retrograde benzpyrene sarcomata. Two (163: 1 and 163: 2) are showing almost normal turnover rates, while ^appreciably lower values were found for the remaining 3 sarcomata. Desoxyribosenucleic Acid Formation in Sarcomata of Colchicine Treated Rats In view of the effect of colchicine on the mitotic process, it was of interest to investigate the effect of colchicine treatment on the turnover of desoxyribosenucleic acid, and to determine if it is possible to obtain similar effects by colchicine treatment as obtained by irradiation. The following experiments were carried out: (a) Rats weighing 138 and 111 gm respectively were injected with 50 y colchicine. Injection was repeated after a lapse of 6 days, and one day later labelled sodiumphosphate was administered by subcutaneous injection. The rat was killed, as in all our other experiments, 2 hours later, and the desoxyribosenucleic acid of the sarcoma, weighing 23 gm, and the inorganic P of sarcoma and plasma were isolated. (b) In these experiments 50 7 colchicin ewere injected subcutaneously, one day before the administration of ^^p to rats weighing 112, 126, 165 and 125 gm respectively. (c) In this experiment 150 y colchicine were administered on three consecutive days. One day later ^sp ^yas injected. (d) ^^P was administered 3 days after injecting 50 y colchicine. Weight of rats was 120 and 155 gm resp. Weight of the only slightly necrotic sarcoma was 9 and 7 gm respectively. NUCLEIC ACID IX RETROGRADE SARCOMATA 741 Table 7. — Colchicine Treated Rats Specific Activity of Desoxyribosenucleic Acid expressed in Percentage of the Specific Activity of the Inorganic P from the .sarcomata and plasma: Experiment (particulars see above) Sarcoma Plasma (a,) 1.78 2.69 2.01 1.52 1.06 1.63 1.46 1.52 1.05 1.68 (b) I II Ill IV 1.94 1.94 1.47 1.14 (c) 2.10 (d) I II (e) 1.44 1.98 0.99 \^/ Mean value 1.66 1.62 (e) 50 y colchicine were administered both 7 days before, and 3 days before the injection of labelled phosphate. Weight of rats was 97 and 135gmresp.atthe start, 105 and 150 gm at the end of the experiment. Weight of sarcomata 5 and 4 gm resp. with hard necrotic inclusions. The results obtained are seen in Table 7. The mean value obtained for the extent of renewal of desoxyribose- nucleic acid in the colchicine treated rats is about ^s lower than the corresponding value for the controls (cf. Table 3) ; the individual values vary greatl3^ The variation in the "plasma scale" is less than it is in the "sarcoma scale", thus indicating a somewhat enhanced phosphate permeabily of the sarcomata in colchicine treated rats. In some of our experiments, we studied the combined effect of Roentgen radiation colchicine injection on nucleic acid foriration. Table 8 denotes an experiment in which (a) Colchicine was administrated 1 day before the injection of 32p. (b) Colchicine was administered; the following day the rat was irra- diated with 30 r per minute for 10 minutes, ^^p then injected and the rat killed 2 hours later. (c) The same procedure was followed as in (b), but without colchicine treatment. (d) The experimental conditions were those of (b) with the sole excep- tion that the rat was irradiated with only 15 r per minute, for 10 min. The results obtained were seen in Table 8. The combined effect of colchicine and X-rays is, as shown by the above figures, no greater than is the effect of X-rays alone in influencing the turnover rate of desoxyribosenucleic acid of the sarcoma. In view 742 ADVENTURES IX RADIOISOTOPE RESEARCH of the results obtained by Levine/^^ who found a combination of X-ray and colchicine treatment of roottips to be very effective in retarding the growth of ^/ZmmCepa, a more detailed investigation of such a combined effect on the nucleic acid turnover in animal tissues may be of interest. Table 8. — Percentage New Formation of Desoxyribosenxjcleic Acid in Colchicine Treated and Irradiated Rats Percentage Specific Act- Percentage Specific Act- ivity of Nucleic Acid P: vity of Nucleic Acid P: Specific Activity of in- Specific Activity of in- organic sarcoma P organic plasma P (a) 1.85 1.36 (b) 1.43 1.25 (c) 1.43 1.53 (d) 1.61 1.61 Replacement of Irradiation with Roentgen Rays by Radiation Emitted by Injected Radio-Elements By injecting very substantial amounts of ^^P we can expect to find a reduced formation of labelled desoxyribosenucleic acid. 1 microcurie per gm tissue produces in the course of 2 hours an ionisation corresponding to 3.5 r units(2).To produce 175 r units in the course of 2 hours we have to administer about 50 microcuries per gm, or 7.5 millicuries, to a rat weigh- ing 150 gm. In this calculation the excretion of some of ^^P administered during the experiment taking 2 hours is disregarded. We have not at our disposal sufficiently strong radioactive prepara- tions to carry out such an experiment. In one experiment, however, we injected 3.9 millicuries of radiosodium 5 hours before administering a tracer dose of ^^P. The mean radiosodium activity per gm tissue of the rat weighing 190 gm was in this experiment 20.5 microcurie. The /j-rays of the radiosodium acted upon the tissue for 5 + 2 = 7 hours producing an ionisation corresponding to 250 r units. Radiosodium emits, l)eside /5-rays, also y-rays, which are only partly absorbed into the rat's body. The ionisation produced by these rays in the rat can be estimated as corresponding roughly to a total dose of about 60 r in the course of 7 hours, bringing the total dose up to about 310 r. The radiosodium was administered by subcutaneous injection in tlie form of 1.2 ml physiological sodiumchloride solution. In the control experiment the same volume of non radioactive sodiumchloride was ^'i>M. Levine, Cancer Res. 3, 107 (1943). ^2) L. D. Marinelli, Amer. J. Roentgenol. 47, 210 (1942). NUCLEIC ACID IX RETROGRADE SARCOMATA 743 injected 5 hours before the administration of a tracer dose of ^-P. The results obtained are seen in Table 9. T.\BLE 9. — Effect of Administration of Ra- diosodium of the formation of labelled Desoxyribosenuci-eic Acid in the Liver in the Course of 2 Hr. Specific Activity of Nucleic Acid P expressed in Percenta£referred. Summary The percentage new formation of desoxyribonucleic acid phosphorus is only slightly smaller in the spontaneously regressive Jensen sarcoma of the rat than in the growing tumour. This fact indicates that the enzyme mechanism, respon- sible for the incorporation of phosphate into the desoxyiibonucleic acid molecule, is hardly disturbed in the regresssive sarcoma. Treatment of rats with colchicine influences the rate of formation of desoxy- ribosenucleic acid phosphorus to a minor extent, A somewhat lower formation rate of desoxyribosenucleic acid phosphorus is observed in grafted benzpyicne tumouis, than in Jensen saicoma. The rate of formation of labelled desoxyribosenucleic acid under uninterrupted irradiaton during the whole experiment, previously reported to be very low, is to be explained by a disturbance in the circulation of the rat, and not by a correspondingly low rate of formation of labelled dosoxyribospnucleic acid. Originally published in Ark. Kemi. 19A, No. 13 (1945). 74. THE INDIRECT EFFECT OF X-RAYS ON THE JENSEN-SARCOMA L. Ahlstrom, H. Euler and G. Hevesy From the Institute for Research in Organic Chemistry, Stockholm Several observations have been made that besides the direct effect of X-rays there is also an indirect effect which is able to influence the division of cells. Kok and Vorlaender(^) observed, for example, an indire